HK40063948A - Systems and methods for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications - Google Patents

Description

Systems and methods for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications

The present application is a divisional application of an invention patent application, filed on the mother as 2015, 11/04, application No. CN201580030686.1, entitled "system and method for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications".

Technical Field

Embodiments of the present invention generally relate to the field of replication and amplification of nucleic acid molecules. More specifically, certain embodiments of the present invention relate to the replication of DNA molecules from biological samples using rolling circle replication. Other embodiments of the invention relate to the amplification of DNA molecules from biological samples using rolling circle amplification. Certain embodiments of the invention can be used to characterize sequence variations in a genome derived from a biological sample. Certain embodiments of the invention can be used to perform molecular enumeration of whole chromosomes or portions thereof derived from a biological sample. Certain embodiments of the invention may be used to characterize haplotype structure in a genome derived from a biological sample. Certain embodiments of the invention may be applied to sample preparation and analysis in genome science, biomedical research, diagnostic assays, and vaccine and therapeutic development.

Background

Genome-wide techniques, such as high-density genotyping arrays and next-generation sequencing (NGS), can identify sequence variations, particularly Single Nucleotide Polymorphisms (SNPs) and Single Nucleotide Variants (SNVs), of a given individual or species, which are collectively referred to herein as "sequence variants". However, current methods are unable to determine the combination of those sequence variants on the same DNA molecule. Determining the combination of sequence variants is referred to as "phasing" and the particular combination of sequence variants on the same DNA molecule is referred to as a "haplotype". For example, a human individual is diploid, each individual cell containing two sets of autosomes inherited from each parent. Characterization of the haplotype status of a given individual is important for locating disease genes, elucidating the history of the population, and studying the balance of cis-acting and trans-acting variants in phenotypic expression.

There are three general methods of determining haplotype information: (i) group inference; (ii) parent inference; and (iii) molecular haplotype analysis. The most common method for phasing haplotypes is to use inferential and statistical methods from data obtained from the population genotypes or the parental genotypes. However, haplotype information across the entire genome cannot be resolved computationally, particularly when linkage disequilibrium is low for a given chromosomal region and for rare variants. On the other hand, parental inference methods rely on the principle of genetic inheritance of sequence variations in the context of family pedigrees. While effective when performed correctly, many biological samples lack sufficient lineage information or require appropriate family samples to infer the haplotype status of a given sample of interest.

Various molecular haplotype analysis methods are known to overcome the limitations of the calculation-based methods. These molecular methods include various strategies to isolate a single DNA molecule or group of single DNA molecules, followed by genotyping or sequencing to determine the haplotype structure of a given biological sample. One such strategy involves constructing a large insert clone (i.e., F cosmid) library. These clones are then diluted in individual wells of a multiwell plate (i.e., 96-well plate or 384-well plate) to form a template library, barcoded to track specific clones to individual wells, and characterized by genotyping or sequencing methods.

The challenge of phasing haplotypes of individual chromosomes or portions thereof has been reduced to characterizing smaller DNA fragments (i.e., from hundreds of megabases to tens of kilobases to hundreds of kilobases in size) in dilution wells in microtiter plates. It has also been reported to size DNA fragments or to use genomic DNA instead of forming large insert clones, followed by dilution, amplification by whole genome methods, formation of template libraries, and sequencing to determine the haplotype of a given sample. Whole chromosomes or portions thereof can also be isolated by flow sorting methods or microdissection methods, followed by dilution, amplification by genome-wide methods, formation of template libraries, and genotyping or sequencing to determine the haplotype structure of a given genome from a biological sample. All of these methods require a high level of technical expertise and the formation of large numbers of individual template libraries (in the order of hundreds) in phasing the haplotypes of a given biological sample.

Most imaging systems are not capable of detecting a single fluorescence event and therefore have to amplify DNA molecules in the sample. There are currently three next-generation sequencing methods: (i) emulsion pcr (empcr); (ii) solid phase amplification; and (iii) solution-based rolling circle replication. For all of these methods, genomic DNA is typically fragmented using standard physical shearing techniques to form a library of DNA fragments. There are exceptions in which fragmentation may not be required. For example, some biological sources, such as plasma or serum obtained from cancer patients or pregnant women, contain circulating cell-free genomic DNA fragments that are typically present in a size of less than 1,000 base pairs (bp), and in some cases less than 500 bp. An intermediate step of size selection is then performed, depending on whether a universal priming site is required, followed by ligation of the adaptor sequence containing the universal priming site to the end of the DNA fragment. A limited number of PCR cycles were performed using common PCR primers. These three methods differ in this step, but in all cases, these clonal amplification methods are limited to replicating or amplifying small fragments that are typically less than 1,000bp in size, and in more typical examples, 700bp or less. For example, the solid-phase amplification method of Illumina can amplify only a DNA fragment having a size of 700bp at most. This size limitation limits the ability to assemble the human genome de novo.

A significant disadvantage of current genome-wide technologies, and NGS in particular, is the reliance on sequence reads derived from short template libraries, which are then clonally amplified in a massively parallel fashion. Importantly, current paired-end library construction methods essentially destroy the ability to readily identify large complex structural changes that exist in the normal human genome and appear to be particularly important in the development of many diseases. Genomic structural variation may represent a driving force in early tumorigenesis and cancer progression, disease susceptibility, and therapy resistance. Sequence reads derived from short template libraries make it very difficult to fully resolve novel, repetitive, and disease-altered sequences via de novo assembly. Thus, most whole genome sequencing efforts still rely on aligning sequence reads to a reference genome. Thus, NGS datasets may contain large stretches of human genomic sequences that have not yet been characterized, and the understanding of disease mechanisms may be biased by the lack of genomic structural information.

Most whole genome sequencing efforts still rely on aligning sequence reads to a reference genome. Although alignment experiments can capture a large fraction of sequence variants, large templates of about 10kb to 100kb are required to resolve a large fraction of structural variants and/or to provide phasing of haplotypes across the entire human genome. Many molecular biology and computer software techniques have been used to overcome size limitations. Despite some improvements, the drawback is the complexity of the biological workflow and the significant increase in costs associated with reagents, labor, and computer hardware.

The formation of DNA circles by ligating the ends of linear nucleic acid fragments is a very inefficient method, requiring significant amounts of starting material from a biological sample. The problems associated with forming loops by bringing the distal ends of a given DNA fragment close to each other have been recognized in the art since the 80's of the 20 th century. For example, one problem associated with forming loops by ligating the ends of DNA fragments together is a competing reaction between an "intramolecular" ligation event (i.e., a DNA loop of the same DNA fragment) and an "intermolecular" ligation event (i.e., the ligation of two or more DNA fragments, referred to as a concatemer). Another problem associated with forming loops by ligating the ends of DNA fragments together is that larger DNA fragments must be further diluted to achieve reasonable efficiency of forming intramolecular loops as compared to smaller DNA fragments.

There is a need in the art for innovative methods that combine the formation of large DNA circles (i.e., large insert sequence clones of 5kb to 7kb or larger for Sanger sequencing) with the high-throughput replication or amplification properties of next generation sequencing methods. Certain embodiments of the present invention overcome the size limitation of forming DNA circles from large DNA fragments by forming DNA circles in a size-independent manner. Other embodiments of the invention overcome the size limitations of templates that directly amplify >1 kilobases by incorporating size-independent DNA loops, by forming and replicating or amplifying large insert sequence templates that can be used for many genome science applications. The present invention also overcomes the complexity and associated higher cost of the researchers working with current methods by providing a simpler workflow for preparing large insert sequence templates using dumbbell loops and improved methods for rolling circle replication and rolling circle amplification to form multiple copies for sequencing applications. Certain embodiments of the present invention also overcome the limitations of genotyping and sequencing applications that require separate allele-discriminating primers for a set of different heterologous nucleic acid sequences by providing a simpler workflow for preparing templates that rely on universal primer sequences.

Disclosure of Invention

One embodiment of the invention is a method of replicating at least one DNA molecule. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of replicating at least one DNA molecule. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated fragmented nucleic acid molecules with an exonuclease; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one DNA molecule. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of detecting at least one replicated dumbbell template. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template; and detecting the at least one replicated dumbbell template. In another embodiment, said step of detecting said at least one replicated dumbbell template consists of sequencing said at least one replicated dumbbell template.

Another embodiment of the invention is a method of detecting at least one replicated dumbbell template. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated fragmented nucleic acid molecules with an exonuclease; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template; and detecting the at least one replicated dumbbell template. In another embodiment, said step of detecting said at least one replicated dumbbell template consists of sequencing said at least one replicated dumbbell template.

In certain embodiments, the step of detecting the at least one replicated dumbbell template comprises contacting the at least one replicated dumbbell template with an oligonucleotide probe. In certain embodiments, the oligonucleotide probe is a labeled oligonucleotide probe. In certain embodiments, the oligonucleotide probe is a labeled DNA probe. In certain embodiments, the oligonucleotide probe is linked to a fluorophore.

Another embodiment of the invention is a method of detecting at least one amplified DNA molecule. The method comprises the following steps: fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified DNA molecule; and detecting the at least one amplified DNA molecule. In another embodiment, the step of detecting the at least one amplified DNA molecule consists of sequencing the at least one amplified DNA molecule.

In certain embodiments, the step of detecting the at least one amplified dumbbell template comprises contacting the at least one amplified dumbbell template with an oligonucleotide probe. In certain embodiments, the oligonucleotide probe is a labeled oligonucleotide probe. In certain embodiments, the oligonucleotide probe is a labeled DNA probe. In certain embodiments, the oligonucleotide probe is linked to a fluorophore.

Another embodiment of the invention is a method of replicating at least one DNA molecule. The method comprises the following steps: isolating at least one DNA molecule from the sample; fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one DNA molecule. The method comprises the following steps: isolating at least one DNA molecule from the sample; fragmenting at least one DNA molecule to form at least one fragmented DNA molecule; ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified DNA molecule.

Another embodiment of the invention is a method of replicating at least one DNA molecule. The method comprises the following steps: isolating at least one DNA molecule from the sample; ligating one or more hairpin structures to each end of the at least one DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one DNA molecule. The method comprises the following steps: isolating at least one DNA molecule from the sample; ligating one or more hairpin structures to each end of the at least one DNA molecule to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified DNA molecule.

Another embodiment of the invention is a method of detecting at least one amplified dumbbell template. The method comprises fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule to form at least one dumbbell template; purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated fragmented nucleic acid molecules with an exonuclease; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template; and detecting the at least one amplified dumbbell template.

Another embodiment of the invention is a method of amplifying at least one nucleic acid molecule. The method comprises isolating at least one nucleic acid molecule from a sample; ligating one or more hairpin structures to each end of the at least one nucleic acid molecule to form at least one dumbbell template; purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated fragmented nucleic acid molecules with an exonuclease; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

Embodiments of the invention also include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a polymerase and at least one primer substantially complementary to a region of the at least one dumbbell template for replicating the at least one dumbbell template to form at least one replicated dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a replicator and at least one primer substantially complementary to a region of said at least one dumbbell template for replicating said at least one dumbbell template to form at least one replicated dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a polymerase and at least two primers substantially complementary to at least two regions of the at least one dumbbell template for amplifying the at least one dumbbell template to form at least one amplified dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a replicator and at least two primers substantially complementary to at least two regions of said at least one dumbbell template for amplifying said at least one dumbbell template to form at least one amplified dumbbell template.

Drawings

So that the manner in which the features and advantages of the invention, as well as others which will become apparent, can be understood in more detail, embodiments of the invention will be described in more detail with reference to the embodiments thereof which are illustrated in the appended drawings, which form a part of this specification. It is to be noted, however, that the appended drawings illustrate only various embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other effective embodiments.

Fig. 1 is a schematic diagram of an exemplary method of rolling circle replication of a dumbbell template according to one embodiment of the invention.

FIG. 2 is an image of an agarose gel analysis of a rolling circle product produced from a dumbbell template according to one embodiment of the invention.

Fig. 3 is a schematic diagram of an exemplary method of rolling circle replication of a dumbbell template according to one embodiment of the invention.

FIG. 4 is an image of agarose gel analysis of dumbbell templates and their rolling circle products produced according to one embodiment of the invention.

FIG. 5 is an image of agarose gel analysis of dumbbell templates and their rolling circle products produced according to one embodiment of the invention.

FIG. 6 is an image of an agarose gel analysis of a dumbbell template generated according to one embodiment of the invention.

FIG. 7 is an image of an agarose gel analysis of a dumbbell template generated according to one embodiment of the invention.

FIG. 8 is an image of an agarose gel analysis of a rolling circle product produced according to an embodiment of the invention.

FIG. 9 is an image of an agarose gel analysis of a rolling circle product produced according to an embodiment of the invention.

FIG. 10 is a diagram showing detection of hairpin structures by fluorescence according to one embodiment of the invention.

Fig. 11A and 11B are images of an exemplary device according to some embodiments of the present invention.

Detailed Description

Before describing embodiments of the present invention in detail, a number of terms used in the context of embodiments of the present invention will be defined. In addition to these terms, other terms are defined elsewhere in the specification as desired. Unless otherwise explicitly defined herein, technical terms used in the present specification will have their art-recognized meanings.

In order to more readily facilitate an understanding of the present invention, the meanings of the terms used herein will become apparent from the context of the specification in view of the common usage of the various terms and the explicit definitions provided below. As used herein, the terms "comprising", "containing", "including" and "such as" are used in their open, non-limiting sense.

By "amplified dumbbell template" is meant a nucleic acid molecule containing one or more hairpin structures that results in multiple copies of the target sequence as a result of rolling circle amplification.

By "contacting" is meant a process of introducing a substance by any means to facilitate an interaction with another substance. By way of example and not limitation, a dumbbell template can be contacted with one or more substantially complementary primers to facilitate one or more hybridization processes to form one or more duplex regions capable of participating in rolling circle replication or rolling circle amplification.

By "detecting a nucleic acid molecule" is meant using an analytical method that can determine the presence of a nucleic acid of interest or can determine more detailed information about the nucleic acid sequence, changes in the nucleic acid sequence when compared to a reference sequence, or the presence or absence of one or more copies of the nucleic acid sequence.

By "dumbbell template" is meant an in vitro replicative or in vitro amplifiable nucleic acid molecule that is linear in structure and topologically circular, having one or more hairpin structures. When denatured or substantially denatured, the dumbbell template exists as a circular single-stranded nucleic acid molecule. Dumbbell templates differ from in vivo replicating circular double-stranded DNA such as, but not limited to, plasmids, cosmids, F-cosmids, bacterial artificial chromosomes, and yeast artificial chromosomes, which are formed by means of cloning vector technology. Unlike these circular double stranded DNAs, which replicate independently in appropriate host cells, the dumbbell template does not require reproductive replication in such host cells.

By "end of a fragmented nucleic acid molecule" is meant one or more terminal nucleotide residues that are or are to be made capable of participating in a ligation reaction. In certain embodiments, one or more nucleic acid molecules may contain functional ends that are capable of, or are to be enabled to, a ligation reaction to allow one or more hairpin structures to be ligated to each end of the nucleic acid molecule. By way of example and not limitation, the terminal nucleotide at the 5 'terminus contains a phosphate group and the terminal nucleotide at the 3' terminus contains a hydroxyl group.

By "fragmented nucleic acid molecule" is meant any larger nucleic acid molecule that is changed by the fragmentation process to any smaller nucleic acid molecule.

By "fragmenting" is meant the use of a chemical or biochemical agent to fragment a nucleic acid molecule in a sequence-independent manner (i.e., randomly) or in a sequence-specific manner. For example, nucleic acids can be randomly fragmented by: enzymatic methods using dnase I, endonuclease V, or transposase; using physical methods such as shearing, sonication, or nebulization, the latter of which passes a nucleic acid solution through a small orifice; or using mechanical forces such as, without limitation, acoustic methods, in particular adaptive focusing acoustic methods. Random nucleic acid fragments can be prepared by PCR using random primers. Nucleic acids can also be fragmented by sequence specific methods, such as, without limitation, using restriction endonucleases and multiplex PCR. The collection of fragments resulting from the fragmentation process of one or more larger nucleic acid molecules is referred to as a library.

By "hairpin structure" is meant a nucleic acid molecule within which two or more partial sequences are complementary or substantially complementary to each other, thereby resulting in the formation of a partially double-stranded region and one or more internal single-stranded regions. The hairpin structure may also contain two or more nucleic acid molecules linked together by a linker and two or more partial sequences of the two or more nucleic acid molecules are complementary or substantially complementary to each other, thereby resulting in the formation of a partially double-stranded region and one or more internal single-stranded regions.

"isolating a nucleic acid molecule" means the process of obtaining a nucleic acid molecule from a sample.

"linker" means to covalently link two or more nucleic acid molecules by an enzymatic agent, such as, without limitation, DNA ligase or RNA ligase; or chemical agents such as, without limitation, condensation reactions using water-soluble carbodiimides or cyanogen bromides, and standard procedures associated with automated DNA synthesis techniques, to produce natural nucleic acid backbone structures, modified nucleic acid backbone structures, and combinations thereof. The native nucleic acid backbone structure is for example, without limitation, composed of one or more standard phosphodiester bonds between nucleotide residues. Modified nucleic acid backbone structures are for example, without limitation, composed of one or more modified phosphodiester linkages, such as replacement of non-bridging oxygen atoms by nitrogen atoms (i.e., phosphoramidate linkages) or sulfur atoms (i.e., phosphorothioate linkages), replacement of bridging oxygen atoms by sulfur atoms (i.e., phosphorothioate linkages), replacement of phosphodiester linkages by peptide linkages (i.e., peptide nucleic acids or PNAs), or formation of one or more additional covalent linkages (i.e., locked nucleic acids or LNAs) having an additional bond between the 2 '-oxygen and the 4' -carbon of the ribose. The modified linkages may each be one type of modification or may be any combination of two or more types of modifications and further may comprise one or more standard phosphodiester linkages.

"linker" means one or more divalent groups (connecting members) that serve as a molecular bridge for covalent bonding between two other nucleic acid molecules. A linker may contain one or more linking members and one or more types of linking members. Exemplary connection members include: -C (O) NH-, -C (O) O-, -NH-, -S-, -S (O) n- (where n is 0, 1 or 2), -O-, -OP (O) OH) O-, -OP (O)^-) O-, alkanediyl, alkenediyl, alkynediyl, aryldiyl, heteroaryldiyl, or a combination thereof. Some linkers have side chains or side chain functional groups (or both). The pendant moieties can be hydrophilic modifiers (i.e., chemical groups that increase the water solubility of the linker), such as, without limitation, solubilizing groups, e.g., -SO₃H、-SO₃ ^-、CO₂H or CO₂ ^-。

By "nucleic acid molecule" is meantAny single-or double-stranded nucleic acid molecule comprising standard canonical bases, highly modified bases, non-natural bases, or any combination of these bases. By way of example and not limitation, a nucleic acid molecule contains four canonical DNA bases, i.e., adenine, cytosine, guanine, and thymine; or four canonical RNA bases, namely adenine, cytosine, guanine, and uracil. When the nucleoside contains a 2' -deoxyribosyl group, uracil may be substituted for thymine. Nucleic acid molecules can be converted from RNA to DNA and from DNA to RNA. By way of example and not limitation, reverse transcriptase may be used to form mRNA into complementary DNA (cDNA), and RNA polymerase may be used to form DNA into RNA. The nucleic acid molecule may also contain one or more highly modified bases such as, but not limited to, 5-hydroxymethyluracil, 5-hydroxyuracil, alpha-putrescine thymine, 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine, N⁴-methylcytosine, 2-aminoadenine, alpha-carbamoylmethyladenine, N⁶-methyladenine, inosine, xanthine, hypoxanthine, 2, 6-diaminopurine, and N⁷-methylguanine. The nucleic acid molecule may also contain one or more non-natural bases such as, but not limited to, 7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG). Nucleic acid molecules containing only canonical bases, highly modified bases, non-natural bases, or any combination of these bases, can also contain, for example and without limitation, linkages in which each bond between nucleotide residues can consist of a standard phosphodiester linkage, and in addition, can contain one or more modified linkages, such as without limitation, substitution of a non-bridging oxygen atom by a nitrogen atom (i.e., an phosphoramidate linkage), by a sulfur atom (i.e., a phosphorothioate linkage), or by an alkyl or aryl group (i.e., an alkylphosphonate or arylphosphonate); the bridging oxygen atom is replaced by a sulfur atom (i.e., a phosphorothioate); phosphodiester bonds are replaced by peptide bonds (i.e., peptide nucleic acids or PNA); or form one or more additional covalent bonds (i.e., locked nucleic acids or LNAs) having additional bonds between the 2 '-oxygen and the 4' -carbon of the ribose sugar. The term "2' -deoxyribonucleic acid molecule" means a nucleic acid molecule"identical, with the proviso that the 2 '-carbon atom of the 2' -deoxyribosyl group contains at least one hydrogen atom. The term "ribonucleic acid molecule" means the same as the term "nucleic acid molecule", with the limitation that the 2' -carbon atom of the ribosyl group contains at least one hydroxyl group.

"nucleic acid sequence" means the order of canonical bases, highly modified bases, non-natural bases, or any combination of these bases present in a nucleic acid molecule.

By "performing" is meant providing all necessary components, reagents, and conditions that enable a chemical or biochemical reaction to proceed to obtain a desired product.

By "purified" is meant that substantially all of the undesired components are separated from the desired components of a given mixture. By way of example and not limitation, purification of dumbbell templates refers to a process that removes unwanted nucleic acid molecules that have not been successfully ligated to form dumbbell templates of any given size range.

By "replicated dumbbell template" is meant one nucleic acid molecule containing one or more hairpin structures that results in multiple copies of the target sequence as a result of rolling circle replication.

"Rolling circle amplification" or "RCA" means a biochemical process using two or more primers in which, in addition to the original dumbbell-shaped template, the copied nucleic acid molecule is also used as a template in subsequent rounds of amplification to prepare further copies of the starting nucleic acid molecule.

"Rolling circle replication" or "RCR" means a biochemical process using one or more primers in which the copied nucleic acid molecule is not used as a template in subsequent rounds of replication to make further copies of the starting nucleic acid molecule. In certain embodiments, when the dumbbell template is a positive strand, rolling circle replication produces more copies of the negative strand. In certain embodiments, when the dumbbell template is a negative strand, rolling circle replication produces more copies of the positive strand. Replication, as used herein, is distinct from amplification which utilizes copies of nucleic acid in subsequent rounds of amplification to make more copies of the starting nucleic acid molecule.

By "sample" is meant material obtained from a biological sample or synthetically produced source containing a nucleic acid molecule of interest. In certain embodiments, the sample is a biological material containing the desired nucleic acids for which data or information is sought. The sample can include at least one cell suspected of containing the target nucleic acid molecule, a fetal cell, a cell culture, a tissue specimen, blood, serum, plasma, saliva, urine, tears, vaginal secretions, sweat, lymph, cerebrospinal fluid, mucosal secretions, peritoneal fluid, ascites, fecal matter, body exudates, cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryos, lysates, extracts, solutions, or reaction mixtures. Samples may also include non-human sources, such as non-human primates, rodents, and other mammals, pathogenic species, including viruses, bacteria, and fungi. In certain embodiments, the sample may also include isolates from environmental sources for the detection of pathogenic species in human and non-human species as well as blood, water, air, soil, food, and for the identification of all organisms in the sample without any a priori knowledge. In certain embodiments, the sample may contain degraded nucleic acid molecules. Nucleic acid molecules may have nicks, breaks, or modifications resulting from exposure to physical forces, such as shear forces; harsh environments, such as heat or ultraviolet light; chemical degradation processes, such as can be used for clinical or forensic analysis; biodegradation processes due to microorganisms or aging; purification or separation techniques; or a combination thereof.

"sequencing" means any biochemical method that can identify the order of nucleotides from a replicated dumbbell template or an amplified dumbbell template.

By "substantially complementary primer" is meant a nucleic acid molecule that forms a stable double-stranded duplex with another nucleic acid molecule, even though one or more bases of the nucleic acid sequence within the duplex region do not base pair with the other nucleic acid sequence.

The basic structure of single-stranded nucleic acid molecules and double-stranded nucleic acid molecules is determined by base pair interactions. For example, the formation of base pairs between complementary or substantially complementary nucleotides on two opposing strands will cause the two strands to wind around each other to form a double helix structure. This is referred to as intermolecular base pairing of complementary nucleotides of two or more nucleic acid molecule strands. The term "nucleotide" is broadly defined herein as a unit consisting of a sugar, a base, and one or more phosphate groups, for which the sugar, for example and without limitation, consists of: ribose, a modified ribose having an additional chemical group attached to one or more atoms of the ribosyl group, 2' -deoxyribose, or a modified 2' -deoxyribose having an additional chemical group attached to one or more atoms of the 2' -deoxyribosyl group; and for it, the base is for example, without limitation, composed of canonical bases, highly modified bases, or non-natural bases, as described above in the definition of nucleic acid molecule. Base pairing of complementary nucleotides or substantially complementary nucleotides can also occur on the same DNA strand molecule, which is referred to as intramolecular base pairing of complementary nucleotides or substantially complementary nucleotides.

Hairpin structures can be formed by intramolecular base pairing of complementary nucleotides or substantially complementary nucleotides of a given nucleic acid molecule, which can form a stem-loop structure. The stem portion of the hairpin structure is formed by hybridization of complementary nucleotides or substantially complementary nucleotide sequences to form a double-stranded segment. The loop region of the hairpin structure is the result of the unpaired segment of the nucleotide sequence. The stability of the hairpin structure depends on the length of the stem region, the nucleic acid sequence composition, and the degree of base pair complementarity or substantial complementarity. For example, a stretch of five complementary nucleotides may be considered more stable than a stretch of three complementary nucleotides, or a stretch of complementary nucleotides consisting essentially of guanine and cytosine may be considered more stable than a stretch of complementary nucleotides consisting essentially of adenine and thymine (DNA) or uracil (RNA). Modified nucleotides, examples of which include, but are not limited to, inosine, xanthine, hypoxanthine, 2, 6-diaminopurine, N, may be substituted to alter the stability of the double-stranded stem region of these natural bases⁶-methyladenine, 5-methylcytosine, 7-deazapurine, 5-hydroxymethylpyrimidine. Modified nucleotides may also be includedIncluding the many modified bases present in the RNA species. Naturally occurring stem-loop structures are found primarily in RNA species such as transfer RNA (trna), microrna precursors, ribozymes, and their equivalents.

Nucleic acid hairpin structures can be created by deliberate design, using methods for making synthetic oligonucleotides. Oligonucleotides are widely used as primers for DNA sequencing and PCR, probes for screening and detection experiments, and linkers or adapters for cloning purposes. Short oligonucleotides ranging from 15 nucleotides to 25 nucleotides can be used directly without purification. Since the stepwise yield is less than 100%, the longer oligonucleotides need to be purified by high performance liquid chromatography or HPLC, or by preparative gel electrophoresis to remove the fraction of failed oligonucleotides, also referred to as n-1, n-2, etc. products. In certain embodiments, the nucleic acid hairpin has approximately 100 bases.

Depending on the nature of the experiment, a given hairpin structure can be designed to contain the desired stability of a double stranded duplex, which is achieved by: substitution with one or more highly modified or unnatural bases and/or one or more backbone linkages as discussed herein; or include other synthetic bases such as 7-deaza-7-hydroxypurine, isoC and isoG, or their equivalents; and forming, for example and without limitation, RNA-DNA, PNA-RNA, PNA-PNA, LNA-DNA, LNA-RNA, LNA-LNA double stranded duplexes. Synthetically designed hairpin structures can be used in a variety of molecular biology techniques, such as, without limitation, as priming sites for DNA polymerases by ligating hairpins to the ends of DNA fragments, as detection moieties for probes to identify sequences of interest, and to generate topologically circular DNA molecules from linear fragments. In certain embodiments, the 5' -end of one or more hairpin structures will be phosphorylated, for example and without limitation, using T₄The polynucleotide kinase is phosphorylated to facilitate efficient ligation to the ends of one or more fragmented nucleic acid molecules using a ligation agent.

In certain embodiments, amplified or replicated dumbbell templates can be detected using oligonucleotide probes. The oligonucleotide probe may be a labeled oligonucleotide probe. The oligonucleotide probe may be a labeled DNA probe. In certain embodiments, the oligonucleotide probe may be linked to one or more of a fluorophore, a chromophore, a radioisotope, an enzyme, or a luminescent compound, or a combination thereof.

Certain hairpin structures have also been used as oligonucleotide probes. Some DNA probes, also known as molecular beacons, are oligonucleotides designed to contain internal probe sequences with two ends that are complementary to each other. Under appropriate conditions, the ends hybridize together to form a stem-loop structure. The probe sequence is contained within the loop portion of the molecular beacon and is independent of the stem arm. A fluorescent dye is attached to one end of the stem and a non-fluorescent quenching moiety or "quencher" is attached to the other end of the stem. In the stem-loop configuration, the hybridization arm holds the fluorescent dye and the quencher in close proximity, thereby causing quenching of the fluorescent dye signal by the well-known Fluorescence Resonance Energy Transfer (FRET) process. When the probe sequence within the loop structure finds its intended target sequence and hybridizes to it, the stem structure is disrupted to facilitate a longer and more stable probe-target duplex. Probe hybridization causes the fluorescent dye and quencher to separate (i.e., now lose proximity) so the dye can now fluoresce upon exposure to the appropriate excitation source of the detector. Molecular beacons have been used in a number of molecular biology techniques, such as real-time PCR, to identify allelic differences.

In certain embodiments, a hairpin structure may be formed by using two or more nucleic acid molecules, which are then linked to form a single hairpin structure. Two or more nucleic acid molecules can be linked together using a linking reagent to form a hairpin structure. Linkers can also be used to chemically link two or more nucleic acid molecules together to form a hairpin structure. In certain embodiments, the 5' -end of one or more hairpin structures will be phosphorylated, for example and without limitation, using T₄The polynucleotide kinase is phosphorylated to facilitate efficient ligation to the ends of one or more fragmented nucleic acid molecules using a ligation agent.

In certain embodiments, functionally important information may be present in the stem region of the hairpin structure. In certain embodiments, functionally important information may be present in the loop region of the hairpin structure. Functionally important information may include, for example and without limitation, sequences necessary for in vitro replication, in vitro amplification, unique identification (i.e., barcodes), and detection. In certain embodiments, where functionally important information is present in the loop region of the hairpin structure, the stem region can be as little as four or six base pairs in length. In certain embodiments in which functionally important information is present in the stem region of the hairpin structure, the loop region may be as little as one or two bases in length.

The paired template library is prepared by circularizing sheared genomic DNA that has been selected for a given size, such as 2kb, so that the ends that were previously distant from each other are brought closer together. The circle is then cleaved into linear DNA fragments by mechanical or physical means. Those DNA fragments containing ligated distal ends, called ligated fragments, are used to form the matched template. A "linker fragment" is a DNA molecule that contains the distal end of a larger DNA molecule in combination with a selectable marker, and is formed by first preparing a DNA loop, fragmenting the DNA loop, and selecting for fragments that contain the selectable marker.

By way of example and not limitation, the method of forming loops involves partial digestion of high molecular weight genomic DNA using a restriction endonuclease, such as Mbo I. Other known 4-base, 6-base, or 8-base "cleavage agents" or equivalents may also be used. DNA fragments at very low concentrations and combined with small selectable markers are ligated together to form covalent DNA loops. Thus, a circular DNA molecule is produced having a selectable marker flanked by the two distal ends of the DNA fragment. The library of ligated fragments is formed by digesting the DNA loops with different restriction endonucleases, such as EcoRI, and then selecting for the tagged fragments that are flanked by those distal ends. The ligation fragment libraries are used in genetic and physical mapping experiments and sequencing applications.

More generally, several factors should be considered when optimizing the ratio of connecting fragments that are prone to "intramolecular" ligation events (i.e., DNA loops of the same nucleic acid molecule) relative to "intermolecular" ligation events (i.e., ligation of two or more nucleic acid molecules called concatemers). The ratio depends on two parameters: the effective local molarity (j) experienced by one end of a molecule by the other end of the same molecule and the molarity (i) of the ends of all other DNA molecules. The parameter j may be determined according to Jacobs-Stockmayer equation:

j＝3.55×10^-8M/kb^3/2

where kb is the length of the nucleic acid molecule in kilobase pairs (kpb). For a given ligation reaction, the percentage of intramolecular events is determined by the ratio of j/(i + j). That is, larger nucleic acid molecules must be further diluted to achieve reasonable efficiency of forming intramolecular loops as compared to smaller nucleic acid molecules. In order to incorporate a selectable marker with reasonable probability during intramolecular ligation, its molar concentration should be approximately equal to j. However, even under very dilute ligation conditions, the probability of forming intermolecular ligation species will still exist, resulting in a mixture of intramolecular loops and linear concatemers of two or more nucleic acid molecules. There are a number of technical problems associated with the formation of large circular nucleic acid molecules, including: (a) generating and processing very large nucleic acid molecules without fragmenting them into smaller nucleic acid molecules; (b) identifying an appropriate selection marker to enrich for ligated fragments; and (c) large amounts of starting nucleic acid material are required to form a complete representative nucleic acid library. There are very well established methods in the art for processing large nucleic acid molecules, such as pulsed field gel electrophoresis, and alternative strategies, such as biotin/avidin or streptavidin systems, have been used to improve the selection of ligated fragments. However, the problem with the large amount of starting nucleic acid material required has not been adequately solved. Thus, the strategy of forming nucleic acid loops by intramolecular ligation events is rarely applicable when considering the analysis of precious biological samples present in limited amounts, such as biopsy samples obtained during surgical procedures or free circulating DNA obtained from whole blood, plasma or serum.

There are many methods for isolating nucleic acids from a sample. Once isolated, one or more nucleic acid molecules may be fragmented into smaller fragments by a fragmentation process, such as, without limitation, in a non-sequence-specific manner or a sequence-specific manner. Non-sequence specific or random fragmentation processes are expected to produce a uniform or substantially uniform distribution of fragmented nucleic acid molecules along a given genome of interest. By way of example and not limitation, 1,000,000 fragmented nucleic acid molecules may be localized to 1,000 positions (i.e., windows) of equal size, with each window having a count of 1,000 localized fragmented nucleic acid molecules. In certain embodiments, data obtained from a uniform or substantially uniform distribution of fragmented nucleic acid molecules along a given genome of interest may show a bias towards certain data types relative to other data types, such as, without limitation, GC content of a given region of the genome under study. In certain embodiments, the nucleic acid molecule is enzymatically fragmented using dnase I which non-specifically fragments double stranded DNA. The products of fragmentation are 5' -phosphorylated di-, tri-, and oligonucleotides with different sizes. DNase I in the presence of Mn²⁺、Mg²⁺And Ca²⁺But has the best activity in buffers without other salts in the buffer. Fragmentation using DNase I will generally result in random digestion of double stranded DNA, when based on Mn²⁺When used in the presence of the buffer of (3), the blunt-ended double-stranded DNA fragments predominate. Even when using Mn-based²⁺The fragmented nucleic acid molecule may still contain a 5 '-overhanging end of one or more single-stranded nucleotides of unknown sequence extending beyond the end of the other nucleic acid strand of the fragmented duplex (referred to herein as a "5' -end overhang") and a 3 '-overhanging end of one or more single-stranded nucleotides of unknown sequence extending beyond the end of the other nucleic acid strand of the fragmented duplex (referred to herein as a "3' -end overhang"). The range of fragment sizes of the library after dnase I digestion depends on several factors, such as, but not limited to, (I) the amount (units) of dnase I used in the reaction; (ii) of reaction(ii) temperature; and (iii) the time of the reaction.

In certain embodiments, the nucleic acid molecule is fragmented in a non-sequence specific manner using physical means or mechanical means. By way of example and not limitation, nucleic acid molecules can be fragmented using nebulization, which cleaves double-stranded nucleic acid molecules into smaller fragments. The range of fragment sizes of the library after nebulization depends on several factors, such as, but not limited to, (i) the pressure applied to the nebulizer; and (ii) the time of the shearing process. The library of sheared fragments contains a variety of end types, including blunt ends, 5 '-end overhangs, and 3' -end overhangs. The ends of one or more fragmented nucleic acid molecules may be directly ligated to adaptors using a linker to form dumbbell templates or be enabled to ligate dumbbell templates, in the case of using a random fragmentation method, see below.

Isolated nucleic acid molecules from a sample can also be fragmented into smaller fragments by a sequence-specific fragmentation process, for example and without limitation, using one or more restriction endonucleases. Sequence-specific fragmentation processes are expected to produce an uneven or substantially uneven distribution of fragmented nucleic acid molecules along a given genome of interest. For whole genome studies, the sequence-specific fragmentation process may not be optimal, as a certain fraction of genomic regions that may be significant are expected to have a low frequency of restriction endonuclease cleavage sites. The distribution of cleavage sites depends on the type and number of restriction endonucleases used for a given fragmentation process. Regions with a low frequency of cleavage sites will lead to an insufficient representation of genomic information. There are several advantages to using a sequence specific fragmentation method, for example, without limitation, targeting a subset of the genome of interest, which reduces work, cost, and data analysis, and the ends of the fragmented nucleic acid molecules will be defined as blunt-ended or defined 5 '-end overhang nucleic acid sequences and defined 3' -end overhang nucleic acid sequences. The overhanging ends with a defined nucleic acid sequence are called "sticky ends". In certain embodiments, two or more restriction endonucleases can be used to form smaller fragments each having a different sticky end sequence at each end. By way of example and not limitation, an isolated nucleic acid molecule is digested with two restriction endonucleases (i.e., EcoRI and BamHI) which will yield three different sticky end types (i.e., both ends containing the same 5 '-overhang sequence 5' -AATT [ EcoRI ] or 5 '-overhang sequence 5' -GATC [ BamHI ] or both ends containing different sticky ends (i.e., one end having the 5 '-overhang sequence 5' -AATT and the other end having the 5 '-overhang sequence 5' -GATC)). One hairpin structure having a complementary cohesive end of 5'-AATT can be ligated to the fragment containing the EcoRI cohesive end using a linker, and a different hairpin structure having a complementary cohesive end of 5' -GATC can be ligated to the fragment containing the BamHI cohesive end using a linker. Affinity vectors containing complementary sequences of different hairpin structures can be used to enrich dumbbell templates containing different hairpin structures, see examples for more details.

Isolated nucleic acid molecules from a sample can also be formed into smaller fragments by a sequence-specific fragmentation process, such as, without limitation, using multiplex PCR. By way of example and not limitation, two or more PCR primer sets can be designed to specifically amplify two or more target regions comprising a nucleic acid molecule. In addition to designing target-specific nucleic acid sequences comprising primers, additional nucleic acid sequences with functionally important information may also include, for example and without limitation, one or more restriction endonuclease cleavage sites and a unique identifier (i.e., a barcode). In certain embodiments, one or more forward PCR primers may contain a given restriction endonuclease cleavage site, and one or more reverse PCR primers may contain a different restriction endonuclease cleavage site. Upon contacting the amplified PCR product with the respective restriction endonucleases, each of which recognizes and cleaves its cleavage site, the ends of the amplified PCR product may contain different sticky ends that can be used to join two different hairpin structures in a predictable manner. By way of example and not limitation, all forward PCR primers contain an EcoRI restriction endonuclease cleavage site and all reverse PCR primers contain a BamHI restriction endonuclease cleavage site, except for the target-specific nucleic acid sequence. After multiplex PCR using two or more PCR primer sets, restriction endonuclease digestion of the amplified PCR products with EcoRI and BamHI will yield forward primer ends with a 5 '-overhang sequence of 5' -AATT and reverse primer ends with a 5 '-overhang sequence of 5' -GATC. One hairpin structure having a complementary cohesive end of 5'-AATT can be ligated only to the forward primer end using a linker, and a different hairpin structure having a complementary cohesive end of 5' -GATC can be ligated only to the reverse primer end using a linker. In some embodiments, isolated nucleic acid molecules from a sample may not require any fragmentation process, as these isolated nucleic acid molecules may be sufficiently fragmented to form a dumbbell-shaped template. For example, and without limitation, isolated nucleic acid molecules from serum or plasma, whole blood obtained from pregnant women or cancer patients are sufficiently fragmented in vivo, and thus additional fragmentation may not be required to form the dumbbell-shaped template. In certain embodiments, the sample may be obtained from a cancer patient. In certain embodiments, the sample may be obtained from a pregnant individual. In certain embodiments, the sample may be obtained from a pathological specimen. In certain embodiments, the sample may be obtained from a formalin-fixed paraffin-embedded (FFPE) specimen. In certain embodiments, the sample may be obtained from an environmental sample. In certain embodiments, the nucleic acid molecule may have a length in the range of 100bp to 100 kbp.

The library of isolated in vivo fragmented nucleic acid molecules contains a variety of end types, including blunt ends, 5 '-end overhangs, and 3' -end overhangs. The ends of the fragmented nucleic acid molecules may be directly ligated to adaptors using a linker to form ligated dumbbell templates or first processed to enable the ends to form ligated dumbbell templates, see below.

Fragmentation with blunt ends can be enhanced by using polishing methods, for example, without limitation, by using polymerases exhibiting 3' -exonuclease activityPercent of nucleic acid molecules of (a). By way of example and not limitation, such polymerases can include T₄DNA polymerase, Klenow DNA polymerase (Klenow DNA polymerase), or Pfu DNA polymerase. The 3 '-exonuclease activity of these DNA polymerases functions by removing one or more single-stranded nucleotides of unknown or known sequence from the 3' -end overhang to form a blunt-end fragmented nucleic acid molecule. Blunt-ended fragmented nucleic acid molecules are also formed by blunting the 5 '-end overhang by enzymatically incorporating complementary nucleotides into the recessed 3' -end strand. In certain embodiments, the 5' -end of one or more fragmented nucleic acid molecules may be phosphorylated, for example and without limitation, using T₄The polynucleotide kinase is phosphorylated to facilitate ligation to the ends of one or more hairpin structures using a linker to efficiently form a dumbbell template.

In certain embodiments, after blunting and phosphorylating the fragmented nucleic acid molecules, the double-stranded oligonucleotide adaptors can be designed to introduce functionally important information, such as, without limitation, replication, amplification, and/or unique identification (i.e., barcode) sequences, as well as to provide any given sticky end sequence. The latter sequence can be used to facilitate efficient dumbbell template formation with 5' -phosphorylated hairpin structures with complementary sticky end sequences using linkers. In certain embodiments, transposases and transposon complexes can be used to fragment one or more nucleic acid molecules and simultaneously insert functionally important information, such as, without limitation, replication, amplification, and/or unique identification (i.e., barcode) sequences as well as providing any given restriction endonuclease cleavage site at the insertion site capable of forming cohesive end sequences. In certain embodiments, the ends of the fragmented nucleic acid molecules may be modified by other means, such as, without limitation, by adding 2 '-deoxyadenosine (dA) nucleotides to the 3' -ends of blunt-ended fragmented nucleic acid molecules. By way of example and not limitation, a DNA polymerase lacking 3 '-exonuclease activity, such as klenow 3' -exo (-) DNA polymerase and Taq DNA polymerase, can add 2 '-deoxyadenosine triphosphate to the 3' -end of a blunt-ended fragmented nucleic acid molecule to obtain a 3 '-end overhang with one nucleotide of 2' -deoxyadenosine monophosphate. The dA tailing method also facilitates efficient dumbbell template construction using a linker with a 5' -phosphorylated hairpin structure with a complementary 3' -terminal overhang of 2' -thymidine monophosphate nucleotide. In certain embodiments, blunt-end fragmented nucleic acid molecules may also be used directly to form dumbbell templates using linkers to link 5' -phosphorylated hairpin structures with corresponding blunt ends.

Unlike current methods of forming nucleic acid loops by a strategy of intramolecular ligation events that is rarely applicable when considering analysis of a limited amount of precious biological samples, the methods of the present invention greatly improve the efficiency of forming nucleic acid loops. For example, and without limitation, the strategy of forming a nucleic acid loop through an intramolecular ligation event requires tens of micrograms of starting material, but yields an approximate efficiency of one percent (1%) or less in forming the desired nucleic acid loop. The problems associated with intra-molecular ligation strategies are further exacerbated because this approach is dependent on the size of the nucleic acid molecule and is inversely proportional to ligation efficiency. That is, larger size nucleic acid molecules form fewer loops by the intra-molecular ligation method because reaction conditions require increasingly dilute concentrations in proportion to the length of the nucleic acid molecule. On the other hand, the method of forming the dumbbell template described in the present invention is very efficient because the method does not depend on an intramolecular linking method. In contrast, dumbbell-shaped templates are formed by intermolecular events, wherein the efficiency of ligation of nucleic acid molecules to hairpin structures can be made very efficient. The ligation reaction can proceed to completion or substantially near completion because the concentration of hairpin structures can be sufficiently high (i.e., 100-fold) above the concentration of nucleic acid molecules. The ligation reaction is also independent or substantially independent of the size of the one or more nucleic acid molecules, since dumbbell templates of 1,000bp size can be formed as efficiently as dumbbell templates of 5,000bp size or 10,000bp size or even 100,000bp size, or even more than 100,000 bp. In certain embodiments of the invention, efficient dual hairpin dumbbell templates can be constructed from genomic DNA in size increments of 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, and 10.0 kb. In certain embodiments, the dumbbell template can then be replicated or amplified using a rolling circle mechanism in a homogeneous reaction solution. In certain embodiments, the dumbbell templates can also be replicated or amplified by introducing the dumbbell templates onto a substrate in a limiting dilution using a rolling circle mechanism using one or more solid phase binding primers in a heterogeneous reaction solution such that the one or more replicated dumbbell templates or amplified dumbbell templates are spatially and spectrally resolvable to detect nucleic acid molecules.

In certain embodiments, the dumbbell template is a plus-strand nucleic acid molecule containing one or more hairpin structures. In certain embodiments, a dumbbell template may also be formed whereby each end of a single-stranded nucleic acid molecule is linked to a hairpin structure, whereby one hairpin structure may serve as a primer to extend and copy the single-stranded nucleic acid molecule to the other end of the hairpin structure. After the joining step, a dumbbell-shaped template is formed. In certain embodiments of the invention, the linear duplex region may be melted, for example and without limitation, by thermal, chemical, or enzymatic means, and the dumbbell template may be converted into a fully open single stranded loop. In certain embodiments, two different hairpin structures having different nucleic acid sequences with unique restriction endonuclease cleavage sites can be used to form a dumbbell template. These circular templates can be replicated using a rolling circle mechanism to form multiple copies of the target sequence. Following the RCR step, the linear concatemer can be digested with an appropriate restriction endonuclease to generate monomeric units of the target sequence, which are then terminally linked together to form multiple copies of the circular target sequence. In certain other embodiments of the invention, dumbbell templates can be used to transcribe RNA molecules of a gene of interest. Dumbbell templates containing one or more RNA promoter sequences are generated and these closed single-stranded nucleic acid loops are used as templates for in vitro transcription of RNA molecules of the gene of interest.

In certain embodiments, exonucleases can be used to remove unwanted nucleic acid molecules that are not successfully ligated to form the dumbbell template. These undesired nucleic acid molecules may have one or more 5 '-ends or 3' -ends, which may be in the form of blunt ends, 5 '-protruding ends and/or 3' -protruding ends, or may be present in single stranded form. These undesired nucleic acid molecules include, but are not limited to, non-fragmented and fragmented nucleic acid molecules, oligonucleotides that may not form hairpin structures, and unligated hairpin structures. Exonuclease III (also known as Exo III) catalyzes the stepwise removal of single nucleotides from the 3' -hydroxyl end of double-stranded DNA. A limited number of nucleotides are removed during each binding event, allowing coordinated progressive deletions to occur within the population of DNA molecules. Preferred substrates for Exo III are nucleic acid molecules containing blunt-ended or 5' -protruding ends, although the enzyme also acts at nicks in double-stranded DNA to create single-stranded gaps. Exo III is not active on single stranded DNA and therefore the 3' -protruding end is resistant to cleavage. The degree of resistance depends on the length of the extension, with extensions of four bases or more being substantially resistant to cleavage. This property can be exploited to generate unidirectional deletions from linear molecules with one resistant end (3 '-overhanging end) and one sensitive end (blunt or 5' -overhanging end). The activity of exonuclease III depends in part on the helical structure and shows sequence dependence (C > a ═ T > G). Temperature, salt concentration and ratio of enzyme to DNA greatly affect the enzyme activity, requiring the reaction conditions to be tailored for specific applications. Exonuclease VII (also known as Exo VII) cleaves single-stranded DNA in the 5'→ 3' direction and the 3'→ 5' direction. This enzyme is not active on linear or circular double stranded DNA. In forming the dumbbell template, it can be used to remove single-stranded oligonucleotide primers and hairpins from the completed PCR reaction and post-ligation reaction. Exonuclease VII is non-metal dependent on the digestion of single stranded DNA. Exo III and Exo VII can be used in combination to remove unwanted nucleic acid molecules that are not successfully ligated to form the dumbbell template.

The matrix may comprise any material, such as, without limitation, a solid material, a semi-solid material (i.e., [ i ] a composite of a solid support and a gel or matrix material or [ ii ] a linear or cross-linked polyacrylamide, cellulose, cross-linked agarose, and polyethylene glycol), or a fluid or liquid material. The matrix may also comprise any material of any size and shape, such as, without limitation, square, trapezoidal, spherical, spheroidal, tubular, spherical, rod-shaped, or octahedral. The matrix should contain properties compatible with the present invention (i.e., exhibit minimal interference with the replication process, amplification process, or detection process). In certain embodiments, the substrate is non-porous. In certain embodiments, the matrix is porous. In certain embodiments, the matrix may comprise a hydrophilic porous matrix, such as a hydrogel. In certain embodiments, solid materials include, for example and without limitation, glass materials (i.e., borosilicate, controlled pore glass, fused silica, or germanium-doped silica), silicon, zirconia, titania, polymeric materials (i.e., polystyrene, cross-linked polystyrene, polyacrylate, polymethacrylate, polydimethylsiloxane, polyethylene, polyvinyl fluoride, polyoxyethylene, polypropylene, polyacrylamide, polyamides (e.g., nylon), dextran, cross-linked dextran, latex, cyclic olefin polymers, cyclic olefin copolymers, and other copolymers and grafts thereof), or metallic materials. The solid matrix may consist of, for example and without limitation: one or more membranes, planar surfaces, substantially planar surfaces, non-planar surfaces, microtiter plates, spherical beads, non-spherical beads, optical fibers containing spherical beads, optical fibers containing non-spherical beads, semiconductor devices containing spherical beads, semiconductor devices containing non-spherical beads, slides having one or more wells for non-spherical beads, filters, test strips, slides, coverslips, or test tubes. In certain embodiments, semi-solid materials include, for example and without limitation, linear or cross-linked polyacrylamides, cellulose, cross-linked agarose, and polyethylene glycol.

One or more primers can be attached to the substrate by any suitable means. In certain embodiments, the attachment of one or more primers to a substrate is mediated, for example and without limitation, by covalent bonding, hydrogen bonding (i.e., whereby the primer hybridizes to another complementary oligonucleotide covalently attached to the substrate and still performs a replicative or amplifiable function), Van Der Waal force (Van Der Waal force), physisorption, hydrophobic interactions, ionic interactions, or affinity interactions (i.e., a binding pair such as biotin/streptavidin or antigen/antibody). In certain embodiments, one member of the binding pair is attached to a substrate and the other member of the binding pair is attached to one or more primers. Attachment of the one or more primers to the substrate occurs via interaction of the two members of the binding pair.

The order in which one or more primers are attached to the substrate can be in any arrangement, which is broadly defined as a "primer array," for example and without limitation, in a random array, by random assignment in a patterned array, or by a known material patterned in an ordered array. A primer array that replicates a dumbbell template by a rolling circle mechanism is broadly defined as an "replicated dumbbell template array". A primer array for amplifying a dumbbell template by a rolling circle mechanism is broadly defined as an "amplified dumbbell template array". By design, it is contemplated that the patterned and ordered arrays provide an array of dumbbell-shaped templates or an array of amplified dumbbell-shaped templates that are spatially and spectrally resolvable for detection of nucleic acid molecules. In certain embodiments of random arrays, one or more primers may be covalently bonded to a substrate to form a high density plateau of immobilized primers on a planar or substantially planar surface. One or more primers may be attached by any means, such as, without limitation, by methods involving dropping, spraying, electroplating or spreading a solution, emulsion, aerosol, vapor, or dry formulation. By introducing the dumbbell template onto the substrate in a limiting dilution, one or more primers will contact the dumbbell template, thereby enabling the rolling circle mechanism to produce one or more replicated dumbbell templates (i.e., replicated dumbbell template arrays) or amplified dumbbell templates (i.e., amplified dumbbell template arrays) that are spatially and spectrally resolvable in the presence of the polymerase for detection of the nucleic acid molecule. In certain embodiments of random distribution in a patterned array, one or more primers can be covalently bonded to a substrate to form a high density of immobilized primers on one or more spherical or non-spherical beads. By introducing the dumbbell template onto the substrate in a limiting dilution using an oil-in-water emulsion system, one or more primers will contact the dumbbell template, thereby enabling the rolling circle mechanism to generate one or more replicated dumbbell templates or amplified dumbbell templates. In certain embodiments, the replicated dumbbell-shaped templated beads or amplified dumbbell-shaped templated beads can be enriched to remove those beads that failed to replicate or amplify the dumbbell-shaped template based on Poisson statistics (Poisson statistics) that distributed the individual molecules. The replicated dumbbell-shaped templated beads or amplified dumbbell-shaped templated beads can then be randomly distributed in an ordered pattern on a planar or substantially planar slide substrate, optical fiber substrate, or semiconductor device substrate containing wells, recesses, or other containers, vessels, features, or locations, with or without enrichment. In certain other embodiments of random distribution in a patterned array, one or more preformed hydrophilic features (i.e., spots) on the surface may be surrounded by a hydrophobic surface to covalently bond one or more primers to the substrate. By way of example and not limitation, the patterned array may be formed on a surface modified silicon substrate having a grid patterned array of about 300 nanometer spots etched by photolithography. By introducing the dumbbell template onto the patterned substrate in a limiting dilution, the primers will contact the dumbbell template, thereby enabling the rolling circle mechanism to generate one or more replicated dumbbell templates or amplified dumbbell templates. In certain embodiments, the preformed hydrophilic spots can be made small enough to accommodate even just one replicated dumbbell template or amplified dumbbell template. Since a single molecule produces a substantial portion of template-free spots based on poisson statistical distribution, additional rounds of distribution, contact, and rolling can be used to increase the density of replicated dumbbell templates or amplified dumbbell templates on the substrate after the rolling circle procedure. Certain embodiments of "knows" patterned in an ordered array, one or more known primers can be printed (i.e., spotted on the array) or prepared in situ at addressable locations on the substrate. By introducing the dumbbell template onto the patterned substrate in a limiting dilution, one or more primers will contact the dumbbell template, thereby enabling the rolling circle mechanism to generate one or more replicated dumbbell templates or amplified dumbbell templates.

Polymerase chain reaction ("PCR") is used to specifically amplify small numbers of nucleic acid molecules, producing thousands to millions of copies of a target sequence of interest. In general, PCR involves repeated heating to denature or melt duplex strands, cooling to allow primer hybridization, and then heating again (usually at an optimal temperature for DNA polymerase, but below the denaturation temperature) to allow amplification of the template sequence in vitro (i.e., in vitro). The DNA polymerase copies or synthesizes a complementary strand from the single-stranded template. In order for this enzymatic reaction to occur, a partial double-stranded stretch of DNA is required. Typically, the primer hybridizes to a complementary region of the single-stranded template. The DNA polymerase synthesizes the nascent strand in the 5 'to 3' direction to form double-stranded DNA. Multiplex PCR allows for the simultaneous amplification of multiple target regions and has been used to detect the deletion of one or more coding exons (these exons are gene sequences that are transcribed into messenger rna (mrna) and translated into one or more proteins) in X-linked disorders; such X-linked disorders include Duchenne muscular dystrophy and Lesch-Nyhan syndrome. In the alternative, PCR can be used to amplify the entire pool of nucleic acids present in the starting mixture, thereby causing amplification of any given subset of nucleic acids, but not targeted enrichment. This is accomplished by ligating a common sequence or common adaptor to the ends of the fragments, and amplifying the fragments by denaturing the fragments, hybridizing common primers having sequences complementary to the common adaptor, and copying the DNA fragments. This type of PCR is called "Universal PCR".

Bacteriophages (bacteriophages/phase), such as Φ X174, M13, λ, and some viruses can replicate their corresponding genomes through a "rolling circle" mechanism. The entire genome is replicated by copying from a circular template. Unlike PCR, the rolling circle mechanism can be performed isothermally (i.e., without the need for heating or cooling cycles).

The rolling circle method has been used for replication (i.e., using copy-onlyOne or more primers for the original dumbbell template of shellfish) or an in vitro method of amplifying (i.e., using two or more primers that copy both the original dumbbell template and the copy of the dumbbell template) a nucleic acid molecule of interest. For example, circular synthetic oligonucleotide templates having a size in the range of 34 to 52 bases have been replicated using the E.coli Pol I DNA polymerase and a single oligonucleotide primer using a rolling circle mechanism. The rolling circle mechanism using similarly sized circles in the 26 to 74 range uses a variety of polymerases including E.coli Pol I, Klenow DNA polymerase, and T₄A DNA polymerase.

In certain embodiments, the DNA loop may be formed as a "padlock probe". The main disadvantage of using the padlock method is the size limitation of forming circular nucleic acid molecules, such as, without limitation, a 46 nucleotide loop for targeting the CFTR G542X locus. These locking loops can be used to use DNA polymerases, e.g.DNA polymerase, Bst DNA polymerase, and Vent (exo-) DNA polymerase form hundreds of target copies in only a few minutes. In certain embodiments, the padlock loops may use two primers in a rolling circle amplification mechanism, which not only enables copying of the template loop (i.e., the negative strand), but also enables copying of the newly synthesized positive strand or strands. RCA methods using two different padlock loops with 46 nucleotides may be used for genotyping applications, such as, without limitation, detection of wild-type and mutant sequences against the CFTR G542X locus. Another disadvantage of RCA-lock methods for genotyping is that the locus of each mutation determined requires a separate allele-discriminating primer.

Rolling circle amplification has been performed using random hexamers (i.e., more than two primers) anduse of DNA polymerase in Sanger sequencing applications for solution-based modeling using traditional clonal sources, such as plasmids and phages, with sizes ranging from 5kb to 7kb in size as DNA loopsAnd (4) preparing a plate. The disadvantage of using traditional cloning methods to form DNA loops is the need to propagate such DNA loops via an appropriate cellular host. The dumbbell template of the present invention overcomes this limitation. Rolling circle replication using chimeric DNA templates has been used in methods of sequencing by ligation. The template preparation method uses a complex series of directed adaptor ligations and type IIs restriction endonuclease digestions to form small DNA circles with a size of about 300bp by the intramolecular ligation method, which are replicated in solution using a single primer and a phi29 DNA polymerase to form "DNA nanospheres". These nanospheres are then adsorbed onto a patterned substrate to perform their side-by-side sequencing method. The main limitation of the nanosphere approach is the small amount of genomic DNA sequence available in the chimeric template loop (i.e., 76bp is the actual target sequence and the remaining 222bp is the adaptor sequence) and the need for intramolecular ligation. The present invention overcomes the limitations of complex methods of constructing small circular templates by intramolecular ligation methods by providing a simpler workflow using dumbbell-shaped templates that can be replicated or amplified in a rolling circle mechanism.

Polymerases and reverse transcriptases useful in the rolling circle mechanism generally exhibit strand displacement properties, which is the ability to displace "downstream" nucleic acid strands encountered by the enzyme during nucleic acid synthesis. These strand displacing enzymes also lack 5' -exonuclease activity. Any strand displacement polymerase or reverse transcriptase can be used in rolling circle replication or rolling circle amplification, for example, without limitationDNA polymerase, Escherichia coli Pol I, Klenow DNA polymerase, Bst DNA polymerase (large fragment), Bsm DNA polymerase (large fragment), Bsu DNA polymerase (large fragment), Vent (exo-) DNA polymerase, T₇(exo-) DNA polymerase (T)₇Sequenases), or TopoTaq (a chimeric protein of Taq DNA polymerase and topoisomerase V), and mutant versions of these DNA polymerases, T₇RNA polymerase, T₃RNA polymerase, SP6 RNA polymerase, mutant forms of these RNA polymerases, avian myeloblastosis virus reverse transcriptase, orMoloney murine leukemia virus reverse transcriptase (Moloney murine leukemia virus reverse transcriptase), and mutated versions of these reverse transcriptases, such as Thermoscript reverse transcriptase, SuperScript reverse transcriptase or PrimeScript reverse transcriptase. In addition to strand displacing polymerases and reverse transcriptases, helper proteins may further enhance displacement of downstream nucleic acid strands during nucleic acid synthesis by improving the robustness, fidelity, and/or persistence of the rolling circle mechanism. The strand displacement helper protein may be of any type and includes, for example and without limitation, helicases, single-strand binding proteins, topoisomerases, counter-rotators, and other proteins that stimulate helper proteins, such as, without limitation, E.coli MutL protein or thioredoxin. DNA helicases are useful in vivo to separate or helice two complementary or substantially complementary DNA strands during DNA replication. Helicases may be in the 5 'to 3' direction (e.g., without limitation, phage T)₇Genes 4 helicase, DnaB helicase, and Rho helicase) and in the 3 'to 5' direction (e.g., without limitation, e.g., e.coli UvrD helicase, PcrA, Rep, and NS3 RNA helicase from hepatitis c virus). Helicases may be obtained from any source and include, for example and without limitation, E.coli helicases (i.e., I, II [ UvrD)]III and IV, Rep, DnaB, PrIA and PcrA), phage T₄gp41, bacteriophage T₇Gene 4 helicase, SV40 large T antigen, Rho helicase, yeast RAD helicase, thermostable UvrD helicase from anaerobacterium tengcongensis (t.tengcongensis), and NS3 RNA helicase from hepatitis c virus, as well as mutant versions of these and other helicases. Single-stranded binding proteins bind single-stranded DNA with greater affinity than double-stranded DNA. These proteins bind synergistically, favoring invasion of the single-stranded region and thus destabilize the duplex structure. By way of example and not limitation, single-stranded binding proteins may exhibit helical destabilizing activity by removing secondary structure and may displace hybridized nucleic acid molecules. Single-chain binding proteins can be obtained from any source and include, for example and without limitation, phage T₄Gene 32 protein, RB 49 gene 32 protein, colibacillus single-chain binding protein,Single-chain binding proteins or bacteriophage T₇Gene 2.5, and mutant versions of these and other single-stranded binding proteins, e.g. phage T₇Gene 2.5F 232L.

Rolling circle replication of the dumbbell template can be performed using a highly processive strand displacement polymerase, such as phi29 polymerase. Rolling circle replication can be performed in two steps. First, a size-selected dumbbell template is hybridized with a dumbbell complementary primer under appropriate "hybridization conditions" including temperature, factors such as salt, buffer, and pH, detergents, and organic solvents. Blocking agents, such as Bovine Serum Albumin (BSA) or Denhardt's reagent, may be used as part of the hybridization conditions. Next, the appropriate polymerase or replisome and nucleotide mixture are provided to the first reaction mixture to produce an amplified or replicated dumbbell template. Hybridization and amplification or replication conditions are optimized based on several factors, including but not limited to the length and sequence composition of the stem region of the dumbbell template, hybridization conditions, the specific polymerase or replicator used herein, and the reaction temperature. In certain embodiments, the reaction temperature may be about 10 ℃ to 35 ℃. In other embodiments, the reaction temperature may be about 15 ℃ to 30 ℃. In other embodiments, the reaction temperature may be about 20 ℃ to 25 ℃. In certain embodiments, the temperature is increased for a selected time interval. By way of example and not limitation, the reaction is maintained at 10 ℃ for five minutes, then at 15 ℃ for five minutes, at 20 ℃ for five minutes, then at 25 ℃ for five minutes, and at 30 ℃ for five minutes.

Replication complexes, referred to as "replicates," can be formed in vitro to replicate or amplify larger dumbbell templates (i.e., have more copies of replicated dumbbell templates or amplified dumbbell templates) by making them>1kb、>5kb、>10kb and>50kb in size) to enhance the rolling circle method. Strand displacement helper proteins including helicases, single-strand binding proteins, topoisomerases, and antiporters may be configured in any combination with strand displacement polymerases and reverse transcriptases for formationReplication-competent or amplification-competent replicon complexes in the rolling circle method for dumbbell-shaped templates. In certain embodiments, the combination of a φ 29DNA polymerase and a φ 29 single strand binding protein can enhance the elongation of the rolling circle mechanism by several fold under appropriate reaction conditions. In certain embodiments, a combination of polymerases or reverse transcriptases that rely on the synergistic activity of a helicase and a single strand binding protein may be used in a rolling circle method to replicate dumbbell templates of 10kb or larger or amplify dumbbell templates of 10kb or larger. By way of example and not limitation, T may be used by forming a replicate complex₇Sequencing enzyme, T₇Helicase and T₇The synergistic activity of the single-stranded binding protein amplified the 10kb plasmid.

Certain embodiments of the invention include the efficient formation of 10kb sized dual hairpin dumbbells with a highly sustained solid phase RCR system. In certain aspects, uniquely selected method-specific dual hairpin dumbbell templates are formed in a size-independent and closely distributed manner (i.e., 10kb ± 1kb), allowing informative downstream bioinformatic processing for de novo assembly. The formation of dumbbell templates eliminates the need for large amounts of starting genomic DNA, as these constructs are efficiently prepared with a simple workflow (i.e., intermolecular ligation versus intramolecular ligation). This is an important consideration when using clinical samples, which are usually obtained in minute quantities. Embodiments of the present invention also provide for the development and optimization of solid-phase RCR, relaxing the current size constraints imposed by available polymerases, which represents a technological breakthrough in NGS technology. These innovative large-template high-density arrays would enable true de novo assembly of complex, novel and disease genomes for research, clinical, and diagnostic applications, and also allow for more comprehensive systematic biological studies to study whole genome DNA-DNA, DNA-RNA, and DNA-protein interactions.

The replicated dumbbell templates and amplified dumbbell templates (i.e., replicated dumbbell template arrays or amplified dumbbell template arrays) attached to a matrix can be useful for many different purposes, including, for example and without limitation, all aspects of nucleic acid sequencing (i.e., whole genome de novo sequencing; whole genome re-sequencing for sequence variant detection, structural variant detection, determining the phase and/or molecular count of molecular haplotypes for aneuploidy detection; targeted sequencing of gene sets, whole exomes, or chromosomal regions for sequence variant detection, structural variant detection, determining the phase and/or molecular count of molecular haplotypes for aneuploidy detection; and other targeted sequencing methods such as RNA-seq, Chip-seq, Methyl-seq, etc.; all types of sequencing activities are broadly defined herein as "sequencing"). The replicated dumbbell template arrays and amplified dumbbell template arrays can also be used to form arrays of nucleic acid molecules to study nucleic acid-nucleic acid binding interactions, nucleic acid-protein binding interactions (i.e., fluorescent ligand interaction characterization that quantitatively measures protein-DNA affinity); and nucleic acid molecule expression arrays (i.e., to transcribe 2' -deoxyribonucleic acid molecules into ribonucleic acid molecules, defined herein as "ribonucleic acid template arrays") to study nucleic acid structural/functional relationships. In certain embodiments, the structural/functional arrays can be used to test the effect of small molecule inhibitors or activators or nucleic acid therapeutics that can interfere with one or more structural/functional relationships of the ribonucleic acid template array, such as, without limitation, therapeutic antisense RNA, ribozymes, aptamers, and small interfering RNA; and detecting nucleic acid-nucleic acid binding interactions and nucleic acid-protein binding interactions. In certain embodiments of the invention, the ribonucleic acid template arrays may be further translated into their corresponding amino acid sequences, defined herein as "protein arrays," which may be used, for example and without limitation, to study protein-nucleic acid binding interactions and protein-protein binding interactions; screening for ligands (particularly orphan ligands) specific for one or more related protein receptors; drug screening for small molecule inhibitors or activators or nucleic acid therapeutics that can interfere with one or more structural/functional relationships of the protein array, such as, but not limited to, therapeutic antisense RNA, ribozymes, aptamers, and small interfering RNA.

In certain embodiments, the replicated dumbbell template arrays and amplified dumbbell template arrays can be used for more than one purpose by providing additional information beyond monocular uses, such as, but not limited to, whole genome de novo sequencing followed by detection of nucleic acid-protein binding interactions to identify sequence specific nucleic acid-protein motifs. One advantage of the present invention over DNA arrays that rely on solid phase methods for amplification of fragments of 700bp or less is that large nucleic acid molecules of at least >1kb, or preferably >5kb, or more preferably 10kb, and most preferably 50kb, are replicated or amplified. Replicated dumbbell template arrays and amplified dumbbell template arrays with increased template size can provide further information, such as synergistic remote interaction of two or more nucleic acid-protein binding interaction events along a nucleic acid molecule.

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. These inventions may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

In certain embodiments of the invention, the efficient generation of circular DNA molecules via dumbbell templates, which are compatible with many of the different purposes described above, has been combined with solid phase rolling circle replication to form replicated dumbbell templates of clonally replicated large inserts (having a size of 10 kb). In certain embodiments of the invention, efficient generation of circular DNA molecules via dumbbell templates, which are compatible with many of the different purposes described above, has been combined with solid phase rolling circle amplification to form amplified dumbbell templates of clonally amplified large inserts (having a size of 10 kb). Dumbbell templates are formed efficiently and are independent of fragmented nucleic acid molecule size, overcoming significant limitations in current next generation sequencing methods. The rolling circle replication method or rolling circle amplification method using dumbbell-shaped templates overcomes the major limitation of fragmented nucleic acid molecule size observed in other solid phase amplification methods such as emulsion PCR and solid phase amplification.

One embodiment of the present invention is a method of replicating at least one dumbbell template, the method comprising the steps of: fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one dumbbell template, the method comprising the steps of: fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

Another embodiment of the invention is a method of detecting at least one replicated dumbbell template, the method comprising the steps of: fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template; and detecting the at least one replicated dumbbell template. In another embodiment, said step of detecting said at least one replicated dumbbell template consists of sequencing said at least one replicated dumbbell template.

Another embodiment of the invention is a method of detecting at least one amplified dumbbell template, the method comprising the steps of: fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template; and detecting the at least one amplified dumbbell template. In another embodiment, said step of detecting said at least one amplified dumbbell template consists of sequencing said at least one amplified dumbbell template.

Another embodiment of the invention is a method of replicating at least one dumbbell template, the method comprising the steps of: isolating at least one nucleic acid molecule from the sample; fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one dumbbell template, the method comprising the steps of: isolating at least one nucleic acid molecule from the sample; fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule; ligating one or more hairpin structures to each end of the at least one fragmented nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

Another embodiment of the invention is a method of replicating at least one nucleic acid molecule, the method comprising the steps of: isolating at least one nucleic acid molecule from the sample; ligating one or more hairpin structures to each end of the at least one nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least one substantially complementary primer, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template.

Another embodiment of the invention is a method of amplifying at least one nucleic acid molecule, said method comprising the steps of: isolating at least one nucleic acid molecule from the sample; ligating one or more hairpin structures to each end of the at least one nucleic acid molecule using a linker to form at least one dumbbell template; contacting the at least one dumbbell template with at least two substantially complementary primers, wherein the at least one substantially complementary primer is attached to at least one substrate; and performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified dumbbell template.

While embodiments have been described herein with an emphasis upon the embodiments, it should be understood that within the scope of the appended claims, the embodiments may be practiced other than as specifically described herein. While the invention has been shown in only a few of its forms, it should be apparent to those skilled in the art that it is not so limited, but is susceptible to various changes without departing from the scope of the invention. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Those skilled in the art will recognize that many variations and modifications may be made to the method of practicing the invention without departing from the scope and spirit of the invention. In the drawings and specification, there have been disclosed embodiments of the invention and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. The invention has been described in considerable detail with particular reference to these illustrated embodiments. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention as set forth in the foregoing description. Furthermore, language referring to the order, such as first and second, should be understood in an exemplary sense and not in a limiting sense. For example, one skilled in the art may recognize that certain steps may be combined into a single step.

Examples

The following examples further illustrate the compositions and methods.

Example 1

Demonstrating the size-independent nature of the dumbbell templates containing two different hairpin structures. Sample DNA, i.e., pUC18 vector, was amplified using a set of primers (i.e., forward: 5'-GGA TCC GAA TTC GCT GAA GCC AGT TAC CTT CG (SEQ ID NO:1) and reverse: 5' -GGA TCC GAA TTC AGC CCT CCC GTA TCG TAG TT) (SEQ ID NO:2) to obtain a plasmid having 425 DNA fragmentsThe product of base pairing. The 5' -end of each primer contained a BamHI restriction endonuclease site and an EcoRI restriction endonuclease site. The PCR product was then digested with EcoRI to provide 5' -AATT overhangs and purified with QIAquick PCR purification kit. By heating to 50 ℃ followed by cooling, the oligonucleotide is allowed to self-anneal at the underlined sequence, thereby creating a 5'-AATT overhang to form hairpin structure 1(5' -AATT)GCGAG TTG CGA GTT GTA AAA CGA CGG CCA GT CTCGC(SEQ ID NO: 3)). The loop structure contains the M13 universal primer sequence. Hairpin 1 and pUC18 PCR products were combined at a 10:1 molar ratio, respectively, and incubated with 5 units of T at 37 deg.C₄Polynucleotide kinase treatment for 40 min with 400 cohesive end units of T at 16 ℃₄DNA ligase was ligated for 30 minutes and then inactivated at 65 ℃ for 10 minutes to form a dumbbell template. When denatured, the dumbbell template becomes a single-stranded loop. The dumbbell template was purified using the QIAquick PCR clean-up kit to remove excess unligated hairpin structures.

Rolling circle replication was performed on the dumbbell template using the Reverse Complement (RC) of the M13 primer, as shown in fig. 1. Here, 2. mu.M of M13-RC primer (5'-ACT GGC CGT CGT TTT ACA A (SEQ ID NO:4)) and M13 control primer (5' -TTG TAA AAC GAC GGC CAGT (SEQ ID NO:5)) were annealed to about 10ng of pUC18 dumbbell template, respectively, by heating to 94 ℃ in 20. mu.L reaction in φ 29 reaction buffer containing 200. mu.M dNTP and 200. mu.g/mL BSA for 5 minutes and cooling to 57 ℃ for 1 minute. The reaction was further cooled to 30 ℃ at which time 10 units of φ 29DNA polymerase were added to the primed dumbbell template and incubated for 30 minutes followed by heat inactivation at 65 ℃ for 10 minutes. As a control, normal M13 universal sequencing primers were incubated in a rolling circle replication mix alone. The replicated dumbbell templates were then analyzed by gel electrophoresis. As shown in figure 2, the M13-RCR product (lane 2) produced a high molecular weight product (upper band in the well), whereas the M13 control produced no visible rolling circle replication product. The results in lane 2 confirm that the rolling circle replication method forms a high molecular weight dumbbell template.

The method shown above is simply to ligate the hairpin structure to the fragmented nucleic acid moleculeOne way of terminating the seed. By way of example and not limitation, hairpin structures, such as hairpin structure 2(5' -T) with "T" -overhangs, may also be ligated by TA cloning and blunt end ligationGCGAG TTG CGA GTT GTA AAA CGA CGG CCA GT CTCGC(SEQ ID NO: 6)). A425 bp pUC18 PCR amplicon can also be treated with the NEBNext dA tailing kit to attach "dA" residues at the 3' -end of the DNA fragment. The pUC18 amplicon and hairpin structure 2 were then phosphorylated and ligated together using the methods described above to form a dumbbell template. This approach is integrated into the next generation sequencing library construction approach for the majority of whole genome samples.

Example 2

Genomic DNA may also be used as a starting sample. By way of example and not limitation, purified genomic DNA from HapMap sample NA18507 may be obtained from a karel Cell repertoire (Coriell Cell repositides) and sheared using standard next generation sequencing methods (i.e., using the Covaris E210R apparatus), and then the fragments size selected at size increments of 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, and 10.0 kb. Similarly to the above, DNA samples can be fragmented to produce DNA fragments of different sizes, the starting number of fragments quantified, hpA/hpB ligated using the same conditions, and enriched for hpA-fragment-hpB dumbbell templates. Double-labeled fluorescence microscopy will be used to count co-localized fluorescence signals and compare the number to the total number of fluorescence signals to determine the enrichment factor. The Nikon Eclipse microscopy analysis tool can perform a number of analyses including intensity measurements, co-localization of multiple fluorescence signals, and other analyses as included in the core package project.

In this example, 500ng of normal human genomic DNA (Millipore) was digested with EcoR1 in a dilution restriction enzyme reaction followed by inactivation at 65 ℃. These fragments, which contained a 5' -AATT overhang at either end, were ligated to a stable and unique hairpin structure HP 1. 5' -AATT mutations were obtained by heating to 95 ℃ in high salt buffer followed by rapid cooling on ice, allowing the oligonucleotide to self-anneal at the underlined sequenceOut-ending to form HP1(5' -AATT)GCGAG TTG CGA GTT GTA AAA CGACGG CCA GT CTCGC(SEQ ID NO: 3)). HP1 and digested genomic DNA were combined at a 10:1 molar ratio, respectively, and T of 400 cohesive end units was used at 16 ℃₄DNA ligase was ligated for 30 minutes and then inactivated at 65 ℃ for 10 minutes to form dumbbell templates, see FIG. 3. Dilution digestion and HP1 ligation yielded a diffuse band (smear) of dumbbell template DNA with sizes ranging from about 20kb to 1 kb. The dumbbell templates were gel purified to remove excess unligated and self-ligated HP1 adaptors and size selected to isolate three different fragment sizes, namely 10kb-6kb, 6kb-3kb, and 3kb-2 kb.

The loop structure of HP1 contained the M13 universal primer sequence. RCR was performed on dumbbell templates (i.e. using one primer) using the Reverse Complement (RC) of the M13 primer, see fig. 3. Here, 2. mu.M of the M13-RC primer and M13 control primer (not shown) were annealed to about 10ng of size-selected genomic DNA dumbbell template, respectively, by heating to 94 ℃ in 20. mu.L of the reaction in φ 29 reaction buffer containing 200. mu.M dNTPs and 200. mu.g/mL BSA for 5 minutes and cooling to 45 ℃ for 2 minutes. The reaction was further cooled to 30 ℃, at which point 10 units of phi29 DNA polymerase were added to the primed loop and incubated for 60 minutes, followed by heat inactivation at 65 ℃ for 10 minutes. The starting and final material were analyzed by agarose gel electrophoresis. As shown in figure 4, EcoR1 digested genomic DNA ligated to HP1 was loaded into the wells of lane 1. Size-selected and purified dumbbell templates were loaded into the wells of lane 2, lane 3, and lane 4. The RCR products loaded in lanes 5,6, and 7 appear to be immobile complexes that remain in the wells after gel electrophoresis. The expected M13-RC RCR product produced a high molecular weight product (upper band in the well of FIG. 4, Lane 5, Lane 6, and Lane 7), while the M13 control produced no visible RCR product (not shown). The results in lanes 5,6, and 7 of FIG. 4 confirm the RCR method for forming high molecular weight DNA using dumbbell templates.

Example 3

Also from largeThe fragmented dA-tailed genomic DNA of (a) forms a replicated dumbbell-shaped template. Here, the hairpins were ligated by TA cloning and blunt end ligation. The TA cloning method integrates well into most current NGS platforms. We have designed hairpin 2(HP2) (5' -/Phos-CTTTTTCTTTCTTTTCT GGGTTGCGTCTGTTCGTCT AGAAAAGAAAGAAAAAGT (SEQ ID NO: 7)). Human genomic DNA (500ng) was fragmented using a Covaris G-tube to achieve a well-defined fragment length population as shown in lanes 1 and 2 of figure 5. This genomic DNA was then end-repaired and dA tailed using the end preparation module of the NEBNext Ultra DNA library preparation kit. HP2 was self-annealed similarly to HP1 and ligated to repaired genomic DNA using Blunt/TA Ligase master Mix (Blunt/TA Ligase Mater Mix) (5:1 molar ratio). Exonucleases III and VII were used to remove excess HP2 and unligated genomic DNA.

The resulting dumbbell template was purified using Qiaex ii beads and the RCR reaction using a unique primer (5' -AAAAAAA CAGACGCAACCC (SEQ ID NO:8)) was performed similarly to the previously described reaction. As shown in FIG. 5, the two fragmented genomic DNA populations showed the highly tunable fragmentation capacity of the Covaris G-tube (lanes 1 and 2). The RCR product produced from the end-repaired dA-tailed HP2 ligated fragment remained as a highly immobile complex that remained in the well after gel electrophoresis (lanes 3 and 4).

Example 4

In another experiment, about 20. mu.L of high molecular weight normal human genomic DNA (gDNA) (100 ng/. mu.L) was combined with 130. mu.L of HPLC grade H₂O combined and a total of 150. mu.L were pipetted into a Covaris G-tube. The tubes were first centrifuged at 5600RCF (i.e., relative centrifugal force) for 1 minute, then the orientation of the G-tubes was reversed and centrifuged at 5600RCF for 1 minute. This resulted in a fragment of about 8 kilobases to 10 kilobases of genomic DNA, as shown in lane 2 of FIG. 6.

Genomic DNA samples can also be fragmented using a variety of methods known in the art, including but not limited to enzymatic fragmentation using New England Biolabs (NEB), a fragmenting enzyme, a nuclease, and a restriction enzyme; fragmentation using mechanical forces, such as needle shearing via a small gauge needle, sonication, point-sink shearing, nebulization, acoustic fragmentation, and transposome-mediated fragmentation.

The ends of the fragmented DNA are then prepared for ligation with appropriate adaptors using one of a variety of means, such as removal or incorporation of nucleotides at the 5' -and 3' -ends of the overhang, 5' phosphorylation, and dA tailing. About 55.5. mu.L of the suspension prepared as above in H₂Fragmented gDNA in O was combined with 6.5. mu.L of 10 × terminal repair buffer (NEB) and 3. mu.L of terminal preparation enzyme cocktail (NEB) and aliquoted into thermocycler microtubes. This reaction mixture was then incubated at 20 ℃ for 30 minutes followed by incubation at 65 ℃ for 30 minutes. The reaction was cooled by placing the reaction tube on ice or at 4 ℃ and was ready for the subsequent step.

Hairpin adaptors are formed from linear oligonucleotides. Lyophilized adaptors were subjected to HPLC H₂The O was recovered to 100. mu.M. The following components were combined in a microcentrifuge tube: mu.L of 100. mu.M adaptor stock, 5. mu.L of 10 × terminal repair buffer (NEB), 1. mu.L of 500mM NaCl, and 34. mu.L of HPLC H₂And O. The mixture was incubated at 95 ℃ for 15 minutes and then immediately transferred to 4 ℃.

A dumbbell template is formed by ligating hairpin adaptors to each end of the fragmented end-repaired gDNA. Combining the following components to form a sample reaction mixture: 65 μ L of fragmented gDNA with repaired ends as described above, 3 μ L of 20 μ M adapter prepared as described above, 15 μ L of blunt-ended/TA ligation master mix (NEB) and 3 μ L of HPLC H₂And O. This ligation reaction was allowed to proceed at 20 ℃ for 1 to 16 hours and then immediately moved to 4 ℃.

The unligated adaptors and unligated fragmented DNA are subjected to exonuclease digestion. Combining the following components to form a sample reaction mixture: 1. mu.L of 10 Xexonuclease VII buffer (NEB), 1. mu.L of exonuclease VII, 1. mu.L of exonuclease III, and 7. mu.L of HPLC H₂And O. Adding the mixture to a mixture containing a dumbbell-shaped template,Unligated adaptors, and fragmented DNA with free ends. The resulting reaction mixture was incubated at 37 ℃ for 1 hour, then at 95 ℃ for 10 minutes, and then transferred back to 4 ℃.

FIG. 6 is an example of agarose gel analysis of DNA products prepared as described above. Lane 1 shows genomic DNA without fragmentation; lane 2 shows fragmented DNA after fragmentation in a Covaris G-tube; lane 3 shows the product formed after ligating adaptors to 1 μ g of fragmented DNA; lane 4 shows the product formed after ligating an adaptor to 500ng of fragmented DNA; lane 5 shows the product formed after subjecting 1 μ g of fragmented DNA to a ligation reaction without any adaptors; lane 6 shows the product formed after the ligation reaction was performed without fragmented DNA but with adaptors alone; lane 7 shows the products formed after exonuclease digestion of the products obtained by ligating adaptors to 1 μ g of fragmented DNA; lane 8 shows the products formed after exonuclease digestion of the product obtained by ligating an adaptor to 500ng of fragmented DNA with Exo III and Exo VII; lane 9 shows the products formed after exonuclease digestion of fragmented DNA that is subjected to ligation reaction without any adaptors with Exo III and Exo VII; lane 10 shows the products formed after exonuclease digestion with Exo III and Exo VII of the products obtained by ligation reactions performed without fragmented DNA but with adaptors only. Lane 11 shows a digestion control of fragmented genomic DNA and adaptors not ligated.

The DNA samples were also concentrated to remove salts and to concentrate exonuclease resistant dumbbell templates. Using about 4. mu.L of HPLC H₂O the volume of the reaction mixture after exonuclease digestion was adjusted to 100. mu.L of solution. Approximately 10. mu.L of 3M sodium acetate (pH 5.2) and 5. mu.L of glycogen (20mg/mL) were added to the solution, followed by 115. mu.L of cold 100% isopropanol. Refrigerating the reaction mixture at-20 deg.C>For 1 hour, and then centrifuged at 10RCF for 20 minutes at room temperature. Sucking supernatantAnd the precipitate was washed with 70% ethanol. The final pellet was dried for about 15 minutes and then resuspended in 30. mu.L of 10. mu.M Tris-HCl (pH 8.0).

FIG. 7 is an example of agarose gel analysis of the DNA product prepared as described above. Lane 1 shows the product formed after ligation of adaptor 10.1 to fragmented DNA and subsequent ethanol precipitation; lane 2 shows the product formed after ligation of adaptor 2.1 to the fragmented DNA and subsequent ethanol precipitation; lane 3 shows the product formed after subjecting the fragmented DNA to a ligation reaction without adaptors and subsequent ethanol precipitation; lane 4 shows that no product is formed after only subjecting the adaptor 10.1 to the ligation reaction without fragmented DNA and subsequent ethanol precipitation; lane 5 shows that no product is formed after only adaptor 2.1 has been subjected to the ligation reaction without fragmented DNA and subsequent ethanol precipitation; lane 6 shows that no product is formed after ligation reaction without fragmented DNA and adaptor and subsequent ethanol precipitation.

The dumbbell template is also sized. Exonuclease resistant dumbbell templates of the desired size are separated by agarose gel electrophoresis to minimize the residue of any unwanted products, such as adaptor-adaptor ligation products. A0.8% (w/v) 1 XTAE agarose gel was prepared. Concentrated dumbbell templates containing appropriate amounts of DNA loading dye were prepared and approximately 20. mu.L of the concentrated dumbbell templates were loaded onto an agarose gel. After a time sufficient to isolate the product, the gel was stained with SybrSafe gel stain and visualized on a light box. Gel sections containing dumbbell templates of the desired size range were cut using a sterile scalpel. Dumbbell templates were isolated using the Qiaex ii isolation protocol and resuspended in 30. mu.L of H₂And (4) in O.

Rolling circle replication of the dumbbell template was then performed using a highly processive strand displacing polymerase. The first reaction mixture was prepared with the following ingredients: mu.L of size-selected dumbbell template, 1.5. mu.L of 10 XPhi 29 polymerase buffer (NEB)) 1. mu.L of dumbbell template complementary primer, 0.5. mu.L of Bovine Serum Albumin (BSA) -100mg/mL, and 7. mu.L of HPLC H₂And O. The reaction mixture was incubated at 95 ℃ for 10 minutes, cooled to 45 ℃ for 5 minutes, and then further cooled to 20 ℃. The second reaction mixture was prepared using the following ingredients: mu.L of 10 XPhi 29 polymerase buffer (NEB), 5. mu.L of 10mM dNTP mix, 0.5. mu.L of phi29 polymerase, and 3.5. mu.L of HPLC H₂And O. The first and second reaction mixtures after treatment as described above were combined and incubated at 25 ℃ for 1-4 hours. The resulting mixture was then heated to 65 ℃ for 20 minutes to inactivate the polymerase.

The rolling circle replication products were then analyzed by agarose gel electrophoresis. Due to their high molecular weight, these rolling circle replication products are present in the wells after electrophoresis without entering the gel. Some additional early termination products were also visible.

FIG. 8 is an example of agarose gel analysis of DNA products prepared by rolling circle replication of the products analyzed in FIG. 6. Lane 1 shows a low efficiency rolling circle reaction of the product excised from lane 7 of figure 6. Lane 2 shows the rolling circle product obtained after rolling circle replication of the product excised from lane 8 of fig. 6, which is a highly immobilized complex retained in the well after gel electrophoresis. Lane 3 shows that no rolling circle product was obtained after rolling circle replication of the product excised from lane 9 of figure 6. Lane 4 shows that no rolling circle product was obtained after rolling circle replication of the product excised from lane 10 of figure 6. Lane 5 shows that the rolling circle reaction performed in the absence of DNA did not result in a rolling circle product. Lane 6 shows that the rolling circle reaction with the fragmented DNA product did not obtain a rolling circle product, indicating no random priming from the fragmented DNA.

FIG. 9 is an example of agarose gel analysis of DNA products prepared by rolling circle replication of the products analyzed in FIG. 7. Lane 1 shows the rolling circle product obtained after rolling circle replication of the size-selected product analyzed in lane 1 of fig. 7. The immobile complexes remaining in the wells after gel electrophoresis indicate successful RCR products. Lane 2 shows the rolling circle product obtained after rolling circle replication of the size-selected product analyzed in lane 2 of fig. 7. Lane 3 shows that no rolling circle product was obtained after rolling circle replication of the size-selected product analyzed in lane 3 of fig. 7. Lane 4 shows that no rolling circle product was obtained after rolling circle replication of the size-selected product analyzed in lane 4 of fig. 7. Lane 5 shows that no rolling circle product was obtained after rolling circle replication of the size-selected product analyzed in lane 5 of fig. 7. Lane 6 shows that no rolling circle product was obtained after rolling circle replication of the size-selected product analyzed in lane 6 of fig. 7. Lanes 7, 8 and 9 show that no rolling circle product was obtained in the control reaction where fragmented DNA was provided to the rolling circle reaction without ligation (lane 7); providing fragmented DNA to the rolling circle reaction without primers (lane 8); and no fragmented DNA was provided to the rolling circle reaction without ligation (lane 9).

Example 5

Rolling circle replication products can also be detected by using molecular probes or beacons directed against the complementary regions of the hairpin sequence of the dumbbell template. To demonstrate the feasibility of this approach, a titration series of H3 hairpin adaptors were formed at concentrations ranging from 0 μ M to 5 μ M. Stock solutions of 10 μ M H3 hairpins were serially diluted to achieve 2-fold test concentrations. Hairpin adaptor 3(H3) has the following sequence:

5'PO₄-AATTG CGAGC TATGA CCATG ATTAC GCCAC TGGCC GTCGT TTTAC AACTC GC(SEQ ID NO:9)

for example, a 10 μ M stock solution is diluted in half to achieve a 5 μ M test sample, a 5 μ M stock solution is diluted in half to achieve a 2.5 μ M test sample, and so on. These represent twice the actual tested concentration (2 ×). Approximately 5. mu.L of 2 XH 3 adaptor concentrate was then mixed with 1. mu.L of NEB phi29 reaction buffer 10 ×, 1. mu.L of 200. mu.M beacon 2, and 3. mu.L of HPLC H₂And (4) combining. Molecular Beacon 2 has the following sequence, and "5, 6-FAM" is 5-FAM and6-mixture of FAM isomers and "IABKFQ" is an Iowa Black quencher:

5'-/5,6-FAM/CGGAGTTGCGAGTTGTAAAACGACGGCCAGTCTCCG/3-IABkFQ(SEQ ID NO:10)

in setting up this reaction mixture, the concentration of H3 in the test sample was reduced to the final 1 × measured concentration. The reaction mixture was then heated to 98 ℃ on a hot plate, maintained at this temperature for ten minutes, and then slowly cooled on a bench top. All reaction and thermal cycling steps were performed with the lights off and with the reaction tubes covered with tinfoil to prevent loss of signal from the beacon. Once cooled to room temperature, the reaction was ready for reading on a Molecular Devices SpectraMax Gemini XPS fluorescence microplate reader. Specifically, spectrafrop microplate slides were used to facilitate measurement of very small volumes. Approximately 2. mu.L from each titration reaction was added to the micro-volume slide. Once inserted into the machine, the following procedure was run at room temperature:

excitation wavelength: 495nm

Emission wavelength: 520nm

6 flashes/readings

Raw data is collected and processed as shown in fig. 10. RLU (relative luminescence units) readings of 0 μ M were subtracted from all samples to normalize by eliminating background fluorescence.

TABLE 1

μM[H3]	5	2.5	1.25	0.625	0.3125	0.15625	0
								RFU	134.896	118.257	96.351	66.297	53.12	52.34	46.489
Is adjusted by	88.407	71.768	49.862	19.808	6.631	5.851	0

Example 6

Experiments can be designed to determine the efficiency of making dumbbell templates independent of fragment length size. The main limitation of current large fragment NGS library construction methods is the formation of paired templates by circularizing the ends of long DNA fragments. In theory, the ability to efficiently form dumbbell templates should be size independent. These dumbbell templates can have various sizes including, for example and without limitation, 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, or 10.0 kb. These fragment sizes can be generated by first using PCR, by using human BAC DNA to design primers that target the same genomic region. This method would allow the use of real-time PCR to quantify the copy number of dumbbell templates of different sizes. In one embodiment, real-time PCR reagents for the TCF7L2 rs7903146 allele have been developed, which were designed using the custom TaqMan assay website of Life Technologies (Life Technologies). The 5' -primer sequence was 5' -CCT CAAACC TAG CAC AGC TGT TAT (SEQ ID NO:11), the 3' -primer sequence was 5' -TGAAAACTAAGG GTG CCT CAT ACG (SEQ ID NO:12), and the probe sequence was 5' -CTT TTT AGATA [ C/T ] TAT ATA ATT TAA (SEQ ID NO: 13). In other embodiments, fragments of different sizes can be generated, the initial number of amplicons quantified, the hairpin structure 2 ligated using the same conditions, and then the copy number of the dumbbell template quantified using real-time PCR.

In other experiments, a defined population of fragments can be formed primarily via two methods, Covaris G-tube and NEB fragmenting enzyme. Dumbbell templates will then be prepared and isolated according to the TA cloning method described herein. Molecular beacons that hybridize to hairpin sequences can be used to quantify the number of dumbbell templates of different sizes using a fluorescent plate reader. These experiments demonstrate that dumbbell templates can be efficiently formed independent of the fragment size of the starting genomic DNA sample. In addition, these molecular beacons can also be used to quantify RCR products and reaction efficiency.

Example 7

Efficient insertion of dumbbell templates in NGS paired-end sequencing platforms requires the presence of unique primers or hairpins on each end of the DNA template. This would be achieved via standard end repair/dA tailing methods, followed by ligation of two unique hairpin oligonucleotides (i.e., hpA and hpB) each containing a unique universal replication/sequencing priming site and a molecular beacon site. After hairpin ligation, we expected a population consisting of 25% hpA-fragment-hpA, 50% hpA-fragment-hpB, and 25% hpB-fragment-hpB. The desired enrichment can be achieved by capture probe chromatography by first passing the ligation product through a column containing the reverse complement of hairpin A, thereby capturing hpA-fragment-hpA template and hpA-fragment-hpB template, but not hpB-fragment-hpB templateForm hpA-fragment-hpB. After elution, the partially enriched sample was then passed through a second column containing the reverse complement of hairpin B, thereby capturing hpA-fragment-hpB template, but not hpA-fragment-hpA template. This dual hairpin approach will be demonstrated using a method similar to that outlined above; populations of uniquely sized DNA fragments surrounding 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, and 10.0kb will be formed, subjected to size selection, purification, end repair, and dA tailing. These were then ligated to the hpA hairpin and hpB hairpin and double labeled, and the hpA-fragment-hpB dumbbell template would be enriched using the technique described above. Will proceed using phi29 DNA polymerase and T₇Preliminary experiments with replica systems to evaluate the dependence of replica copy number on dumbbell template size using solution-based molecular beacons.

Example 8

The conditions for rolling circle amplification can be optimized to include the appropriate DNA polymerase, replication factors, and reaction conditions such that at least 1,000-fold replication of a 10kb dumbbell template can be supported. Fold replication of 1,000 copies was targeted, since this is the equivalent number of short templates of clonal amplification achieved on the Illumina cBot instrument, and therefore, similar levels of fluorescence signal measured during the sequencing process can be expected. A panel of DNA polymerases can be employed for long synthesis, including commercially available DNA polymerases: (LongAmp, Bst 2.0, Q5, and T₇DNA polymerase), and at least 12 non-commercially available proprietary families A, B, and DDNA polymerase. In addition, a set of replication cofactors may be used as replication enhancers. Helper proteins may be added to increase the efficiency of production of the desired rolling circle product, including but not limited to processivity clamp and clamp loader complex that increase DNA polymerase persistence, single-stranded binding proteins that stabilize single-stranded DNA regions, helicases that separate double-stranded DNA prior to DNA polymerase, flap endonucleases for resolving flap DNA structures, andDNA ligase to seal the DNA nicks. These factors are interchangeable with DNA polymerases from within the same family and can be tested using the appropriate DNA polymerase partner. For example, a core cofactor from archaea thermophila species 9 ° N (archaeon thermochoccus sp.9 ° N) will be used with family B DNA polymerases, while family ADNA polymerases will be tested using e. The replicated dumbbell template DNA will be measured using quantitative PCR. The qPCR probes can target hairpin regions of a 2kb dumbbell template and a 10kb dumbbell template formed as described herein. In the case of replication-triggered dumbbell templates, probes can be bound to each segment of the synthetic hairpin region. Thus, probe intensity can be used to indicate copy number when compared to a series of standard diluted hairpin templates (fig. 11). In addition to qPCR, the length of the amplified product can also be monitored by alkaline agarose gel electrophoresis. Alkaline agarose gel electrophoresis separates DNA into single strands and accurately measures total replication product length.

Example 9

In one embodiment, an optimal density of functionalized primers for rolling circle replication is attached to the glass surface of a custom designed flow cell. A custom cut adhesive gasket sandwiched between two slides was designed as shown in fig. 11A. The replicon was attached to the bottom side of the cover slip. The cover glass has inlet/outlet ports secured with a nano-port fitting. The gasket here is a 3M double-sided tape with microchannels and is placed on top of a standard microscope slide. Such a design matches the necessary optical, chemical and mechanical properties with practical needs such as ease of use, speed of manufacture, simplicity and cost effectiveness.

As shown in fig. 11B, the flow cell design consisted of microchannels formed by sandwiching a 130 μ M thick 3M double-sided adhesive film gasket between a standard 1mm thick 25mm x 75mm borosilicate glass slide (VWR) and a 25mm x 75mm #1.5H borosilicate cover slip (Schott Nexterion). The microfluidic channel gasket layer was cut from 3M double-sided tape using a laser cutter (Universal X-660), and the inlet/outlet holes were grit blasted through the top coversheet layer. The channel is sealed by placing an adhesive gasket on top of the slide and then placing a cover slip on top of the gasket. The resulting channel had a rectangular cross-section 130 μm deep, 3mm wide and 4cm long. A 100 μm inner diameter PEEK tube was connected to the inlet and outlet ports using a nano-port clamp (IDEX Health & Science corporation) as a means for exchanging solutions and reagents within the flow cell.

Pre-synthesized oligonucleotides can be attached to a glass surface by using a chemical strategy. The identification of the optimal carrier chemistry is important because previous studies have demonstrated that certain coupling strategies can affect the performance of hybridization and solid phase PCR applications. In one example, functionalizing the glass surface with a silane reagent, such as 3-aminopropyltriethoxysilane, is the first step. Many chemical coupling strategies involve amino-modified oligonucleotides. The use of these terminal functional groups as a starting point allows the use of systematic methods for the evaluation of different intermediate coupling agents for the attachment of oligonucleotides to glass surfaces. By way of example and not limitation, cyanuric chloride activation has been used to make oligonucleotide sequences 5' -NH₂TTTTTTTTTTTTGTAAAACGACGGCCAGT (SEQ ID NO:14) was attached to the surface of the patch. Other examples may utilize a variety of other activation chemistries, such as 1, 4-phenylene diisothiocyanate and dicarboxylic acid reactions. All of these activation strategies produced similarly good hybridization data. Embodiments of the invention include poly (dT) of varying lengths_nLinker (i.e., n is 0, 10, 20).

In one example, a Nikon Eclipse FN1 microscope may be utilized that uses a broadband LED light source and provides the flexibility to use different fluorescent dyes across the visible and near IR regions. In one example, the hairpin structure 3(5' -T) is used using the dA tailing methodCGCGAG CTCGCG(SEQ ID NO:15)) to form a pUC18 dumbbell template. Molecular beacon 2(5' -FAM-CGGAG CTCCGIowa Black (SEQ ID NO:16)) to determine the solid phase rolling circle replication reaction in the flow cell described above. Underlined sequences indicate double-stranded stem regions, the first boxed sequence indicates probe sequence, and the second boxed sequence indicates primer sequence to be bound to the immobilized M13 primer sequence. Since molecular beacons should produce low background fluorescence, sufficient rolling circle replication to produce surface-bound replicons will produce good signal-to-noise ratios (SNR). The dilution of the 0.5kb dumbbell template can be determined empirically to target replicon densities of 25k to 50k per field of view (FOV).

In certain embodiments, surface effects may inhibit some reactions and may require the use of passivating agents, such as polyvinylpyrrolidone or high molecular weight PEG. In some instances, low yields of phosphorothioate primers can be utilized, since the φ 29DNA polymerase can exhibit significant exonuclease activity on single stranded DNA.

Example 10

The reagents and conditions gleaned from the previous examples can be applied to fragmented human genomic DNA to demonstrate the robust ability to form NGS-compatible clonal replication clusters from a 10kb dumbbell template. In some examples, a 10kb dumbbell template can produce 1,000 copies of the target sequence. A variety of DNA polymerases are effective in rolling circle replication methods, including but not limited toBst, and Vent (exo-) DNA polymerase. Recently, a mutant φ 29DNA polymerase was identified to increase DNA synthesis yield by several fold and is commercially available from Sygnis. In some instances, a replicator complex may be used to use T₇Sequencing enzyme, T₇Helicase and T₇The synergistic activity of the single-chain binding proteins replicated a 10kb dumbbell template. In certain examples, dumbbell templates (at size increments of 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, and 10.0 kb) can be used for solution assays and analyzed for TCF7L2 using real-time PCR testing. In some instances, it is desirable to provide,helper proteins may be included, including for example and without limitation other helicases, single-chain binding proteins, thioredoxins, topoisomerases, counter-rotating enzymes, or any combination thereof to improve the efficiency and accuracy of rolling circle replication methods.

In certain examples, hairpin structure 3 can be used to form 0.5kb, 1.0kb, 2.5kb, 5.0kb, 7.5kb, or 10.0kb dumbbell templates and tested with several of the optimal conditions identified in solution-based real-time PCR assays. Following the rolling circle replication method, the replicated dumbbell templates can be probed using molecular beacon 2 and analyzed by fluorescence microscopy to determine the signal intensity of the replicated dumbbell templates. In certain embodiments, rolling circle replication can be performed using a dual hairpin dumbbell template as a real world template isolated from HapMap sample NA 18507.

Embodiments of the invention may also include one or more hairpin structures, enzymes, other nucleotides, and protein reagents packaged into kits for performing the methods and producing the compositions described herein. Reagents for performing the methods and detecting the presence of rolling circle products as described herein may be provided separately or may be co-packaged in the form of a kit. For example, a kit can be prepared to include one or more primers, one or more labeled nucleoside triphosphates, and associated enzymes for performing the various steps of the methods described herein. The kit can also include a packaged combination of one or more affinity-tagged hairpin structures and one or more corresponding solid supports to purify the dumbbell template. The arrangement of reagents within the containers of the kit will depend on the particular reagents involved. Each reagent may be packaged in a single container, but various combinations are possible. Embodiments of the invention may also include a kit containing one or more oligonucleotides to form one or more hairpin structures; a set of components for ligation comprising a ligase, a cofactor, and a suitable buffer; and a set of components for replication, including substantially complementary primers, enzymes to perform the various steps described herein, cofactors, and appropriate buffers.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a first set of components for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template, wherein the components of the first set contain one or more of a ligase, a cofactor, a ligase-appropriate buffer, and a combination thereof; (ii) a second set of components for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules, wherein the components of the second set contain one or more of an exonuclease, an exonuclease compatible buffer, and combinations thereof; and a third set of components for replicating the at least one dumbbell template to form at least one amplified dumbbell template, wherein the components of the third set contain a polymerase or replisome, nucleotides, a cofactor, and at least one primer substantially complementary to a region of the at least one dumbbell template.

Embodiments of the invention also include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a polymerase and at least one primer substantially complementary to a region of the at least one dumbbell template for replicating the at least one dumbbell template to form at least one replicated dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a replicator and at least one primer substantially complementary to a region of said at least one dumbbell template for replicating said at least one dumbbell template to form at least one replicated dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a polymerase and at least two primers substantially complementary to at least two regions of the at least one dumbbell template for amplifying the at least one dumbbell template to form at least one amplified dumbbell template.

Certain embodiments of the invention include a kit comprising at least one oligonucleotide capable of forming a hairpin structure; a ligase for ligating the hairpin structure to at least one nucleic acid molecule from a sample to form at least one dumbbell template; an exonuclease for purifying the at least one dumbbell template by digesting any unligated hairpin structures and any unligated nucleic acid molecules; and a replicator and at least two primers substantially complementary to at least two regions of said at least one dumbbell template for amplifying said at least one dumbbell template to form at least one amplified dumbbell template.

Furthermore, the foregoing has outlined rather broadly certain objects, features and technical advantages of the present invention and the detailed description so that the embodiments of the invention that form the subject matter of certain claims of the invention may be better understood in view of the features and advantages of the invention as described herein. It should be appreciated that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized that such equivalent constructions do not depart from the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the foregoing description when considered in connection with the accompanying figures. It is to be expressly understood, however, that such description and drawings are provided for the purpose of illustration and description only and are not intended as a definition of the limits of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made within the spirit and scope of the invention as described in the foregoing specification.

Sequence listing

<110> Reidwort biosciences Co

<120> systems and methods for clonal replication and amplification of nucleic acid molecules for genomic and therapeutic applications

<130> REDV.P0004WO

<150> US 61/978,823

<151> 2014-04-11

<160> 16

<170> PatentIn version 3.5

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aattgcgagt tgcgagttgt aaaacgacgg ccagtctcgc 40

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actggccgtc gttttacaa 19

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tgcgagttgc gagttgtaaa acgacggcca gtctcgc 37

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<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<400> 11

cctcaaacct agcacagctg ttat 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<400> 12

tgaaaactaa gggtgcctca tacg 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<220>

<221> misc_feature

<222> (12)..(12)

<223> n = C or T

<400> 13

ctttttagat antatataat ttaa 24

<210> 14

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<400> 14

tttttttttt ttgtaaaacg acggccagt 29

<210> 15

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<400> 15

tcgcgagcta tgaccatgat tacgccactg gccgtcgttt tacaactcgc g 51

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic oligonucleotide

<400> 16

cggagctatg accatgatta cgccctccg 29

Claims (8)

1. A method of replicating or amplifying at least one nucleic acid molecule, the method comprising:

(a) ligating a plurality of hairpin structures to each end of at least one double-stranded nucleic acid molecule to form at least one dumbbell template, wherein the double-stranded nucleic acid molecule is isolated from the sample with a minimum length of greater than 5 kilobases;

(b) contacting the at least one dumbbell template with at least one substantially complementary primer in a replication method or with at least two substantially complementary primers in an amplification method;

wherein the at least one substantially complementary primer is attached to at least one substrate; and

(c) performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated or amplified dumbbell template.

2. The method of claim 1, wherein the double-stranded nucleic acid molecule has a length greater than 10 kilobases.

3. The method of claim 1, wherein the double-stranded nucleic acid molecule has a length of at least 20 kilobases.

4. The method of any one of claims 1 to 3, wherein:

prior to the ligating step, the method further comprises fragmenting the at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule having a minimum length of greater than 5 kilobases, greater than 10 kilobases, or at least 20 kilobases.

5. The amplification method of any one of claims 1 to 3, wherein:

prior to the contacting step, the method further comprises purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated nucleic acid molecules with an exonuclease.

6. A method of detecting at least one replicated or amplified dumbbell template, the method comprising:

(a) fragmenting at least one nucleic acid molecule to form at least one fragmented nucleic acid molecule having a minimum length of greater than 5 kilobases, greater than 10 kilobases, or at least 20 kilobases;

(b) replicating or amplifying the fragmented nucleic acid molecules according to the replication method of claim 1; and

(c) detecting the at least one replicated or amplified dumbbell template, wherein the detecting comprises a next generation sequencing (or NGS) method involving clonally replicated templates.

7. The method of claim 6, wherein the step of detecting the at least one replicated or amplified dumbbell template comprises contacting the at least one replicated or amplified dumbbell template with a DNA probe.

8. The method of claim 6, wherein:

prior to the contacting step, the method further comprises purifying the at least one dumbbell template by treating any unligated hairpin structures and any unligated fragmented nucleic acid molecules with an exonuclease.