Ligase-assisted nucleic acid circularization and amplification

Abstract

To provide, e.g., efficient methods for non-specifically amplifying short DNA sequences in a single reaction vessel without any intervening cleaning steps.SOLUTION: Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of linear chromosomal DNA in a single tube using ligation-assisted DNA amplification is also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention generally relates to a method for amplifying linear nucleic acid sequences by rolling circle amplification in a single reactor without intervening isolation and / or purification steps. The method involves generating single-stranded DNA circles from single- or double-stranded linear DNA by template-independent single-stranded DNA ligation, followed by rolling circle amplification using specialized primer sequences. The method further relates to whole-genome amplification of linear chromosomal DNA in a single reactor by rolling circle amplification using a random primer mixture, followed by its detection. [Background technology]

[0002] DNA amplification is the process of replicating a target double-stranded DNA (dsDNA) to generate multiple copies of it. Because the individual strands of dsDNA are antiparallel and complementary, each strand can serve as a template strand to generate its complementary strand. The template strand is preserved as a whole or as a truncated fragment, and the complementary strand is constructed from deoxynucleoside triphosphates (dNTPs) by DNA polymerase. Complementary strand synthesis proceeds in the 5'→3' direction, starting from the 3' end of a primer sequence that hybridizes to the template strand.

[0003] Whole genome amplification (WGA) involves nonspecific amplification of target DNA. WGA is often achieved by multiple displacement amplification (MDA), which uses random oligonucleotide primers to initiate DNA synthesis at multiple locations in the target DNA in conjunction with a high-fidelity DNA polymerase with strand displacement activity (e.g., Phi29 polymerase). Even though currently available commercial WGA systems, such as the GenomiPhi (GE Healthcare, USA) and RepliG (Qiagen) kits, provide optimal results with high-molecular-weight target DNA, these systems perform poorly when the target DNA is short and / or highly fragmented. When target DNA is fragmented and the sequence length is less than approximately 1,000 nucleotides, amplification of the target DNA using conventional methods results in reduced amplification rates, loss of critical sequences, especially near the ends of the target DNA, and highly sequence-biased amplification. The shorter the template DNA length, the lower the likelihood of multiple primed strands in the MDA reaction. This reduces the amplifiable potential of these shorter fragments. Therefore, an efficient method for non-specifically amplifying short fragmented DNA is highly desirable.

[0004] Ligation-mediated polymerase chain reaction (PCR) has been used to amplify fragmented dsDNA. However, only a small portion of the fragmented DNA is amplified in these reactions, leading to inadequate genome coverage. To efficiently amplify fragmented target dsDNA, they must first be repaired and then concatenated by blunt-end ligation to generate sequences longer than 1,000 base pairs (bp). However, relatively high concentrations of target DNA are often required to facilitate concatenation and subsequent amplification. Circularization of double-stranded target DNA is also used in various nucleic acid-based assays, including MDA, WGA, highly branched rolling circle amplification (RCA), and massively parallel DNA sequencing. To efficiently circularize and amplify fragmented dsDNA, the double-stranded ends of the fragmented DNA must first be repaired, followed by blunt-end ligation to form double-stranded DNA circles. However, circularization of double-stranded DNA fragments less than 500 bp in length is difficult.

[0005] Double-stranded DNA can be denatured to generate single-stranded DNA (ssDNA), which can be further circularized in a template-dependent intramolecular ligation reaction using a ligase. However, prior sequence information of the target DNA is required to perform template-dependent circularization. Template-independent intramolecular ligation of ssDNA has also been documented. For example, TS2126 RNA ligase (commercially available under the trademarks THERMOPHAGE™ RNA Ligase II or THERMOPHAGE™ ssDNA Ligase (Prokaria, Matis, Iceland) or CIRCLIGASE™ ssDNA Ligase (Epicentre Biotechnologies, Wisconsin, USA)) has been used to create digital DNA balls and / or for locus-specific cleavage and amplification of DNA, such as genomic DNA. CIRCLIGASE I™ has a low degree of adenylation (approximately 30%), while CIRCLIGASE II™ contains a substantially adenylated form of TS2126 RNA ligase. Linear single-stranded complementary DNA (cDNA) molecules prepared from the 5'-end fragments of mRNA have also been amplified by rolling circle replication after circularization using TS2126 RNA ligase. By appropriately incorporating a sense RNA polymerase promoter sequence into the cDNA, the circularized cDNA template has been shown to serve as a transcription substrate, thus influencing the amplification of mRNA molecules in biological samples. Furthermore, TS2126 RNA ligase has been used to amplify cDNA ends for random amplification of cDNA ends (RACE). DNA templates for rolling circle amplification have also been generated from limited amounts of fragmented DNA using TS2126 RNA ligase. This method involved denaturing linear fragmented dsDNA to obtain linear ssDNA fragments, ligating the linear ssDNA with CIRCLIGASE™ ssDNA ligase to obtain single-stranded DNA circles, and then amplifying the single-stranded DNA circles using random primers and Phi29 DNA polymerase by RCA. However, even after optimizing the reaction conditions, the amount of single-stranded circular DNA produced was highly variable and sequence-dependent.For example, oligonucleotides containing 5' G and 3' T nucleotides ligated significantly better than their complementary oligonucleotides containing 5' A and 3' C nucleotides under identical ligation conditions. Furthermore, intramolecular ligation efficiency varied between linear ssDNA sequences with identical or very similar sizes but slight differences in nucleotide sequence. This efficiency also varied between linear ssDNA sequences of different sizes (e.g., sequence lengths ranging from 100 bases to several kilobases). Furthermore, all attempts at ligation-amplification reactions necessarily involved intermediate isolation, purification, and / or cleaning steps, thus making the ligation-amplification workflow unwieldy. For example, analysis of forensic samples consisting of fragmented DNA by circularization followed by rolling circle amplification was performed in multiple steps, including 5' DNA phosphorylation, adapter ligation, DNA circularization, and whole genome amplification. Each step reaction was subjected to reaction cleanup before the next step was performed. Furthermore, multistep processes often resulted in loss of template DNA and analytical failure. No amplification advantage was observed when ligation and amplification were performed in a single reactor; rather, components of the carried-over ligation reaction were often found to be inhibitory to the subsequent amplification reaction. Therefore, an efficient method for nonspecifically amplifying short DNA sequences in a single reactor without an intervening cleaning step is highly desirable, especially when representative and averaged whole-genome information is desired. Furthermore, a method for amplifying linear nucleic acid sequences by rolling circle amplification in a single reactor that overcomes the inhibition due to reactants in each of the ligation-amplification reactions is highly desirable. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 022359 Brochure Summary of the Invention

[0007] In some embodiments, a method for amplifying linear chromosomal DNA by rolling circle amplification is provided. The method includes the steps of preparing linear chromosomal DNA, performing intramolecular ligation of the linear chromosomal DNA using a ligase capable of template-independent intramolecular ligation of single-stranded DNA to generate a single-stranded DNA circle, and amplifying the single-stranded DNA circle by rolling circle amplification. The rolling circle amplification uses a random primer mixture containing an oligonucleotide sequence having at least one nucleotide analog. All steps of the method, including the ligation reaction and the rolling circle amplification reaction, are performed in a single reaction vessel without any intervening isolation or purification steps. If the linear chromosomal DNA is in double-stranded form, it is denatured to generate single-stranded DNA before the intramolecular ligation reaction. In some embodiments, this method is used for whole genome amplification of target DNA.

[0008] These and other features, aspects and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows a schematic diagram of one embodiment of ligase-assisted whole genome amplification of fragmented dsDNA. [Figure 2] FIG. 1 shows the size profile of circulating DNA isolated from the plasma of healthy individuals. [Figure 3A] FIG. 1 shows ligase-assisted whole genome amplification of circulating DNA extracted from the subcellular fraction of whole blood using CIRCLIGASE II™. [Figure 3B] FIG. 1 shows ligase-assisted whole genome amplification of circulating DNA extracted from the subcellular fraction of whole blood using T4 DNA ligase. [Figure 3C] FIG. 1 shows ligase-assisted whole genome amplification of circulating DNA extracted from the subcellular fraction of whole blood using E. coli DNA ligase. [Figure 4]FIG. 1 shows the effectiveness of ligase-assisted whole genome amplification for sensitive and balanced DNA amplification of four different CODIS loci. [Figure 5] FIG. 1 shows the effectiveness of ligase-assisted whole genome amplification for sensitive and balanced DNA amplification of 12 different CODIS loci. [Figure 6] FIG. 1 shows the efficiency of ligase-assisted whole genome amplification under different reaction and buffer conditions. [Figure 7] FIG. 1 shows the suppression of amplification of high molecular weight genomic DNA in ligase-assisted whole genome amplification. [Figure 8] FIG. 1 shows a schematic diagram of ligase-assisted whole genome amplification, which involves processing (e.g., end repair) of fragmented DNA with polynucleotide kinase, followed by ligase-assisted amplification of the processed fragmented DNA. [Figure 9] FIG. 1 shows a schematic diagram of a single-tube reaction for ligase-assisted amplification of fragmented DNA using PNK and CIRCLIGASE II™ in the presence of GTP. [Figure 10] FIG. 1 shows a single-tube ligase-assisted amplification reaction using male-female plasma / blood in which the DYS14 male-specific marker is detected using a library made from input DNA. [Figure 11] FIG. 1 shows a schematic diagram of phosphorylation and pre-adenylation of fragmented DNA followed by ligation using a substantially non-adenylated ligase. [Figure 12] FIG. 1 shows the improved efficiency of circularization of pre-adenylated DNA sequences using a substantially non-adenylated ligase. [Figure 13] FIG. 1 shows the improved efficiency of ligase-assisted whole genome amplification when the target DNA sequence is pre-adenylated and when ligation is performed with a non-adenylated ligase. [Figure 14] FIG. 1 shows ligase-assisted whole genome amplification of plasma DNA using CIRCLIGASE II™. [Figure 15]FIG. 1 shows a qualitative analysis of the amplified DNA in terms of coverage depth and uniformity level. [Figure 16] FIG. 1 shows the overall coverage and uniformity observed across the target sequence region using AT hexamers. [Figure 17] FIG. 1 shows the depth of coverage when uracil DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg) are used to repair / remove damage from single-stranded DNA before generating single-stranded DNA circles, or when damage is repaired / removed from the generated single-stranded DNA circles, or when no DNA damage is repaired / removed. [Figure 18] FIG. 1 shows the depth and uniformity of coverage when uracil DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg) are used to repair / remove DNA damage from single-stranded DNA before generating single-stranded DNA circles, when DNA damage is repaired / removed from the generated single-stranded DNA circles, or when no DNA damage repair / removal is performed. [Figure 19] FIG. 1 shows the favorable positive predictive value (PPV) and sensitivity when uracil DNA glycosylase (UDG) and formamidopyrimidine DNA glycosylase (Fpg) are used to repair / remove DNA damage from single-stranded DNA circles before rolling circle amplification, or when DNA damage is repaired / removed from the generated single-stranded DNA circles, or when DNA damage is not repaired / removed. DETAILED DESCRIPTION OF THE INVENTION

[0010] The following detailed description is illustrative and is not intended to limit the invention or its uses. Throughout the specification, illustrations of specific terms should be considered as non-limiting examples. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Approximate terms used herein throughout the specification and claims may be applied to modify any quantitative expression that can vary within acceptable limits without changing its associated basic function. Accordingly, values ​​modified by terms such as "about" are not limited to the precise value specified. Unless otherwise specified, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like, used in the specification and claims are understood to be modified in all instances by the term "about." Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and without any attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques. Where necessary, ranges are provided, including all subranges therebetween. To more clearly and concisely describe and illustrate the subject matter of the claimed invention, the following definitions are provided for specific terms used in the following description and appended claims.

[0011] As used herein, the term "nucleoside" refers to a glycosylamine compound in which a nucleic acid base (nucleobase) is linked to a sugar moiety. "Nucleotide" refers to a nucleoside phosphate. Nucleotides can be represented using the alphabetical letter (letter designation) corresponding to the nucleoside, as listed in Table 1. For example, A means adenosine (a nucleoside containing a nucleobase, adenine), C means cytidine, G means guanosine, U means uridine, and T means thymidine (5-methyluridine). W means either A or T / U, and S means either G or C. N represents a random nucleoside, and dNTP refers to a deoxyribonucleoside triphosphate. N can be any of A, C, G, or T / U. [Table 1]

[0012] As used herein, the term "nucleotide analog" refers to a compound structurally similar to a naturally occurring nucleotide. A nucleotide analog may have an altered phosphate backbone, an altered sugar moiety, an altered nucleobase, or a combination thereof. A nucleotide analog may be a natural nucleotide, a synthetic nucleotide, a modified nucleotide, or a surrogate replacement moiety (e.g., inosine). Generally, nucleotide analogs with altered nucleobases confer, among other things, different base-pairing and base-stacking properties. As used herein, the term "LNA (locked nucleic acid) nucleotide" refers to a nucleotide analog in which the sugar moiety of the nucleotide contains a bicyclic furanose unit locked into a sugar conformation that mimics ribonucleic acid (RNA). The structural change from deoxyribonucleotides (or ribonucleotides) to LNA nucleotides is chemically limited by the introduction of an additional bond between the 2' and 4' carbon atoms (e.g., a 2'-C,4'-C-oxymethylene bond; see, for example, Singh, SK, et al., Chem. Comm., 4, 455-456, 1998, or Koshkin, AA, et al., Tetrahedron, 54, 3607-3630, 1998). In LNA nucleotides, the 2' and 4' positions of the furanose units can be linked by O-methylene (e.g., oxy-LNA: 2'-O, 4'-C-methylene-β-D-ribofuranosyl nucleotides), S-methylene (thio-LNA), or NH-methylene moieties (amino-LNA), and the like. Such bonds limit the conformational freedom of the furanose ring. LNA oligonucleotides show improved hybridization affinity to complementary single-stranded RNA and complementary single-stranded or double-stranded DNA. LNA oligonucleotides can induce A-type (RNA-like) double-stranded conformation. Nucleotide analogs with modified phosphate-sugar backbones (e.g., PNA, LNA) often modify strand properties, such as secondary structure formation. A star (*) symbol before a letter designation indicates that the nucleotide designated by that letter is a phosphorothioate-modified nucleotide. For example, *N represents a phosphorothioate-modified random nucleotide. A plus (+) symbol before a letter designation indicates that the nucleotide designated by that letter is an LNA nucleotide. For example, +A represents an adenosine LNA nucleotide, and +N represents a locked random nucleotide (i.e., a random LNA nucleotide). The letter designation "(at N)" represents a random nucleotide containing the nucleobase 2-amino dA, 2-thio-dT, G, or C.

[0013] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotides. As used herein, the term "nucleic acid" refers to a polymer of nucleotides. As used herein, the term "sequence" refers to the nucleotide sequence of an oligonucleotide or nucleic acid. Throughout this specification, whenever an oligonucleotide or nucleic acid is represented by a series of letters, the nucleotides are in 5'->3' order from left to right. For example, an oligonucleotide represented by the letter sequence (W)x(N)y(S)z (where x=2, y=3, and z=1) represents the oligonucleotide sequence WWNNNS, where W is the 5'-terminal nucleotide and S is the 3'-terminal nucleotide. An oligonucleotide or nucleic acid may be DNA, RNA, or an analog thereof (e.g., a phosphorothioate analog). An oligonucleotide or nucleic acid may also contain modified bases and / or backbones (e.g., modified phosphate linkages or modified sugar moieties). Non-limiting examples of synthetic backbones that confer stability and / or other advantages to nucleic acids include phosphorothioate linkages, peptide nucleic acids, locked nucleic acids, xylose nucleic acids, or analogs thereof.

[0014] As used herein, the term "primer" refers to a short, linear oligonucleotide that hybridizes with a target nucleic acid sequence (e.g., a DNA template to be amplified) to initiate a nucleic acid synthesis reaction. A primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. A primer may contain natural, synthetic, or modified nucleotides. The upper and lower limits of primer length are determined empirically. The lower limit of primer length is the minimum length required to form a stable duplex by hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 nucleotides in length) do not form thermodynamically stable duplexes with the target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of duplex formation in regions other than the predetermined nucleic acid sequence of the target nucleic acid. Generally, suitable primer lengths are within the range of about 3 to about 40 nucleotides.

[0015] As used herein, the term "random primer" refers to a mixture of primer sequences generated by randomizing nucleotides at any given position in an oligonucleotide sequence in such a way that the given position can be composed of any possible nucleotide or its analog (fully randomized). Thus, random primers are random mixtures of oligonucleotide sequences consisting of all possible combinations of nucleotides in the sequence. For example, hexamer random primers can be represented by the sequence NNNNNN or (N)6. Hexamer random DNA primers consist of all possible hexamer combinations of the four DNA nucleotides A, C, G, and T, resulting in a random mixture containing 46 (4,096) unique hexamer DNA oligonucleotide sequences. Random primers can be effectively used to initiate nucleic acid synthesis reactions when the sequence of the target nucleic acid is unknown or for whole genome amplification reactions.

[0016] As used herein, the term "partially constrained primer" refers to a primer in which some of the nucleotides in the oligonucleotide sequence are completely randomized (i.e., the nucleotides are , T / U, C, G, or their analogs) while restricting complete randomization of some other nucleotides (i.e., randomization of nucleotides at certain positions to a lesser extent than the possible combinations A, T / U, C, G, or their analogs). For example, a partially constrained DNA hexamer primer represented by WNNNNN represents a mixture of primer sequences in which the 5'-terminal nucleotide of all sequences in the mixture is A or T. Here, the 5'-terminal nucleotide is constrained to two possible combinations (A or T), as opposed to the maximum four possible combinations (A, T, G, or C) of a fully random DNA primer (NNNNNN). Suitable primer lengths for partially constrained primers may range from about 3 nucleotides to about 15 nucleotides.

[0017] As used herein, the term "partially constrained primer with terminal mismatch primer-dimer structure" refers to a partially constrained primer sequence in which, when two individual primer sequences in a partially constrained primer intermolecularly hybridize with each other with three or more nucleotides of internal homology to form a primer-dimer structure without a recessed end, a primer-dimer structure with a single nucleotide base 3' recessed end, or a primer-dimer structure with two nucleotide bases 3' recessed end, both 3'-terminal nucleotides of the primer-dimer structure have nucleotide mismatches (i.e., non-base-paired nucleotides). For example, a partially constrained pentameric primer represented by WNNNS intermolecularly hybridizes to form a primer-dimer structure without a recessed end, resulting in terminal mismatches at both 3'-terminal nucleotides. The primer-dimer structure has three nucleotides of internal homology (i.e., when a primer-dimer structure without a recessed end is formed by intermolecular hybridization, the three random nucleotides of WNNNS can base-pair with each other). However, this exemplary primer does not result in terminal mismatches when hybridized intermolecularly to form a primer-dimer structure containing a single nucleotide base 3' recessed end. Similarly, a partially constrained hexamer primer, represented by WWNNNS, results in terminal mismatches at both 3'-terminal nucleotides when hybridized intermolecularly to form a primer-dimer structure without a recessed end. Furthermore, this exemplary primer results in terminal mismatches at both 3'-terminal nucleotides, even when hybridized intermolecularly to form a primer-dimer structure with a single nucleotide base 3' recessed end. A partially constrained heptamer primer, represented by WWWNNNS, results in terminal mismatches at both 3'-terminal nucleotides when hybridized intermolecularly to form a primer-dimer structure without a recessed end.Furthermore, this exemplary primer provides terminal mismatches at both 3'-terminal nucleotides when hybridizing intermolecularly to form a primer-dimer structure with a single nucleotide base 3' recessed end or a primer-dimer structure with a two-nucleotide base 3' recessed end.

[0018] As used herein, the term "rolling circle amplification (RCA)" refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., a single-stranded DNA circle) by a rolling circle mechanism. A rolling circle amplification reaction is initiated by hybridization of a primer to a circular, often single-stranded, nucleic acid template. A nucleic acid polymerase then extends the primer hybridized to the circular nucleic acid template by continuously advancing around the circular nucleic acid template, replicating the nucleic acid template sequence over and over again (the rolling circle mechanism). Rolling circle amplification generally produces concatemers containing tandem repeats of the circular nucleic acid template sequence. Rolling circle amplification can be linear RCA (LRCA), which exhibits a linear amplification rate (e.g., RCA using a single specific primer), or exponential RCA (ERCA), which exhibits an exponential amplification rate. Rolling circle amplification can also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA), leading to hyperbranched concatemers. For example, In dual-primed RCA, one primer may be complementary to a circular nucleic acid template, as in linear RCA, while the other may be complementary to the tandem repeat unit nucleic acid sequence of the RCA product. As a result, dual-primed RCA can proceed as a chain reaction with an exponential (geometric) amplification rate characterized by a branched cascade of multiple hybridization, primer extension, and strand displacement events involving both primers. This often generates a set of distinct concatemeric double-stranded nucleic acid amplification products. Rolling circle amplification may be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0019] As used herein, multiple displacement amplification (MDA) refers to a nucleic acid amplification method in which amplification involves annealing primers to denatured nucleic acids followed by strand-displacement nucleic acid synthesis. As nucleic acids are synthesized by strand displacement, the number of priming events gradually increases, forming a network of hyperbranched nucleic acid structures. MDA is highly useful for whole genome amplification to generate high-molecular-weight DNA with limited sequence bias from small amounts of genomic DNA samples. Strand-displacement nucleic acid polymerases that have strand-displacement activity in addition to their nucleic acid synthesis activity, such as Phi29 DNA polymerase or large fragments of Bst DNA polymerase, can be used in MDA. MDA is often performed under isothermal reaction conditions using random primers to achieve amplification with limited sequence bias.

[0020] As used herein, the term "pre-adenylation ligase" refers to a ligase in its adenylated form. The adenylated form of the ligase is capable of intramolecular ligation of linear ssDNA molecules having a 5' phosphoryl group and a 3' hydroxyl group in the absence of ATP or dATP. Ligation using a pre-adenylation ligase refers to a ligation reaction in which a high percentage of the ligase molecules used in the reaction are in the adenylated form. Generally, more than 60% of the ligase molecules may be in their adenylated form. In some embodiments, when a ligation reaction is performed using a pre-adenylation ligase, more than 70% of the ligase molecules used in the reaction may be in their adenylated form. In some other embodiments, when a ligation reaction is performed using a pre-adenylation ligase, more than 80%, 90%, or 95% of the ligase molecules used in the reaction may be in their adenylated form.

[0021] As used herein, the term "adenylation enzyme" refers to an enzyme capable of adenylating a nucleic acid sequence to produce a 5'-adenylated nucleic acid. As used herein, a 5'-adenylated nucleic acid refers to a nucleic acid sequence having a hydroxyl group at its 3'-end and an adenylated terminal nucleotide at its 5'-end. For example, 5'-adenylated DNA (AppDNA) refers to a DNA sequence that is adenylated at its 5'-end and has a hydroxyl group at its 3'-end.

[0022] As used herein, the term "non-adenylated ligase" refers to a ligase in its non-adenylated form. The non-adenylated form of the ligase is capable of intramolecular ligation of linear 5'-adenylated ssDNA molecules having a 3' hydroxyl group in the absence of ATP or dATP. Ligation using a non-adenylated ligase refers to a ligation reaction in which a high percentage of the ligase molecules used in the reaction are in the non-adenylated form. Generally, more than 60% of the ligase molecules may be in the non-adenylated form. In some embodiments, when a ligation reaction is performed using a non-adenylated ligase, more than 70% of the ligase molecules used in the reaction may be in the non-adenylated form. In some other embodiments, when a ligation reaction is performed using a non-adenylated ligase, more than 80%, 90%, or 95% of the ligase molecules used in the reaction may be in the non-adenylated form.

[0023] As used herein, the term "melting temperature" (Tm) of a primer-template nucleic acid duplex refers to the temperature at which one half of the duplex separates into single-stranded molecules. The quality of a primer can be measured by its Tm. Primer length and sequence are important determinants in designing the parameters for successful amplification. The melting temperature of a primer-template nucleic acid duplex increases with primer length and with increasing GC content. Monovalent and divalent salt concentrations (e.g., K+, Mg2+, K+), temperature, and the presence of chemical denaturants can affect the Tm of a primer-template nucleic acid duplex and can be used to alter the stability of the primer-template nucleic acid duplex. For example, DNA duplex stability typically increases with higher salt concentrations but decreases as a function of elevated temperature or in the presence of denaturants. For example, high concentrations of salt (e.g., NaCl) increase the Tm of a primer-target DNA duplex because Na+ ions can shield the negative charge of the phosphodiester backbone, thereby reducing the electrostatic repulsion of DNA strands. On the other hand, higher temperatures (approaching or exceeding the Tm of the primer-target DNA hybrid under the buffer conditions used) decrease duplex stability and DNA hybridization efficiency. The Tm of any defined sequence depends on the combined effects of duplex length, GC content, salt concentration, denaturant concentration, and buffer composition, including pH. Furthermore, because hybridization is required during DNA amplification reactions, buffer compatibility with enzyme activity is also a major concern. For optimal enzyme activity, conditions for primer-template hybridization must not only be achieved, but also for enzyme stability and activity. In some cases, optimal enzyme activity may occur under conditions where primer-target hybridization is suboptimal. By incorporating modifications to the primer conditions that result in Tm, primer-target hybridization can be improved by modifying the Tm of the duplex under the conditions used.

[0024] In some embodiments, a method is provided for generating single-stranded DNA circles from linear DNA by incubating with a suitable ligase capable of template-independent intramolecular ligation of the single-stranded DNA. The linear DNA may be linear chromosomal DNA, cell-free circulating DNA, very old DNA or DNA degraded by environmental exposure, or formalin-fixed DNA. In some embodiments, the linear DNA may be fragmented linear DNA. The length of the fragmented linear DNA may range from 15 nucleotides to 21,000 nucleotides. The linear DNA may contain a sequence that already has a ligatable end, or it may contain a sequence that has a non-ligatable end. In one embodiment, the linear DNA may contain a sequence that already has a ligatable end. For example, the linear DNA may already have a phosphate group at the 5' end and a hydroxyl group at the 3' end. Such a DNA sequence is amenable to intramolecular ligation by incubation with a suitable ligase. In some embodiments, a method for generating single-stranded DNA circles from linear chromosomal DNA is provided, the method comprising incubating linear chromosomal DNA with a ligase capable of template-independent intramolecular ligation of single-stranded DNA to generate single-stranded DNA circles. In some embodiments, a pre-adenylation ligase is used in the ligation reaction. Any pre-adenylation ligase capable of joining single-stranded DNA sequences in a template-independent manner may be used. In some embodiments, a substantially adenylated form of TS2126 RNA ligase is used in the template-independent intramolecular ligation reaction. If the linear chromosomal DNA is in double-stranded form, it must be denatured before the intramolecular ligation reaction. The ligation reaction may be performed in the absence of ATP and / or dATP.

[0025] In some embodiments, the linear DNA may contain a sequence with an end that cannot be ligated. For example, the linear DNA may have either a 5' hydroxyl group or a 3' phosphoryl group, or both. In some embodiments, the method includes the steps of: preparing a linear DNA; end-repairing the linear DNA by incubating it with polynucleotide kinase (PNK) in the presence of a phosphate donor to generate a ligatable DNA sequence with a phosphate group at the 5' end and a hydroxyl group at the 3' end; and performing intramolecular ligation of the ligatable DNA sequence with a ligase to generate a single-stranded DNA circle. End repair may include phosphorylating the 5'-terminal nucleotide, dephosphorylating the 3'-terminal nucleotide, or both to generate a ligatable DNA sequence. If the end-repaired ligatable DNA is in double-stranded form, it must be denatured before the intramolecular ligation reaction. In some embodiments, DNA is denatured before the PNK reaction. Phosphorylation or dephosphorylation of single-stranded DNA is usually more efficient than phosphorylation or dephosphorylation of double-stranded blunt ends or 5' recessed ends. The phosphate donor and its concentration in the reaction mixture are selected so that it does not inhibit the subsequent intramolecular ligation reaction. For example, any suitable phosphate donor other than adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) may be used in the end repair reaction using PNK. Suitable phosphate donors include, but are not limited to, guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), or deoxythymine triphosphate (dTTP). In some embodiments, a pre-adenylation ligase is used in the ligation reaction. Any pre-adenylation ligase capable of template-independent single-stranded DNA sequences can be used. In some embodiments, a substantially adenylated form of TS2126 RNA ligase is used in the template-independent intramolecular ligation reaction. The kinase reaction and ligation reaction are performed in the absence of ATP and / or dATP. All steps of this method are performed in a single reaction vessel without any intervening isolation or purification steps. The individual steps of this method can be performed simultaneously or sequentially without intermediate purification or isolation steps. For example, PNK can be added to a reaction vessel (e.g., an Eppendorf tube) containing a nucleic acid solution containing linear target DNA together with GTP to promote end repair of the linear target DNA. Any PNK with 5' phosphorylation and 3' phosphatase activity (e.g., T4 PNK) can be used in the end repair reaction.A combination of PNKs, each with a 5' phosphorylating or 3' phosphatase, may be used in the end-repair reaction. Once the kinase reaction is complete, a pre-adenylation ligase can be added to the same reaction vessel to facilitate the intramolecular ligation reaction.

[0026] Linear DNA can be double-stranded or single-stranded DNA of natural or synthetic origin. DNA can be obtained from a biological sample (e.g., a sample obtained from a biological subject) or found in vivo or in vitro from an unknown entity (e.g., DNA obtained during a forensic investigation). For example, it can be obtained from, but is not limited to, a biological subject's bodily fluid (e.g., blood, plasma, serum, urine, milk, cerebrospinal fluid, pleural fluid, lymph, tears, sputum, saliva, stool, pulmonary aspirate, throat or genital swab), organ, tissue, cell culture, cell fraction, section (e.g., cross-section of an organ or tissue), or cells isolated from a biological subject or from a specific region (e.g., a region containing diseased cells or circulating tumor cells). A biological sample containing or suspected to contain target linear DNA (i.e., the linear DNA of interest) can be a sample of eukaryotic, prokaryotic, viral, or bacteriophage origin. For example, target linear DNA can be obtained from insects, protozoa, birds, fish, reptiles, mammals (e.g., rats, mice, cattle, dogs, guinea pigs, or rabbits), or primates (e.g., chimpanzees or humans). Linear DNA can be genomic DNA (e.g., linear chromosomal DNA) or cDNA (complementary DNA). cDNA can be generated from an RNA template (e.g., mRNA, ribosomal RNA) using reverse transcriptase. Linear DNA can be fragmented DNA or have non-ligatable terminal nucleotides. For example, linear DNA can contain 5' hydroxyl groups and / or 3' phosphate groups so that DNA ligase cannot perform intramolecular ligation reactions. Linear DNA can be dispersed in solution or immobilized on a solid support, such as a blot, assay, array, slide, microtiter plate, or ELISA plate. For example, linear DNA can be immobilized on a substrate via primers and then circularized and amplified.

[0027] If the linear DNA is in double-stranded form, it must be denatured to single-stranded form before the intramolecular ligation reaction. This can be achieved by using any of the art-recognized methods for converting dsDNA to ssDNA sequences. For example, dsDNA can be heat-denatured, chemically denatured, or thermochemically denatured. dsDNA can be chemically denatured using a denaturant (e.g., glycerol, ethylene glycol, formamide, urea, or a combination thereof) that lowers the melting temperature of dsDNA. Denaturants can lower the melting temperature by 5–6°C for every 10% (volume / volume) of denaturant added to the reaction mixture. Denaturants or combinations of denaturants (e.g., 10% glycerol and 6–7% ethylene glycol) can comprise 1%, 5%, 10%, 15%, 20%, or 25% of the reaction mixture (volume / volume). Salts, which reduce hybridization stringency, can also be included in the reaction buffer at low concentrations to chemically denature dsDNA at low temperatures. The dsDNA can be heat denatured, for example, by heating the dsDNA at 95°C.

[0028] After the denaturation step, the resulting ssDNA can be treated with a DNA or RNA ligase capable of intramolecular ligation of ssDNA substrates in the absence of a template to form single-stranded DNA circles. Suitable ligases that may be used in the ligation reaction include, but are not limited to, TS2126 RNA ligase, T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, or E. coli DNA ligase. Conversion of linear single-stranded DNA molecules into single-stranded DNA circles is traditionally carried out by template-dependent intramolecular ligation using ligation enzymes such as T4 RNA ligase. However, template-dependent intramolecular ligation of single-stranded DNA or single-stranded RNA has had limited success, especially when circularization of ssDNA molecules is performed on a population of ssDNA molecules of unknown sequence and / or size. Even though bacteriophage T4 RNA ligase I exhibits template-independent intramolecular ligation activity, this activity is too low and inefficient for practical use in generating circular ssDNA molecules from linear ssDNA molecules.

[0029] In some embodiments, the conversion of ssDNA into single-stranded DNA circles is carried out with a thermostable RNA ligase that has good template-independent intramolecular ligation activity toward linear ssDNA and / or ssRNA substrates bearing 5' phosphoryl and 3' hydroxyl groups. The ligase may be substantially in a pre-adenylated form. For example, TS2126 RNA ligase, derived from the Thermus bacteriophage TS2126, which infects the thermophilic bacterium Thermus scotoductus, can be used for template-independent circularization of fragmented linear ssDNA into circular ssDNA. TS2126 RNA ligase is more thermostable (stable up to approximately 75°C) than many mesophilic RNA ligases, such as T4 RNA ligase. The temperature range for TS2126 RNA ligase activity can be greater than approximately 40°C, e.g., from about 50°C to about 75°C. For this reason, TS2126 RNA ligase may be used at higher temperatures, which further reduces undesirable secondary structures in ssDNA. Circularization of linear ssDNA can also be achieved by ligases other than TS2126 RNA ligase or by using any other enzyme with DNA-binding activity, such as topoisomerase. In some embodiments, circularization of fragmented single-stranded DNA molecules is achieved by RNA ligase 1 from the thermophilic archaeon Methanobacterium thermoautotrophicum (Mth RNA ligase), which has high template-independent ligase activity in circularizing linear fragmented ssDNA molecules.

[0030] In some embodiments, the efficiency of ssDNA circularization by TS2126 RNA ligase A method for improving the ligation efficiency is provided. The use of a pH 8.0 HEPES buffer in the ligation reaction improved ligation efficiency. Template-independent ssDNA ligation was inefficient when the reaction was performed in a TRIS buffer (e.g., the 10x reaction buffer suggested by EpiCenter for CIRCLIGASE II™ contains 0.33 M TRIS acetate (pH 7.5), 0.66 M potassium acetate, and 5 mM DTT). Furthermore, manganese, an essential cofactor for the ligation reaction, is rapidly oxidized under alkaline conditions and precipitates in the presence of TRIS. Air oxidation of Mn2+ to Mn3+ can be promoted by anions that can strongly complex Mn3+ ions. For example, when equal volumes of 0.2 mol / L TRIS and 2 mmol / L MnCl2 were mixed, with the pH appropriately adjusted with HCl, the color change was immediate at pH 9.3 (the pH of TRIS base alone); there was an initial lag of approximately 3 minutes at pH 8.5; and it was not detectable within 1 hour at pH values ​​lower than 8.3. Although no reaction occurred at lower pHs, the change observed at higher pHs was not reversed by adding acid. Due to the rapid oxidation of manganese in TRIS buffer, higher concentrations of manganese are essential for the ligation reaction when intramolecular ligation is performed in TRIS buffer (e.g., adding MnCl2 to a final concentration of 2.5 mM). Furthermore, because the manganese concentration continues to decrease over time, it is difficult to accurately predict the working concentration of manganese in the reaction. When ligation and amplification are performed in a single reactor, higher manganese concentrations can lead to higher polymerase error rates during amplification. By replacing TRIS buffer with HEPES buffer in the ligation reaction, effective intramolecular ligation can be achieved with a manganese ion concentration of less than 0.5 mM. In addition to HEPES, any of Good's buffers (see, e.g., Good, Norman et al., Biochemistry, 5(2):467-477, 1966; and Good, Norman et al., Methods Enzymol., 24:53-68, 1972) can be used for intramolecular ligation reactions.In one embodiment, the intramolecular ligation reaction is carried out in 35 mM HEPES buffer (pH=8.0) containing about 2.5 mM MnCl, about 66 mM KOAc, about 0.5 mM DTT, about 0.003% (wt / wt) Tween-20, and about 0.5 M betaine.

[0031] The ssDNA circle in the ligation reaction mixture can be amplified under isothermal conditions by the rolling circle amplification (RCA) method. Amplification reagents containing DNA polymerase, primers, and dNTPs may be added to the same reaction vessel to generate an amplification reaction mixture and initiate the RCA reaction. Individual reagents used in the amplification reaction may be pretreated to remove contaminating nucleic acids. Decontamination of amplification reagents may be performed using any method known in the art. For example, a decontaminated test DNA polymerase, such as decontaminated phi29 DNA polymerase, may be used in the RCA reaction. Decontamination of proofreading DNA may be performed by incubating it with divalent cations in the absence of dNTPs to remove contaminating nucleic acids. A DNA polymerase without proofreading capabilities, such as Bst DNA polymerase, may be used after incubating it with a proofreading DNA polymerase in the presence of divalent cations and in the absence of dNTPs to remove contaminating nucleic acids. Decontamination may also be performed by incubating the amplification reagents with a nuclease, such as DNase. If decontamination is performed using a nuclease, it must be removed or digested before the amplification reaction. The amplification reaction mixture may further include a reagent such as a single-stranded DNA binding protein and / or a suitable amplification reaction buffer. The amplification of the ssDNA circle is performed in the same reaction vessel in which ligation is performed. Isolation or purification of the ssDNA circle and / or removal of the ligase are not required before the amplification reaction. The amplified DNA may be detected by any currently known method for DNA detection.

[0032] RCA may be performed using any DNA polymerase known in the art (e.g., Phi29 DNA polymerase, Bst DNA polymerase). It may be performed using a random primer mixture or specific primers. In some embodiments, random primers are used in the RCA reaction. Primer sequences containing one or more nucleotide analogs (e.g., LNA nucleotides, 2-amino-dA, or 2-thio dT modifications) may be used. In some embodiments, nuclease-resistant primers (e.g., primer sequences containing phosphorothioate groups at appropriate positions) are used in the amplification reaction (e.g., NNNN*N*N). In some embodiments, RCA may be performed by contacting the ssDNA circle with a primer solution containing a random primer mixture to form a nucleic acid template-primer complex; contacting the nucleic acid template-primer complex with DNA polymerase and deoxyribonucleotide triphosphates; and amplifying the nucleic acid template. In some embodiments, the primer solution contains a partially constrained primer, such as WWNNS. The partially constrained primer may have a primer-dimer structure with mismatches at its ends. In some embodiments, a partially constrained primer consisting of the nucleotide sequence (W)x(N)y(S)z, where x, y, and z are integer values ​​independent of one another, with the value of x being 2 or 3, the value of y being 2, 3, or 4, and the value of z being 1 or 2, is used in the RCA reaction. The partially constrained primer may contain one or more nucleotide analogs. In some embodiments, a nuclease-resistant, partially constrained primer containing modified nucleotides and having a terminal mismatch primer-dimer structure is used in the RCA reaction. Suitable primer sequences include, but are not limited to, +W+WNNS, W+W+NNS, +W+WNNNS, W+W+NNNS, W+W+NN*S, +W+WNN*S, W+W+NNN*S, +W+WNNN*S, W+W+N*N*S, +W+WN*N*S, W+W+NN*N*S, or +W+WNN*N*S.In some embodiments, the RCA reaction is performed by contacting the ssDNA circle with a primer solution consisting essentially of a partially constrained primer mixture containing a terminal mismatch primer-dimer structure and amplifying the ssDNA circle. In some other embodiments, the RCA reaction is performed by contacting the ssDNA circle with a primer solution consisting essentially of a partially constrained primer mixture containing nucleotide analogs and amplifying the ssDNA circle. RCA of ssDNA circles generates large amounts of DNA with reduced sequence loss and reduced amplification bias. The entire ssDNA ligation and amplification process can be performed in a single tube without intermediate purification or isolation steps. To avoid non-target amplification, reagents used in ligation and / or nucleic acid amplification (e.g., primer solution, ligation buffer, DNA polymerase) can be pretreated to remove contaminating nucleic acids.

[0033] In some embodiments, a method for amplifying linear chromosomal DNA is provided. This method may be used for whole genome amplification of chromosomal DNA. The linear chromosomal DNA may be cell-free circulating DNA, DNA isolated from a formalin-fixed, paraffin-embedded sample, a forensic DNA sample, or a very old DNA sample. The linear chromosomal DNA may be exposed to environmental conditions or may be fragmented DNA. The method includes (a) providing linear chromosomal DNA, (b) incubating the linear chromosomal DNA with a ligase capable of template-independent intramolecular ligation of single-stranded DNA sequences to generate single-stranded DNA circles, and (c) amplifying the single-stranded DNA circles by rolling circle amplification using a random primer mixture to form an amplified DNA product. All steps of this method are performed in a single reactor without intervening isolation or purification steps. Individual reagents used in the amplification reaction may be pretreated to remove contaminating nucleic acids. Decontamination of the amplification reagents may be performed using any method known in the art. For example, a decontaminated test DNA polymerase, such as decontaminated phi29 DNA polymerase, may be used in the RCA reaction.This may be performed by incubating it with divalent cations in the absence of dNTPs to remove any remaining nucleic acids. The ligase may be TS2126 RNA ligase, T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, E. coli DNA ligase, or a combination thereof. Pre-adenylated TS2126 RNA ligase is used for template-independent intramolecular ligation of single-stranded DNA sequences in exemplary embodiments. The presence of excess salt, ligation reagents, and / or other by-products may inhibit rolling circle amplification of the generated single-stranded DNA circle when a standard random primer mixture is used in the RCA reaction. The random primer mixture used in the single-reactor ligation-assisted whole genome amplification method contains oligonucleotide sequences containing at least one nucleotide analog. The nucleotide analog in the random primer mixture is selected so that it increases the melting temperature (Tm) of the primer, prevents primer-dimer formation, and / or makes the primer resistant to nucleases. For example, in some embodiments, the method incorporates modified nucleobases (e.g., 2-amino-dA) and nucleotide analogs containing LNA to increase the melting temperature of random primer mixtures used for ligase-assisted whole genome amplification in a single reaction vessel. The inclusion of each 2-amino-dA base in the random hexamer primer mixture increases the T by approximately 3°C, while the inclusion of each LNA nucleotide increases the T by 2-8°C. The modified random primer mixture may further comprise a nucleotide analog containing the nucleobase, 2-thio-deoxythymidine (2-thio-dT), where the incorporation of the 2-amino-dA and 2-thio-dT-containing nucleotide analogs prevents primer-dimer formation. Furthermore, inclusion of nucleotide analogs, including 2-amino-dA and 2-thio-dT, improves the ability of the primer to hybridize to the target nucleic acid, as 2-amino-dA forms three hydrogen bonds with unmodified deoxythymidine (dT) and 2-thio-dT forms a normal, stable pair with its unmodified partner, i.e., deoxyadenosine (dA).The use of modified nucleotide analog bases and LNA nucleotides in the random primer mixture allows for the use of a more stringent hybridization buffer, thereby significantly reducing the formation of unwanted nucleic acid duplexes and reducing the occurrence of unwanted non-target nucleic acid amplification. Furthermore, when the primers are modified random primers, a high salt concentration may also be used in the nucleic acid amplification reaction. The random primer mixture is generally used in excess compared to the target linear chromosomal DNA. The random primer mixture may be pretreated with a nuclease such as DNase to remove contaminating nucleic acids. In some embodiments, the linear chromosomal DNA is treated with a DNA repair enzyme before the ligation and amplification reaction. In some embodiments, the linear chromosomal DNA is treated with a DNA repair enzyme before the amplification reaction. In some embodiments, the DNA repair enzyme treatment is performed after the ligation reaction but before the amplification reaction. The treatment may be performed by incubating the ligation mixture with uracil DNA glycosylase, formamidopyrimidine DNA glycosylase, or a mixture thereof. Increased incubation times at elevated temperatures, such as those used for ligation with TS2126 RNA ligase, increase the risk of spontaneous DNA base changes (e.g., DNA base transitions resulting in CT and GA mutations). In particular, single-stranded DNA exhibits a 140-fold faster spontaneous deamination rate than double-stranded DNA. For example, circle sequencing mediated by TS2126 RNA ligase demonstrated that treatment with DNA-modifying enzymes such as uracil DNA glycosylase (UDG) and / or formamidopyrimidine DNA glycosylase (Fpg) effectively suppressed CT and GA mutations. In some embodiments, the individual steps of this method are performed sequentially without intermediate purification or isolation steps. These steps are typically performed in HEPES buffer in the absence of adenosine triphosphate or deoxyadenosine triphosphate.In one embodiment, the amplification reaction is carried out in a buffer solution of about 38 mM HEPES (pH 8.0), about 18 mM MgCl2, about 1 mM TCEP, about 2.5 mM KOAc, about 2.5% PEG-8000, about 0.007% Tween-20, and about 40 uM of at least one nuclease. The PNK reaction is carried out in a buffer containing a random primer mixture containing oligonucleotide sequences with nucleotide analogs. In some embodiments, all steps of this method are carried out simultaneously without intermediate purification or isolation steps. During ligation-assisted whole genome amplification in a single reaction vessel, excess ligation reagents, excess DNA, excess salt, and / or other impurities from the ligation reaction (e.g., unwanted ligation products) may be present in the reaction vessel after the ligation reaction, and the amplification reaction is carried out in the same reaction vessel without removing any of these reagents, salts, DNA, and / or other impurities. In a further embodiment, the linear chromosomal DNA may be fragmented and treated with polynucleotide kinase to generate ligatable DNA before the ligation step. Because the PNK reaction is carried out in the presence of a phosphate donor other than adenosine triphosphate or deoxyadenosine triphosphate, all steps, including the PNK reaction, intramolecular ligation, and RCA amplification, can be carried out in a single reaction vessel without any intervening isolation or purification steps.

[0034] In some embodiments, the random primer mix contains an oligonucleotide sequence containing at least one modified base. In some embodiments, the modified base is either 2-amino-deoxyadenosine (2-amino-dA) or 2-thio-deoxythymidine (2-thio-dT). In some other embodiments, the random primer mix contains an oligonucleotide sequence containing at least one 2-thio-deoxythymidine and at least one 2-thio-deoxythymidine. In one exemplary embodiment, the random primer mix used for whole genome amplification contains oligonucleotides that form selective binding complementary oligonucleotides (SBC oligonucleotides). SBC oligonucleotides are complementary pairs of oligonucleotides containing one or more modified base pairs (i.e., each member oligonucleotide forming a complementary pair is modified with a modified base). Each modified base does not form a stable base pair with its modified partner, but forms a particularly stable base pair with its natural (unmodified) counterpart. Thus, while two complementary SBC oligonucleotides do not form stable duplexes with each other, individual SBC oligonucleotides form highly stable duplexes with unmodified sequences, such as complementary targets. This property allows SBC duplexes to effectively bind to both the sense and antisense strands of DNA or RNA duplex targets.

[0035] In one specific embodiment, the random primer mixture used in whole genome amplification essentially consists of SBC oligonucleotides. For example, to generate a primer mixture essentially consisting of selectively binding complementary pairs, one or more deoxyadenosines of the oligonucleotide sequences in the random primer mixture may be replaced with 2-amino-deoxyadenosine, and one or more deoxythymidines of the oligonucleotide sequences in the random primer mixture may be replaced with 2-thio-deoxythymidine. The incorporation of 2-amino-dA improves the ability of the oligonucleotide to hybridize to its target. A 2-amino-dA nucleotide base forms three hydrogen bonds (H-bonds) with thymine (T), compared to only two H-bonds between unmodified A and T. A 2-amino A:T base pair thus has the same number of H-bonds as a G:C base pair. As a result, when a 2-amino-dA oligonucleotide binds to its unmodified target, the melting temperature (Tm) of the duplex increases by approximately 3°C per added 2-amino-dA residue compared to the unmodified case. Moreover, 2-amino-dA also destabilizes AG wobble mismatches, likely due to steric clashes between the 2-amino on A and the 2-amino on G. Thus, 2-amino-dA modified oligonucleotides exhibit better target specificity than their unmodified counterparts. A superior pair of SBC oligonucleotides can be created by substituting 2-amino-dA for A and 2-thio-dT for T (referred to herein as AT random primers). 2-amino-dA shares only one water bond with 2-thio-dT. Because these modified base pairs form a nucleotide bond, these modified base pairs are very weak and the corresponding duplexes are unstable. However, both 2-amino-dA and 2-thio-dT effectively bind to T and A bases, respectively. Generally, a 20-mer SBC annealed to a 20-mer DNA target exhibits a Tm value 10°C higher than the corresponding DNA-DNA hybrid, while an SBC-SBC hybrid exhibits a Tm value 3°C lower. In addition to 2-amino-dA and 2-thio-dT, the oligonucleotides in the AT random primer mixture may also contain phosphorothioate-modified nucleotides or LNA nucleotides, which can further improve the melting temperature (Tm) of the primer-target duplex, prevent the formation of primer-dimer structures, and / or make the random primer mixture exonuclease-resistant.

[0036] Ligation and subsequent single-tube amplification of linear chromosomal DNA by RCA in a single reactor without intervening isolation and purification steps was inefficient when using standard nuclease-resistant random hexamers (Figure 14). The presence of excess salt, ligation reagents, and / or other by-products inhibited rolling circle amplification of the resulting DNA circles. However, the use of an AT random primer mix containing oligonucleotide sequences containing 2-amino-dA, 2-thio-dT, phosphorothioate-modified nucleotides, and LNA nucleotides surprisingly enabled the ligation-amplification reaction to proceed in a single reactor without intervening isolation and purification steps. While this primer allows the amplification reaction to proceed well under these buffer conditions, standard nuclease-resistant random hexamers cannot. The position of the LNA nucleotide in the primer sequence is selected so that it does not occupy the 3' end of the primer sequence. In some embodiments, each oligonucleotide sequence in the random primer mix contains at least one 2-amino-dA or 2-thio-dT. In one exemplary embodiment, ligase-assisted whole genome amplification by RCA in a single reactor is performed using a random primer mixture containing hexameric oligonucleotide sequences with the general structure +N+N(at N)(at N)(at N)*N. The concentration of the random primer mixture is generally kept higher than the concentration of single-stranded DNA circles during the whole genome amplification method described above to facilitate multiplex random-primed rolling circle amplification.

[0037] A genomic DNA library can be generated using the amplified DNA products of ligation-assisted whole genome amplification. The genomic library can be generated by fragmenting the amplified DNA products. In some embodiments, the fragmented products include a single monomer sequence of the concatemeric amplified DNA product. In some other embodiments, the fragmented products include one or more monomer sequences of the concatemeric amplified DNA product. The amplified DNA products can be further sequenced. Sequencing can be performed using any DNA sequencing technique established in the art, including NextGen sequencing. Because the amplified DNA products are tandem repeats of DNA circles, sequencing of the amplified DNA products can be used to eliminate sequencing errors associated with NextGen sequencing. A major limitation of high-throughput DNA sequencing is the high rate of incorrect base requests. Generating a genomic DNA library by whole genome amplification with ligation-assisted RCA amplification allows for robust downstream computational correction of sequencing errors in the generated genomic DNA library. As linear chromosomal DNA templates are circularized, copied multiple times in tandem by rolling circle polymerase, and then sequenced on any high-throughput sequencing machine, each resulting read can be computationally processed to obtain a consensus sequence of all related copies of the initial sequence. Physically linking the copies ensures that each copy is independently derived from the initial sequence, allowing for efficient formation of a consensus sequence in such circle sequencing protocols. The method of whole genome amplification described herein thus allows for single-tube amplification of the entire genome followed by subsequent amplification. This allows for a simple protocol for error-free sequencing of the prepared genomic DNA library. Genomic DNA libraries can also be used for hybridization-based capture of target genomic DNA. Hybridization-based capture can be performed in solution or on a surface (e.g., microarray-based capture). Solution-based target capture is generally more scalable and economical, especially when a large number of samples are involved. Furthermore, solution-based capture of target DNA results in improved coverage uniformity. The captured target DNA can be further sequenced by targeted resequencing. The target DNA sequence can be selected to be the exome region of genomic DNA to enable exome analysis.

[0038] In some embodiments, a method is provided for amplifying limited amounts of linear, fragmented DNA by multiple displacement amplification (MDA). Conventional MDA methods, when attempted with linear, fragmented DNA, resulted in slow amplification rates and highly sequence-biased amplification. Furthermore, loss of critical sequences was often observed, particularly near the ends of the fragmented DNA. To overcome these limitations, fragmented dsDNA is first converted to ssDNA. The ssDNA is then converted to single-stranded circular DNA (i.e., DNA circles) by a template-independent intramolecular ligation reaction, thereby removing problematic DNA ends. Even ssDNA sequences shorter than 500 bp can be circularized using template-independent intramolecular ligation of ssDNA. Furthermore, when ssDNA ligation is performed in a template-independent manner, prior knowledge of the target sequence is not required to create DNA circles. Prior to circularization, the fragmented DNA can be treated with PNK to repair unligatable ends. After circularizing the fragmented ssDNA, MDA is performed on the circularized DNA. The amplification reaction can be performed under isothermal conditions using the rolling circle amplification (RCA) method. RCA can be performed using commercially available RCA amplification kits, such as the TempliPhi™ RCA Kit (GE Healthcare). TempliPhi™ rolling circle amplification uses random primers containing locked nucleic acids, which provide higher sensitivity and amplification balance. In some embodiments, nuclease-resistant primers are used in the RCA reaction. The methods disclosed herein improve amplification sensitivity, reduce sequence dropout, and enable more balanced amplification. Because template-independent circularization of single-stranded fragmented DNA can be achieved with shorter sequences, even at low concentrations, when ligase-assisted whole genome amplification is used to amplify highly fragmented DNA (e.g., circulating DNA in plasma), more balanced DNA amplification with faster speeds and improved sequence coverage can be achieved. For example, the persistence length of ssDNA can be as low as 15 nucleotides for template-independent circularization of ssDNA.When CIRCLIGASE™ is used in ligation reactions, linear or circular concatemers are rarely produced under standard conditions. Furthermore, both the circularization and amplification reactions can be performed in a single reactor without intermediate purification or isolation steps, thereby reducing the chance of contamination and simplifying the amplification workflow. Ligase-assisted whole genome amplification can be used to analyze, but is not limited to, circulating plasma cell-free DNA, fragmented DNA isolated from formalin-fixed, paraffin-embedded (FFPE) samples, forensic DNA samples damaged by exposure to environmental conditions, or very old DNA samples. The amplified library can be further used for targeted detection of the amplified sequences by qPCR or sequencing.

[0039] The various ligation-assisted whole genome amplification methods described herein, including prior ligation of ssDNA fragments into DNA circles followed by rolling circle amplification, result in preferential amplification of fragmented DNA over high-molecular-weight genomic DNA. For example, plasma preparations containing circulating DNA are often contaminated with genomic DNA released from blood cells during the purification process. Conventional methods of whole genome amplification by MDA amplify both circulating and genomic DNA. In contrast, fragmented circulating DNA molecules are initially ligated into TS2126 RNase A (TS2126 RNase B) and then ligated into TS2126 RNase B (TS2126 RNase B). When circularized DNA molecules were subsequently amplified by RCA using Phi29 DNA polymerase, the circularized DNA was preferentially amplified over high-molecular-weight genomic DNA. Such preferential amplification of fragmented DNA to genomic DNA is particularly suitable for diagnostic applications, as diagnostically relevant DNA can be preferentially amplified for downstream analysis (see Example 4). Furthermore, ligase-assisted whole genome amplification allows for stronger amplification of fragmented DNA when compared to conventional MDA-based whole genome amplification.

[0040] Figure 1 shows a schematic diagram of one embodiment of ligase-assisted whole genome amplification of fragmented dsDNA. The persistence length of double-stranded DNA is very high (approximately 150 bp), and its inherent rigidity makes circularization of fragments less than 500 bp very inefficient. Furthermore, circularization is inefficient (approximately 10.5 bp / turn) for small double-stranded fragmented DNA molecules in the range of approximately 250 bp unless the ends are properly aligned. In contrast, the circularization persistence length of single-stranded fragmented DNA is very small, approximately 15 nucleotides, compared to double-stranded fragmented DNA. As shown in Figure 1, in ligase-assisted whole genome amplification, fragmented dsDNA is first converted into single-stranded DNA circles. This can be achieved by incubating the fragmented double-stranded DNA at 95°C for a sufficient time to denature the dsDNA into single strands. The fragmented ssDNA is then treated with a DNA or RNA ligase capable of template-independent intramolecular ligation of single-stranded DNA substrates to generate single-stranded DNA circles. Non-limiting examples of ligases that can be used for intramolecular ligation include CIRCLIGASE™, T3 DNA ligase, T4 RNA ligase, Mth RNA ligase (MthRnl1), or E. coli ligase. Next, amplification reagents containing DNA polymerase, random primers, and dNTPs are added to initiate the RCA reaction of the single-stranded DNA circles. This ligase-assisted whole genome amplification using RCA generates large amounts of DNA with reduced sequence loss and amplification bias, in contrast to conventional whole genome amplification methods. Therefore, it can be used to amplify and detect even highly fragmented DNA. The entire process of generating single-stranded DNA circles and their subsequent amplification by RCA is performed in a single tube without any intervening purification steps.

[0041] In some embodiments, a single-tube workflow is provided for ligase-assisted whole genome amplification of fragmented DNA, including processing the fragmented DNA to repair unligatable DNA ends. For example, if fragmented single-stranded DNA does not contain a 5' phosphoryl group and a 3' hydroxyl group, it may not be ligated in an intramolecular ligation reaction. The presence of such unligatable DNA sequences can cause amplification bias in ligase-assisted whole genome amplification. For example, as shown schematically in Figure 8, DNA fragments generated by DNase II digestion during cell death may contain a 5' hydroxyl group and a 3' phosphoryl group. Single-stranded DNA fragments resulting from such double-stranded DNA fragments containing a 5' hydroxyl group and a 3' phosphoryl group are not circularized in an intramolecular ligation reaction. Therefore, DNase II-type nicks are poorly represented in whole genome amplification. In some embodiments, the fragmented DNA is treated with a kinase (e.g., T4 polynucleotide kinase, TPK) to phosphorylate the 5' hydroxyl group and / or dephosphorylate the 3' phosphoryl group of the fragmented DNA. Inclusion of a kinase in the reaction allows for efficient circularization of pool fragments that lack a 5' phosphate. Phosphorylation of the 5' ends of the fragmented DNA with a kinase followed by amplification of the fragmented DNA creates a more representative library.

[0042] In some embodiments, phosphorylation repair of fragmented dsDNA can be performed using T4 PNK kinase. Phosphorylation repair can be performed on either fragmented dsDNA or denatured and fragmented ssDNA. If performed, the repaired dsDNA may then be denatured to form linear ssDNA, which can then be circularized using CIRCLIGASE II™ (abbreviated as CLII). CIRCLIGASE II™ contains a substantially adenylated form of TS2126 RNA ligase. Template-independent intramolecular ligation of ssDNA by CIRCLIGASE II™ is inhibited by higher concentrations of ATP or dATP. However, kinase-mediated phosphorylation repair often requires the presence of ATP. Furthermore, it can be difficult to remove ATP from the reaction mixture without damaging the DNA. For example, phosphatase treatment of the reaction mixture to remove ATP also results in dephosphorylation of the DNA (unless the DNA is protected, for example, by pre-adenylation), thus rendering the DNA strands incapable of ligation. As a result, it is often difficult to perform phosphorylation repair of fragmented DNA and generation of ssDNA circles in a single tube without an intervening purification or isolation step. The methods provided herein use GTP, CTP, UTP, or dTTP instead of ATP during the kinase reaction. Because CIRCLIGASE II™ has a high tolerance for GTP or alternative phosphate donors (e.g., CTP or UTP), the kinase repair and ligation steps can be performed in a single reaction vessel without intervening purification and / or isolation steps. The kinase reaction mixture may further contain additional reagents such as manganese salts and betaine (the zwitterionic trimethylglycine). Upon ligation, the ssDNA circle can be amplified. By performing the ligation and amplification reactions with a relatively low concentration of GTP, the single-tube workflow described herein avoids intermittent cleanup steps between enzyme treatments and minimizes DNA template loss (see Figure 9 for a schematic diagram of the single-tube workflow involving kinase repair, ligation, and amplification).

[0043] In some embodiments, an alternative method for generating single-stranded DNA circles from linear DNA is provided. The method uses a DNA pre-adenylation step before an intramolecular ligation step. First, the linear DNA can be incubated with polynucleotide kinase in the presence of ATP to generate ligatable DNA sequences containing a phosphate group at the 5' end and a hydroxyl group at the 3' end. The ligatable DNA sequences are then incubated with an adenylating enzyme in the presence of adenosine triphosphate to generate 5'-adenylated DNA sequences. The 5'-adenylated DNA sequences have a free 3'-hydroxyl group. The ATP concentration in the ligation reaction is selected so that adenylation does not occur at the 3' end of the ligatable DNA sequences. The 5'-adenylated DNA sequences are then incubated with a non-adenylation ligase capable of template-independent intramolecular ligation of the 5'-adenylated DNA sequences to generate single-stranded DNA circles. When an ATP-dependent non-adenylation ligase is used for the intramolecular ligation reaction, ATP may need to be removed from the reaction mixture by treating the reaction mixture with phosphatase prior to the intramolecular ligation reaction. The 5' phosphate of the terminal nucleotide of the DNA (normally removed by phosphatase) is protected from phosphatase treatment due to pre-adenylation. If the DNA is in double-stranded form, it must be denatured prior to the intramolecular ligation reaction. All steps of this method are performed in a single reaction vessel without any intervening isolation or purification steps.

[0044] In some embodiments, an RNA ligase, such as RNA ligase I from the thermophilic archaeon Methanobacterium thermoautotrophicum (Mth RNA ligase 1), is used in the presence of ATP to generate an adenylated form of linear DNA. To generate single-stranded DNA circles, mutants or appropriately engineered ATP-independent ligases that are unable to self-adenylate, deadenylate, and / or transfer adenylyl groups may be used in intramolecular ligation reactions of adenylated linear DNA. For example, a motif V lysine mutant (K246A) of Mth RNA ligase can be used. This mutant cleaves the pre-adenylated substrate completely. It has potent ligation activity. Mth RNA ligase mutants with an alanine substitution for the catalytic lysine in motif I (K97A) may also be used. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates, but it has a two-fold preference for RNA as an acceptor substrate compared with ssDNA of the same sequence. When an ATP-dependent ligase such as TS2126 RNA ligase is used for intramolecular ligation of 5'-adenylated DNA sequences, the ATP in the reaction may need to be removed prior to the ligation reaction.

[0045] In some embodiments, ligase-assisted whole genome amplification using an alternative workflow is provided. A schematic diagram of this workflow is provided in Figure 11. This method involves repairing fragmented DNA with a kinase and pre-adenylating the 5' ends of the fragmented DNA with an RNA ligase or DNA ligase in the presence of ATP prior to ligation and amplification. Fragmented DNA containing sequences with non-ligatable ends (e.g., sequences containing 5' hydroxyl and / or 3' phosphoryl groups) is phosphorylated at the 5' end and dephosphorylated at the 3' end by treatment with a kinase to generate ligatable DNA sequences. The ligatable DNA sequences can then be adenylated using an RNA ligase, such as Mth RNA ligase (MthRnl 1), in the presence of ATP to generate adenylated forms of the fragmented DNA. The ATP is then removed from the reaction mixture by treating the reaction mixture with a phosphatase (e.g., shrimp alkaline phosphatase (SAP)). Any method available in the art for 5' adenylating DNA may be used (e.g., RNA ligase, DNA ligase, or synthetic methods). The pre-adenylated single-stranded linear DNA is then treated with an RNA ligase with a low degree of adenylation, such as CIRCLIGASE I™, to generate DNA circles by intramolecular ligation. The DNA circles are then amplified using RCA. In embodiments in which CIRCLIGASE I™ generates DNA circles by intramolecular ligation, the intramolecular DNA ligation and subsequent amplification reaction are performed in the absence of ATP. Because ATP inhibits the circularization of pre-adenylated ssDNA by CIRCLIGASE I™, removal of ATP from the reaction mixture after kinase treatment and the pre-adenylation reaction is essential. In some embodiments, ATP is converted to adenosine and phosphate by treatment with phosphatase. Even if adenosine is not inhibitory to the circularization reaction, the resulting phosphate may inhibit the intramolecular ligation reaction.The generated phosphate can be further removed from the solution by treating the reaction mixture with a phosphate-sequestering enzyme or a reagent that precipitates or removes phosphate (e.g., a phosphate-binding resin such as LayneRT resin). Phosphate removal can also be achieved by treating the reaction mixture with an enzyme such as maltose phosphorylase, which catalyzes the conversion of maltose to glucose and glucose-1-phosphate, thereby removing the phosphate from the solution. Including a kinase in the reaction allows for the circularization and amplification of pooled DNA fragments that do not contain 5' phosphate and / or 3' hydroxyl groups, thereby generating a more representative library through ligase-assisted amplification. Pre-adenylation of the target DNA facilitates the use of a ligase with a low degree of adenylation (e.g., CIRCLIGASE I™, which is approximately 30% adenylated) for intramolecular ligation reactions. This can be interesting because a ligase with a high degree of adenylation (e.g., CIRCLIGASE II™) will only ligate non-adenylated DNA once. Therefore, a stoichiometric amount of ligase is often required to complete an intramolecular ligation reaction. In contrast, ligases with a low degree of adenylation (such as CIRCLIGASE I™) have a high turnover rate and can act reversibly and catalytically or repeatedly on multiple pre-adenylated DNA molecules. This increases the ligation rate, reduces the amount of ligase required, and potentially allows for increased circularization of more difficult or complex DNA templates.

[0046] In some embodiments, methods for ligase-assisted whole genome amplification are used to amplify and subsequently detect circulating nucleic acids in biological samples, such as whole blood or urine (e.g., circulating DNA from the noncellular fraction of a biological sample). The circulating nucleic acids may originate from apoptotic or necrotic cells or may be actively released from cells. Circulating nucleic acids are naturally highly fragmented because cellular nucleases degrade high-molecular-weight genomic DNA into small, nucleosome-sized fragments. Highly fragmented circulating nucleic acids are often not amenable to conventional nucleic acid amplification methods. Furthermore, circulating nucleic acids are present in very small amounts in the bloodstream. Standard rolling circle amplification (RCA) of double-stranded circulating linear nucleic acids is inefficient and highly biased. Separating circulating nucleic acids into single strands and circularizing them with ligase before rolling circle amplification increases efficiency and reduces bias. To achieve high RCA speed and sensitivity with such dilute DNA templates, an RCA method using primers containing nucleotide analogs and / or LNAs in the presence of excess ligation reagents, salts, and other ligation by-products is used. This improved RCA method has been optimized for trace DNA and single-cell amplification.

[0047] In some embodiments, a method for amplifying circulating DNA from whole blood is provided. Circulating DNA is amplified from a non-cellular fraction of whole blood (e.g., plasma or serum). The method includes collecting the non-cellular fraction of whole blood, collecting circulating DNA (mainly in its natural double-stranded form) from the non-cellular fraction, denaturing the double-stranded DNA to generate linear single-stranded DNA, circularizing the circulating single-stranded DNA molecules to generate single-stranded DNA circles, and amplifying the single-stranded DNA circles by rolling circle amplification. Due to persistence length, it is usually not possible to circularize dsDNA with a sequence length of less than 150 bp, and it is very difficult to circularize dsDNA until the DNA is longer than 200 bp. In contrast, linear ssDNA molecules with a sequence length of 15 nucleotides (nt) or more can be circularized very efficiently by an appropriate ligase as long as the 5' end is phosphorylated and the 3' end is hydroxylated. Circularization of single-stranded DNA to generate single-stranded DNA circles is achieved by using a ligase capable of template-independent intramolecular ligation of single-stranded DNA. In some embodiments, circularization of single-stranded DNA molecules is performed by treating single-stranded linear DNA with an RNA ligase, such as CIRCLIGASE II™.

[0048] In some embodiments, the sensitivity of circulating DNA detection is further enhanced by phosphorylating circulating nucleic acids with polynucleotide kinase (PNK) prior to the ssDNA ligation step and RCA. By incorporating the PNK step into the workflow, the ligase-assisted whole genome amplification method presented herein was able to detect male circulating DNA in female whole blood when spiked at a 1% level (triplicate). Template-independent intramolecular ligation cannot be achieved unless the ssDNA template has a 5' phosphate group and a 3' hydroxyl group. Various conditions generate 5' hydroxyls in DNA (e.g., DNase II enzyme cleavage and phosphatase activity in blood). PNK treatment eliminates this issue and improves the diversity of rolling circle amplified CNA libraries.

[0049] In some embodiments, a kit is provided for generating single-stranded DNA circles from linear DNA. In one embodiment, the kit includes, packaged together, a polynucleotide kinase, a phosphate donor, and a pre-adenylation ligase capable of template-independent intramolecular ligation of ssDNA sequences. The polynucleotide kinase may be T4 PNK. The phosphate donor may be selected from GTP, UTP, CTP, or dTTP. In one embodiment, the kit may include TS2126 ligase, of which more than 60% may be pre-adenylated. The kit may also include buffers (e.g., HEPES), DNA amplification reagents (e.g., DNA polymerase, primers, dNTPs), and the like. ), and other reagents (e.g., MnCl2, betaine) used in generating single-stranded DNA circles by the provided methods. In some embodiments, the kits may include Phi29 DNA polymerase and random / partially constrained primers. In another embodiment, the kits include an adenylating enzyme, a phosphatase, and a non-adenylated ligase packaged together. The kits may further include a polynucleotide kinase and / or a phosphate donor. The adenylating enzyme may be RNA ligase I from Methanobacterium thermoautotrophicum (Mth RNA ligase). The non-adenylated ligase may be a composition of TS2126 ligase, in which more than 60% of the ligase is in its non-adenylated form. The kits may further include instructions for generating single-stranded DNA circles from linear DNA.

[0050] The practice of the invention will be more fully understood from the following examples, which are presented herein for illustrative purposes only and should not be construed as limiting the scope of the invention, which is defined by the appended claims. Some abbreviations used in the Examples section are spelled out as follows: "mg": milligram; "ng": nanogram; "pg": picogram; "fg": femtogram; "mL": milliliter; "mg / mL": milligram per milliliter; "mM": millimole; "mmol": millimole; "pM": picomolar; "pmol": picomole; "μL": microliter; "min.": minute and "h.": hour.

[0051] Example Example 1: Whole genome amplification of circulating nucleic acids from plasma: Circulating DNA was isolated from citrate-phosphate-dextrose (CPD)-stabilized plasma of apparently healthy individuals using a Wako DNA Extractor SP Kit (Wako Pure Chemical Industries, Ltd.). Approximately 1.3 ng was analyzed by electrophoresis on a 2% agarose gel in TBE buffer, stained with SYBR Gold, and visualized with a Typhoon imager. As shown in Figure 2, the majority of the circulating DNA was approximately 180 bp in length. Additional, less abundant sequences were approximately 370 bp in length, and there were also significantly less sequences of higher molecular weight.

[0052] 350 pg of plasma-derived circulating DNA was heated at 95°C to denature the template. The denatured single-stranded DNA template was then treated with RNA or DNA ligase to generate single-stranded DNA circles. ATP-dependent T4 DNA ligase, cell-encoded NAD-dependent E. coli DNA ligase, or thermostable RNA ligase (CIRCLIGASE II™) was used for the ligation reaction. The 100 pg DNA-ligated single-stranded DNA circles were then subjected to whole genome amplification using the GenomiPhi kit (GE Healthcare) with Phi29 DNA polymerase. Amplification was performed using a primer mixture +N + N(at N)(at N)(at N)*N, where "(at N)" represents a random mixture containing 2-amino dA, 2-thio-dT, normal G, and normal C. Real-time amplification was performed by adding a small amount of SYBR Green I to the amplification mixture and monitoring the increase in fluorescent signal over time on a Tecan plate reader (Tecan SNiPer, Amersham-Pharmacia Biotech). For comparison, equivalent concentrations of untreated genomic DNA, untreated plasma DNA, and a sample without DNA template (no template amplification) were included.

[0053] As shown in Figure 3, the amplification rate of untreated fragmented plasma DNA was much lower, indicating an amplification defect, when compared with an equivalent amount of high-molecular-weight genomic DNA. However, when fragmented plasma DNA was pretreated and converted into single-stranded DNA circles using CIRCLIGASE II™, rapid amplification rates were achieved (Figure 3A). The ATP-dependent T4 DNA ligase (Figure 3B) and the cell-encoded NAD-dependent E. coli DNA ligase (Figure 3C) were used to ligate the DNA. Ligases containing the ligase (Figure 3C) were also effective but less efficient at restoring amplification rates for fragmented plasma DNA. In these examples, the relative increase in amplification rate indicates the effectiveness of each of the ligases in promoting intramolecular ligation of single-stranded DNA templates.

[0054] Example 2: Analysis of plasma-derived circulating nucleic acids amplified by ligase-assisted whole genome amplification.

[0055] The amplified DNA generated in Example 1 was further analyzed by quantitative PCR using primers targeting four different CODIS loci (vWA, TPOX, D8S1129, and D13S317) to test the effectiveness of the ligase-assisted whole genome amplification method for promoting sensitive and balanced DNA amplification. These DNA levels were compared to those obtained with unamplified DNA to determine relative expression levels after amplification. As shown in Figure 4, in both instances, amplification of untreated plasma DNA led to loss of sequences or produced DNA that was very poorly representative of the loci tested. In contrast, inclusion of either CIRCLIGASE II™ or T4 DNA ligase in the method prevented loss of sequences at the four loci and produced DNA that was more similar in representation to the amplified high-molecular-weight genomic DNA. In an example using CIRCLIGASE II™ as the single-stranded DNA ligase, of 12 different CODIS loci tested by quantitative PCR (qPCR) using primers targeting 12 different CODIS loci, 11 were recovered after amplification, whereas only 4 were present in the amplified untreated plasma DNA ( FIG. 5 ). In FIG. 5 , the reported Ct values ​​are the average of two replicates. PCR reactions for which the Ct value was undetermined are marked with an “X.”

[0056] Example 3: Optimization of reaction conditions for ligase-assisted whole genome amplification.

[0057] The ligase-assisted DNA amplification reaction was further optimized by optimizing the efficiency of ligation of single-stranded DNA molecules with TS2126 RNA ligase. The presence of metal ions was essential for the ligation reaction, as omitting manganese from the standard manufacturer-recommended buffer reduced the amplification rate to background levels. Untreated genomic DNA and untreated plasma DNA were compared with plasma DNA samples treated with CIRCLIGASE II™ using modified buffer conditions (Figure 6). All buffer conditions contained 33 mM KOAc, 0.5 mM DTT, and 1 M betaine. Where indicated, buffers contained 33 mM TRIS-acetate (pH 7.5) or 33 mM HEPES-KOH (pH 8.0) and either 2.5 mM MgCl or 2.5 mM MnCl. Real-time amplification was performed by adding a small amount of SYBR Green I to the amplification mixture and monitoring the increase in fluorescence over time on a Tecan plate reader. The amplification threshold is the point at which fluorescence rises above background levels (2000 RFU).

[0058] A comparison of the amplification rates of ligase-assisted whole genome amplification reactions (100 pg sample) is shown in Figure 6. While both magnesium and manganese promoted similar effects in the presence of standard TRIS buffer, it was observed that the combination of manganese and magnesium in the presence of HEPES buffer, pH 8.0, was most effective in promoting high amplification rates. The increased circularization efficiency of plasma DNA in this reaction condition due to the HEPES buffer may be due to reduced oxidation of manganese cations in the HEPES buffer.

[0059] Example 4: Suppression of amplification of high molecular weight genomic DNA in ligase-assisted whole genome amplification.

[0060] The amplification rate of whole genome amplification reactions of untreated genomic DNA was compared to genomic DNA samples (100 pg sample) treated with CIRCLIGASE I™ and CIRCLIGASE II™. The results are shown in Figure 7. As shown in Figure 7, the CI of genomic DNA CIRCLIGASE™ treatment produced an inhibitory effect on the amplification rate of high molecular weight genomic DNA (different from the positive effect on plasma DNA). Inhibition was evident for both CIRCLIGASE I™ and CIRCLIGASE II™.

[0061] To investigate whether Phi29-based amplification was suppressed by ligase, untreated genomic DNA was amplified in the presence of active ligase. A small amount of SYBR Green I was added to the amplification mixture, and real-time amplification was performed by monitoring the increase in fluorescence over time on a Tecan plate reader. The amplification threshold was the point at which fluorescence rose above background levels (2000 RFU). It was observed that the suppression of genomic DNA amplification was not due to the presence of active ligase during amplification.

[0062] Because genomic DNA from blood cells often contaminates preparations of circulating nucleic acids and is of little diagnostic value, prioritizing amplification of circulating over high molecular weight genomic DNA may be advantageous for certain applications.

[0063] Example 5: Single-tube amplification of fragmented DNA using ligase-assisted whole genome amplification - Effect of phosphorylation of circular DNA fragments with kinases prior to intramolecular ligation.

[0064] Phosphorylation of circulating DNA fragments with kinases allowed for more sensitive detection of circulating DNA in plasma. Male-female plasma / blood mixing experiments were performed to establish that libraries generated from kinase-treated input DNA were more representative, allowing for more sensitive detection of the DYS14 male-specific marker (Figure 10, 3 / 3 replicates, as opposed to only 1 / 3 detected without phosphorylation). 100 μL blood / plasma mixtures were prepared as follows: 100A: 100% male plasma; 5A-C: male plasma spiked at 5% v / v into female whole blood; 1A-C: male plasma spiked at 1% v / v into female whole blood; and 0A: 100% female blood. Plasma was separated from blood cells by lateral flow through an MF1 membrane (Whatman) and subsequently collected onto a cellulose pad, which was dried and stored overnight. Circular DNA was then isolated from the cellulose pad using a modification of the standard sodium iodide / detergent-based method, the Wako Extractor SP Kit (Wako Pure Chemical Industries, Ltd.). Approximately 1.8 ng of DNA was then treated with or without T4 polynucleotide kinase in the presence of GTP, manganese, and betaine, followed by treatment with CIRCLIGASE II™ to circularize single-stranded DNA fragments. The DNA was then subjected to GenomiPhi whole genome amplification (GE Healthcare), and the products were analyzed by quantitative PCR to assess the detection of two markers: Dys14 (a multicopy gene located on the Y-chromosome that should be detectable only in the male fraction) and D16S539 (a STR locus located on chromosome 16 that should be detectable in both the male and female fractions). The reaction was performed in a single reactor without any intermediate purification or isolation steps during the workflow. This was achieved by performing the phosphorylation reaction at a relatively low concentration of GTP.

[0065] Figure 10 shows that including a kinase in the reaction allowed for circularization and amplification of DNA fragments from the pool that lacked 5' phosphates, thereby creating a more representative library. This included DNA fragments containing 5' hydroxyls, which are specifically generated by DNase II digestion during cell death. Using male-female plasma / blood mixing experiments, we demonstrated that libraries generated from kinase-treated input DNA were more representative, allowing for more sensitive detection of the DYS14 male-specific marker (3 / 3 replicates, compared to only 1 / 3 detected without phosphorylation).

[0066] Example 6: Effect of pre-adenylation of fragmented DNA prior to circularization reaction.

[0067] The circularization efficiency of phosphorylated or pre-adenylated small DNA fragments in 40 minutes was evaluated with different amounts of CIRCLIGASE™ enzyme. 2.5 pmol of a 64-mer oligonucleotide containing either a phosphate group or adenylation at the 5' position was treated with increasing amounts of CIRCLIGASE I™ or CIRCLIGASE II™ for 40 minutes at 60°C. The circularization rate was determined by scanning the intensity of the bands at the linear and circular positions. As shown in Figure 12, pre-adenylation of fragmented DNA improved the ligation and amplification rates. In Figure 12, P-64mer represents a 5'-phosphorylated 64-nt oligonucleotide; ad-64 represents a pre-adenylated 64-nt oligonucleotide. Pre-adenylated DNA circularized more rapidly than standard phosphorylated DNA. Furthermore, the less adenylated ligation enzyme catalyzed the ligation of a molar excess of substrate, indicating that the ligase has multiple opportunities to join pre-adenylated DNA molecules, which may increase the ligation rate and allow for increased circularization of more difficult templates.

[0068] Example 7: Circularization of 5'-phosphate and 5'-hydroxyl containing oligonucleotides using a pre-adenylation workflow.

[0069] Reactions containing 5 pmol of 64-mer oligonucleotide with either a phosphate or hydroxyl group at the 5' position were treated with 1.25 U of T4 polynucleotide kinase at the indicated location at 37°C. After incubation with 25 pmol of Mth RNA ligase at 65°C, the reactions were treated with 0.25 units of shrimp alkaline phosphatase. Mth RNA ligase is highly sensitive to ATP concentration; at a standard ATP concentration of 100 μM, Mth RNA ligase adenylates most DNA ends. Intramolecular ligation by Mth RNA ligase does not occur at this ATP concentration. The enzyme was heat-inactivated after each incubation. Finally, the reactions were treated with 50 units of CIRCLIGASE I™ at the indicated location and incubated at 60°C for 60 minutes. The circularization rate was determined by scanning the intensity of the bands at the linear and circular positions (Figure 13). P-64mer represents a 5'-phosphorylated 64-nt oligonucleotide; ad-64mer represents a pre-adenylated 64-nt oligonucleotide.

[0070] Figure 11 shows a "single-tube" pre-adenylation workflow in which linear oligonucleotides containing 5'-phosphate or 5'-hydroxyl groups are converted to circular forms. In this "single-tube" process, the substrate is treated sequentially with polynucleotide kinase, Mth RNA ligase, shrimp alkaline phosphatase, and CIRCLIGASE I™ without intermediate purification steps.

[0071] Example 8: Kinetics of whole genome amplification of plasma-derived fragmented nucleic acids: Plasma DNA was isolated from apparently healthy individuals using a Wako DNA Extractor SP Kit (Wako Pure Chemical Industries, Ltd.). 1 ng of purified plasma DNA was heated at 95°C to denature the template. The denatured single-stranded DNA template was then treated with RNA ligase (CIRCLIGASE II™, Epicentre) to generate single-stranded DNA circles. For the ligation reaction, the plasma DNA was incubated with a ligation reaction mixture (6 μL) containing 50 mM HEPES, pH 8.0, 66 mM KOAc, 0.5 mM DTT, 1 M betaine, and 30 U CIRCLIGASE II™ (Epicentre) at 60°C for 2 hours. The ligase was then heat-inactivated by incubating the reaction mixture at 80°C for 10 minutes. The single-stranded DNA circles were then subjected to whole genome amplification using random-primed rolling circle whole genome amplification with phi29 DNA polymerase. The single-stranded DNA circle can be ligated without any intermediate DNA purification. The amplification reaction mixture was amplified in the same reaction vessel by adjusting the reaction mixture to the following conditions: 20 mM MgCl, 1 mM TCEP, 0.01% Tween-20, 2.5% PEG-8000, 40 μM AT random hexamer primer mix, 20 ng / μL Phi29 polymerase, and 50 mM HEPES (pH 8.0) to a final volume of 20 μL. The amplification reaction mixture was incubated at 30°C for 10 hours, followed by heat inactivation of the polymerase at 65°C for 20 minutes. Real-time amplification was performed by adding a small amount of SYBR Green I to the amplification mixture and monitoring the increase in fluorescent signal over time on a Tecan plate reader (Tecan SNiPer, Amersham-Pharmacia Biotech). Amplification was performed using a random primer mixture with the sequence +N+N(at N)(at N)(at N)*N (AT random hexamer), where "at N" represents a random mixture containing 2-amino dA, 2-thio-dT, normal G, and normal C.

[0072] For comparison, equivalent concentrations of plasma DNA were amplified with standard random hexamers (NNNN*N*N) and a sample containing no DNA template (no template control) using the same protocol as above. As shown in Figure 14, amplification reactions performed with the AT random hexamer primer mix surprisingly exhibited faster rates and produced significantly higher amplification product DNA yields compared to standard random hexamers. The DNA yield for amplification using the AT random hexamers was 2.25 μg, compared to 0.84 μg using standard random hexamers. The DNA yield was zero for the "no template control" (NTC) reaction. As shown in Figure 14, rapid amplification rates were achieved when plasma DNA was ligated and converted into single-stranded DNA circles using CIRCLIGASE II™ when random primers containing modified nucleotides, such as AT random hexamers, were used (Figure 3). Single-tube ligation and amplification reactions contain carryover components from DNA ligation reactions, including betaine, potassium acetate, and manganese, which are generally known to have an inhibitory effect on amplification. However, when the reaction was performed in the presence of AT random hexamers containing modified nucleotides, this inhibitory effect on amplification was surprisingly minimal. The relative increase in amplification rate, as illustrated in Figure 14, demonstrates the effectiveness of a random primer mixture containing at least one modified nucleotide in promoting intramolecular ligation of a single-stranded DNA template followed by a rolling circle amplification reaction in the same reaction vessel without the need for an intervening isolation and purification step.

[0073] Example 9: Analysis of plasma-derived nucleic acids amplified by ligase-assisted whole genome amplification.

[0074] The amplified DNA products produced in Example 8 were purified by ethanol precipitation and subjected to sequencing reactions to determine the quality of ligase-assisted whole genome amplification using AT random hexamer primers. Sequencing was performed using the Ion Ampliseq Comprehensive Cancer Panel single-end targeted sequencing, using Ion Torrent PGM 318 chips and a read length of 200 bp. As illustrated in Figure 15, the DNA products amplified using AT random hexamers are of higher quality than DNA amplified using standard random hexamers. The percentage of recovered bases in the DNA amplified using AT hexamers is closer to that obtained from bulk unamplified plasma DNA. As shown in Table 2, the coverage depth and uniformity level of the DNA amplified using AT hexamers is closer to that obtained from bulk unamplified plasma DNA. [Table 2]

[0075] The higher overall coverage and uniformity observed across the target sequence region using AT random hexamers also resulted in higher coverage depth in regions containing clinically relevant single nucleotide polymorphisms (SNPs) measured at known ClinVar mutation sites (Figure 16, where labeling indicates average depth of coverage, 1x depth, 15x depth, etc.). This figure shows that at all cutoff levels, AT primers cover a greater proportion of ClinVar mutation regions than random primers. In contrast, one-step reactions that directly amplify plasma DNA without a circularization step provided very poor sequence coverage, with variable coverage depth in these regions and insufficient coverage at ClinVar mutation sites.

[0076] Example 10: Single-tube FFPE tissue extraction, DNA circularization, repair, and genome amplification for targeted resequencing.

[0077] Deparaffinization of FFPE tissue slides was performed by incubating them in a 65°C oven for 1 hour. The deparaffinized slides were washed twice with HISTOCHOICE™ Clean Agent (AMRESCO, Catalog No. H103) for 5 minutes. The slides were washed consecutively with 100% ethanol (twice for 5 minutes each), 75% ethanol (once for 5 minutes), and 50% ethanol (once for 5 minutes). The slides were then rinsed with nuclease-free water and air-dried. Antigens were retrieved from the slides using citrate-TRIS antigen retrieval (AR) buffer. Citrate-AR (pH 6.0) and TRIS-AR buffer (pH 8.5) were preheated at 70°C for 20 minutes. The slides were placed in a jar containing the preheated citrate-AR, and the jar was placed in a pressure cooker at 110°C for 4 minutes, followed by a 75°C pressure cooker for 20 minutes. The slides were then transferred to preheated TRIS-AR buffer and held for 20 minutes. The jar was allowed to cool at room temperature for 10 minutes, and the slides were briefly washed with water and then air-dried. The FFTE tissues were then digested with proteinase K. For the digestion, 0.6 μL of 2 mg / mL proteinase K digestion solution was used per 1 mm2 area of ​​tissue (e.g., for a 4 mm x 6 mm tissue section, approximately 15 μL of proteinase K digestion solution was used). The proteinase K digestion solution was prepared by mixing 5 μL of 20 mg / mL proteinase (Invitrogen # AM2548) with 5 μL of tissue digestion buffer (30 mM HEPES (pH 8.0), 1 mM EDTA, 0.5% SDS, and 0.01% Tween-20). First, 0.5 μL of proteinase K digestion solution was added to the slide to wet the tissue. Using an ethanol-wiped razor blade, the tissue was scraped and transferred to a 0.2 mL tube. The remainder of the 100 μg / mL Proteinase K digestion solution was added to the tube and incubated at 50° C. for 2 hours or longer until the slurry was clear. The slurry was cooled to room temperature and 2 μL of crude extract was set aside for DNA concentration measurement (Quant-iT™ DNA Assay Kit, High Sensitivity (Invitrogen# Q-33120)).

[0078] To inactivate the digestion mixture, 5 μL of crude extract (containing 40 ng of DNA) is treated with proteinase K inhibitor (0.6 μL of 5 mM proteinase K inhibitor (EMD Millipore # 539470), 3.3 μL of 9.1% α-cyclodextrin, (Sigma #C4680)). The sample is then treated with three different combinations:

[0079] REV10 Protocol—To the extract, 1.1 μL of 5 M betaine, 1.1 μL of 10× circularization buffer (350 mM HEPES (pH 8.0), 25 mM MnCl2, 660 mM KOAc, 5 mM DTT, and 0.03% Tween-20), and nuclease-free water up to 10.56 μL (total volume 11 μL) were added. The mixture was incubated at room temperature for 10 minutes. The reaction mixture was then heated to approximately 95°C for 3 minutes, followed by quick chilling on ice. To this was added 0.44 μL of CIRCLIGASE II™ (Epicentre #CL9025K) for a final reaction volume of 11 μL. The reaction mixture was incubated in a thermocycler at 60°C for 8 hours, followed by 80°C for 10 minutes to inactivate the enzyme.

[0080] REV11 Protocol - Repair reaction components were added to the extract by adding 1.1 μL of 10x Repair Buffer (0.03% Tween-20, 100 mM MgCl, 6 mM DTT), 0.77 μL Repair / Lesion Removal Mix (0.6 μL UDG (5 U / μL), 0.3 μL Fpg (8 U / μL), 0.15 μL Endo IV (10 U / μL) (New England Biolabs)), and nuclease-free water to a final volume of 11 μL. The reaction mixture was incubated in a thermocycler at 37°C for 30 minutes, followed by 85°C for 15 minutes to inactivate the enzyme. To this was added 1.5 μL of 5 M betaine, 1.5 μL of 10× circularization buffer (350 mM HEPES (pH 8.0), 25 mM MnCl2, 660 mM KOAc, 5 mM DTT, and 0.03% Tween-20) and nuclease-free water (total volume 14.4 μL). The reaction mixture was heated to approximately 95°C for 3 minutes and then quickly chilled on ice. To this was added 0.6 μL of CIRCLIGASE II™ (Epicentre #CL9025K) for a final reaction volume of 15 μL. The reaction mixture was incubated in a 60°C heat block for 8 hours, then at 80°C for 10 minutes to inactivate the enzyme.

[0081] To the REV12 protocol extract, 1.1 μL of 5 M betaine, 1.1 μL of 10× circularization buffer (350 mM HEPES (pH 8.0), 25 mM MnCl2, 660 mM KOAc, 5 mM DTT, and 0.03% Tween-20), and nuclease-free water were added (total volume 10.56 μL). The mixture was incubated at room temperature for 10 minutes. The reaction mixture was then heated to approximately 95°C for 3 minutes, followed by quick chilling on ice. To this was added 0.44 μL of CIRCLIGASE II™ (Epicentre #CL9025K) for a final reaction volume of 11 μL. The reaction mixture was incubated in a thermocycler at 60°C for 8 hours, followed by 80°C for 10 minutes to inactivate the enzyme. The entire circularization mix (11 μL) was used in a repair / damage removal reaction by adding 1.5 μL of 10x repair buffer (0.03% Tween-20, 100 mM MgCl, 6 mM DTT), 1.05 μL repair mix (0.6 μL UDG (5 U / μL), 0.3 μL Fpg (8 U / μL), 0.15 μL Endo IV (10 U / μL) (New England Biolabs)), and 1.45 μL of nuclease-free water to a final volume of 15 μL. The reaction mixture was incubated at 37 °C in a thermocycler. The enzyme was inactivated by incubation for 30 min followed by 15 min at 85°C.

[0082] For DNA amplification, a cleaning reaction master mix was assembled by mixing 20 μL of 3× Phi29 buffer (114 mM HEPES (pH 8.0), 120 μM AT primer mix 0.021% Tween-20, 54.6 mM MgCl2, 3 mM TCEP, 7.5 mM KOAc, and 7.5% PEG-8000), 0.3 μL of 1:100 CyberGreen I* (LifeTech S-7563), 1.2 μL of Phi29 polymerase (1 mg / mL, GE Healthcare), and 21.1 μL of nuclease-free water to a final volume of 42.6 μL. The cleaning reaction master mix was incubated at 30°C for 1 hour and kept at 4°C until use. The amplification reaction was initiated by adding 2.4 μL of 10 mM dNTPs solution to the cleaning reaction. The entire cleaned reaction mix was immediately added to 15 μL of the repaired mix to obtain a final reaction volume of 60 μL. This was incubated at 30°C for 8–16 hours, followed by heat inactivation of the polymerase at 65°C for 15 minutes. In the real-time setup, data were collected every 10 minutes.

[0083] Whole genome amplification products were purified according to the manufacturer's instructions (SURECLEAN PLUS™, Bioline). Briefly, 60 μL of SURECLEAN PLUS™ was added to 60 μL of WGA product and mixed thoroughly. This was incubated for 30 minutes at room temperature and then centrifuged at maximum speed in a benchtop centrifuge for 30 minutes. The supernatant was removed by aspiration. 120 μL of freshly prepared 70% ethanol was added, vortexed for 10 seconds, and centrifuged at maximum speed for 15 minutes. The supernatant was carefully removed. The washing step was repeated once, followed by air-drying to ensure complete removal of ethanol. The dried pellet was resuspended in 30 μL of 10 mM Tris-HCl (pH 8). 2 μL of purified WGA product was used for DNA concentration measurement (Quant-iT™ dsDNA Broad-Range Assay Kit, Invitrogen # Q-33130). The expected yield was approximately 3 μg.

[0084] DNA samples were analyzed using next-generation sequencing on the MiSeq platform (Illumina). The TruSeq Amplicon-Cancer Panel (TSACP) (Illumina), a highly multiplexed targeted resequencing assay for detecting somatic mutations, was used according to the manufacturer's recommendations. 250 ng of DNA from recently frozen tissue was used as a positive control, and 1,000 ng of rolling-circle amplified total genomic DNA from 40 ng of FFPE DNA, all obtained in duplicate from the REV10, REV11, and REV12 protocols, was used in the sequencing workflow. Coverage depth, sequence target uniformity, and mutation statistics were determined from these sequencing reactions. As shown in Figures 17, 18, and 19, the REV10, 11, and 12 protocols provide excellent coverage depth and coverage uniformity. However, the REV12 protocol, in which the DNA repair / DNA damage removal step is performed after the long DNA circularization step, had improved positive predictive value and improved sensitivity compared to the REV10 and REV11 protocols (Figure 19).

[0085] The claimed invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are selected from all possible embodiments or examples. As such, the foregoing embodiments should be considered in all respects illustrative rather than limiting of the invention described herein. While only certain features of the claimed invention have been illustrated and described herein, it should be understood that one of ordinary skill in the art, having the benefit of this disclosure, will be able to identify, select, optimize, or modify suitable conditions / parameters for using the method in accordance with the principles of the invention suitable for these and other types of applications. The exact use, selection, and choice of variables, such as concentrations, volumes, incubation times, incubation temperatures, and the like, of reagents may depend primarily on the particular application for which it is intended. It is, therefore, to be understood that the appended claims are intended to cover all changes and modifications that fall within the true spirit of the invention. Moreover, all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A method for nucleic acid amplification, said method comprising: (a) providing linear chromosomal DNA; (b) if the linear chromosomal DNA is in double-stranded form, denaturing the linear chromosomal DNA into single-stranded DNA; (c) incubating the linear chromosomal DNA with a ligase capable of template-independent intramolecular ligation of single-stranded DNA sequences to generate single-stranded DNA circles in a ligation mixture; (d) treating the single-stranded DNA circles to modify any damaged nucleobases by incubating the ligation mixture with uracil DNA glycosylase, formamidopyrimidine DNA glycosylase, or a combination thereof; and (e) amplifying the single-stranded DNA circle by rolling circle amplification using a random primer mixture to form an amplified DNA product. Including, the random primer mixture comprises an oligonucleotide sequence comprising at least one nucleotide analog; all of the steps of the process are carried out in a single reactor without any intervening isolation or purification steps; method.

2. 2. The method of claim 1, wherein the at least one nucleotide analog comprises 2-amino-deoxyadenosine and / or 2-thio-deoxythymidine.

3. 2. The method of claim 1, wherein the random primer mixture comprises selectively binding complementary oligonucleotides, each member of the selectively binding complementary oligonucleotides comprising at least one nucleotide comprising 2-amino-deoxyadenosine or at least one nucleotide comprising 2-thio-deoxythymidine.

4. 2. The method of claim 1, wherein the random primer mixture comprises an oligonucleotide sequence comprising phosphorothioate-modified nucleotides, LNA nucleotides, nucleotides containing 2-amino-deoxyadenosine, nucleotides containing 2-thio-deoxythymidine, or a combination thereof.

5. the random primer mixture is a hexamer containing oligonucleotide sequences with the general structure +N+N(at N)(at N)(at N)*N; N represents a random nucleotide containing a nucleobase selected from A, C, G, T, and U; (+N) represents a random locked nucleic acid (LNA)-containing nucleotide; (at N) represents a random nucleotide containing a nucleobase selected from 2-amino dA, 2-thio-dT, G, or C; *N represents phosphorothioate modified random nucleotides, The method of claim 1.

6. 2. The method of claim 1, wherein the concentration of the random primer mixture is greater than the concentration of the single-stranded DNA circles to facilitate multiplex random primed rolling circle amplification.

7. 2. The method of claim 1, wherein the linear chromosomal DNA is selected from the group consisting of cell-free circulating DNA, DNA isolated from formalin-fixed paraffin-embedded samples, forensic DNA samples exposed to environmental conditions, very old DNA samples, and combinations thereof.

8. 2. The method of claim 1, wherein the linear chromosomal DNA is fragmented DNA.

9. 2. The method of claim 1, wherein the ligase is selected from the group consisting of TS2126 RNA ligase, T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, E. coli DNA ligase, and combinations thereof.

10. 10. The method of claim 1, wherein all of said steps of said method are carried out in a HEPES buffer.

11. 2. The method of claim 1, further comprising treating the linear chromosomal DNA with polynucleotide kinase in the presence of a phosphate donor other than adenosine triphosphate or deoxyadenosine triphosphate prior to incubating the linear chromosomal DNA with the ligase to generate ligatable DNA sequences having a phosphate group at the 5' end and a hydroxyl group at the 3' end.

12. 10. The method of claim 1, further comprising sequencing the amplified DNA product.

13. 10. The method of claim 1, further comprising fragmenting the amplified DNA product to create a genomic DNA library.

14. The method of claim 1 , wherein the amplification is whole genome amplification.

15. 10. The method of claim 1, wherein the rolling circle amplification is performed using a decontaminated DNA polymerase.

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