Compositions and methods for determining nucleic acid quality

Abstract

Regarding the determination of nucleic acid quality in a sample, this invention provides compositions and methods for determining nucleic acid quality before library preparation and sequencing. [Solution] A composition and method for determining the quality of nucleic acids in a sample is provided, comprising a first primer set and a second primer set. Furthermore, a composition and method for determining the quality of nucleic acids in a sample is provided, comprising a primer set for amplifying repeating nucleic acid sequences.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Patent Application No. 63 / 002,785, filed Mar. 31, 2020. The disclosure of the prior application is considered a part of the disclosure of this application and is incorporated herein by reference.

[0002] Incorporation of Sequence Listing The data of the attached sequence listing is incorporated herein by reference. The attached sequence listing text file, file name PGDX3150 - 1 WO_SL.txt, was created on Mar. 15, 2021 and is 3,116 bytes. The file can be accessed using Microsoft Word on a computer using the Windows® operating system.

[0003] Background of the Invention Field of the Invention The present invention generally relates to determining the quality of nucleic acids in a sample, and more specifically to determining the quality of nucleic acids prior to library preparation and sequencing.

Background Art

[0004] Background Information Determining the quality of nucleic acid samples, such as DNA, prior to extensive library preparation and sequencing is very useful to avoid wasting time and cost on samples that are too degraded to be analyzed well. Current assays are based on the length or amplifiability of DNA. Neither assay has sufficient robustness and predictability.

[0005] In principle, amplifiability is closest to a functional evaluation of nucleic acids, but since it is performed on a small number of specific fragments, it does not always provide sufficient information. Therefore, there is a need for an assay that provides robust and predictive information based on a wider range of nucleic acid sizes for analysis.

Summary of the Invention

[0006] Summary of the Invention The present invention relates to compositions and methods for determining nucleic acid quality using amplification of repeating nucleic acid sequences.

[0007] In one embodiment, the present invention provides a system for determining the quality of nucleic acids in a sample, comprising: (a) a first primer set comprising a plurality of first forward primers and a plurality of first reverse primers, wherein each first forward primer and each first reverse primer comprises (i) a 3' terminal sequence complementary to a repeat sequence in the nucleic acid and having a first melting temperature, and (ii) a 5' terminal common sequence not present in the nucleic acid and having a second melting temperature, the second melting temperature being higher than the first melting temperature; and (b) a second primer set comprising a plurality of second forward primers and a plurality of second reverse primers, wherein each second forward primer and each second reverse primer comprises a 5' terminal common sequence. In one embodiment, the second melting temperature is about 5°C to 25°C higher than the first melting temperature. In one embodiment, the first melting temperature is about 45°C to 70°C. In one embodiment, the second melting temperature is approximately 60°C to 85°C. In one embodiment, the first primer set comprises an equal number of first forward primers and first reverse primers. In one embodiment, the first primer set comprises an unequal number of first forward primers and first reverse primers. In one embodiment, the second primer set comprises an equal number of second forward primers and second reverse primers. In one embodiment, the second primer set comprises an unequal number of second forward primers and second reverse primers. In one embodiment, the first primer set comprises approximately 1 to 20 first forward primers and approximately 1 to 20 first reverse primers. In one embodiment, the second primer set comprises approximately 1 to 20 second forward primers and approximately 1 to 20 second reverse primers. In one embodiment, the repeating nucleic acid sequence comprises a retrotransposon. In one embodiment, the retrotransposon is an L1 retrotransposon. In one embodiment, the common 5' terminal sequence of each first forward primer includes the sequence of SEQ ID NO: 1. In another embodiment, the common 5' terminal sequence of each first reverse primer includes the sequence of SEQ ID NO: 2.In one embodiment, the 3' terminal sequence of each first forward primer includes the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. In one embodiment, the 3' terminal sequence of each first reverse primer includes the sequence of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof.

[0008] In another embodiment, the present invention relates to a method for determining the quality of nucleic acid in a sample, comprising the steps of (a) preparing a polymerase chain reaction (PCR) mixture, (i) a first primer set comprising a plurality of first forward primers and a plurality of first reverse primers, each first forward primer and each first reverse primer comprising (i) a 3' terminal sequence complementary to a repeat sequence in the nucleic acid and having a first melting temperature, and (ii) a 5' terminal common sequence not present in the nucleic acid and having a second melting temperature, the second melting temperature being higher than the first melting temperature, and (ii) a second primer set comprising a plurality of second forward primers and a plurality of second reverse primers The present invention provides a method comprising the steps of: preparing a polymerase chain reaction (PCR) mixture comprising a lymer set, wherein each second forward primer and each second reverse primer contains a common 5' terminal sequence; (a) performing a first polymerase chain reaction (PCR) on a sample, wherein the first extension step of each cycle of the first PCR is at a temperature approximately equal to the first melting temperature; (b) performing a second polymerase chain reaction (PCR) on a sample, wherein the second extension step of each cycle in the second PCR is at a temperature approximately equal to the second melting temperature; and (c) determining the size range of the amplicon. In one embodiment, the second melting temperature is approximately 5°C to 25°C higher than the first melting temperature. In one embodiment, the first melting temperature is approximately 45°C to 70°C. In one embodiment, the first extension step is approximately 2 minutes. In one embodiment, the second extension step is approximately 1 minute. In one embodiment, the first primer set comprises an equal number of first forward primers and first reverse primers. In one embodiment, the first primer set comprises about 1 to 20 first forward primers and about 1 to 20 first reverse primers. In one embodiment, the second primer set comprises about 1 to 20 second forward primers and about 1 to 20 second reverse primers. In one embodiment, the repeating nucleic acid sequence comprises a retrotransposon. In one embodiment, the retrotransposon is an L1 retrotransposon.In one embodiment, the second PCR includes approximately 10 to 35 cycles.

[0009] In yet another embodiment, the method further includes the step of determining the intensity ratio of the amplicons. In one embodiment, the presence of a predicted amplicon size correlates with nucleic acid quality. In one embodiment, the common 5' terminal sequence of each first forward primer includes the sequence of SEQ ID NO: 1. In one embodiment, the common 5' terminal sequence of each first reverse primer includes the sequence of SEQ ID NO: 2. In one embodiment, the 3' terminal sequence of each first forward primer includes the sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. In one embodiment, the 3' terminal sequence of each first reverse primer includes the sequences of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows an overview of the primers and method for polymerase chain reaction (PCR).

[0011] [Figure 2] Figure 2 shows representative LINE sequences for PCR to determine nucleic acid quality.

[0012] [Figure 3] Figure 3 shows typical primers and primer components for determining nucleic acid quality.

[0013] [Figure 4] Figure 4 shows the predicted amplicon size distribution. [Modes for carrying out the invention]

[0014] Detailed description of the invention This invention is based on the discovery that amplification of human genome repetitive sequences using a small number of primers capable of amplifying hundreds or more different sites within the genome provides robust and accurate information about DNA quality. Such information is useful, for example, for library preparation and next-generation sequencing (NGS).

[0015] A system of oligonucleotide primers for determining nucleic acid quality is provided herein. The system of oligonucleotide primers for determining nucleic acid quality provided herein may include a first set of oligonucleotide primers. The system of oligonucleotide primers for determining nucleic acid quality provided herein may also include a second set of oligonucleotide primers.

[0016] The quality of any nucleic acid can be determined using the oligonucleotide primer system provided herein. Nucleic acids may originate from any sample or any type of sample. For example, a sample may be blood, saliva, plasma, serum, urine, or other biological fluids. Further exemplary biological fluids include serous fluid, lymph, cerebrospinal fluid, mucosal secretions, vaginal fluid, ascites fluid, pleural fluid, pericardial fluid, peritoneal fluid, and peritoneal fluid. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a cell sample or a single cell. Fresh or preserved samples may be used, including, for example, frozen preserved samples, formalin-fixed paraffin-embedded (FFPE) samples, and samples preserved by any other method.

[0017] Samples may originate from normal, healthy subjects. Samples may also originate from subjects with disease or disorder. The quality of nucleic acids in samples originating from any subject with disease or disorder can be determined using the system of primers provided herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the sample is a fluid sample originating from a subject with cancer. In some embodiments, the sample is a tissue or cell sample originating from a healthy subject or a subject with or suspected of having cancer. Cancer samples may originate from solid tumors or liquid tumors. Cancers may include kidney cancer, renal cancer, bladder cancer, prostate cancer, uterine cancer, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, pancreatic cancer, thyroid cancer, melanoma, skin cancer, head and neck cancer, brain cancer, hematopoietic cancer, leukemia, lymphoma, bone cancer, muscle cancer, sarcoma, and rhabdomyosarcoma. The disease or disorder may be an infectious disease, such as a viral infection, bacterial infection, fungal infection, or parasitic infection.

[0018] The quality of nucleic acids can be determined in a sample using the primer system of the present invention for determining nucleic acid quality, as provided herein. Before determining nucleic acid quality, nucleic acids can also be extracted, isolated, or purified from the sample. Any suitable method for extraction, isolation, or purification can be used. Exemplary methods include phenol-chloroform extraction, guanidinium-thiocyanate-phenol-chloroform extraction, gel purification, and the use of columns and beads. Commercially available kits can be used for the extraction, isolation, or purification of nucleic acids.

[0019] The quality of nucleic acids derived from any organism or species can be determined using the primer system provided herein. For example, the quality of nucleic acids derived from any animal, plant, or microorganism can be determined using the primer system provided herein. The quality of nucleic acids derived from any mammal can be determined, including nucleic acids derived from humans, rodents (including mice, rats, hamsters, and guinea pigs), livestock including cats, dogs, rabbits, cattle, horses, goats, sheep, and pigs, as well as primates (including monkeys, chimpanzees, orangutans, and gorillas). The quality of nucleic acids derived from any other animal can be determined using the primer system provided herein, including nucleic acids derived from reptiles, birds, amphibians, bony fish, cartilaginous fish, and invertebrates, for example. For example, the quality of nucleic acids derived from any angiosperms, gymnosperms, ferns and related organisms, hornworts, liverworts, mosses, and green algae can be determined. Examples of microorganisms include single-celled eukaryotes or prokaryotes, such as bacteria, archaea, protists, protists, and fungi, as well as viruses and viroids.

[0020] The quality of any type of nucleic acid can be determined using, for example, a system of primers provided herein, including DNA, RNA, and nucleic acid fragments. Examples of DNA sources include chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), and any fragments thereof. DNA can be prepared by reverse transcription of RNA by any suitable method, and the DNA quality can be determined using the system of primers provided herein. After determining the nucleic acid quality, the nucleic acids can be used, for example, to prepare a nucleic acid library. In some embodiments, the library is a genomic library. A nucleic acid library can be prepared by, for example, end repair, A-tailing, and adapter ligation, by attaching a set or subset of oligonucleotides to nucleic acid molecules, which may contain one or more barcodes for identification. Nucleic acids and nucleic acid libraries can be analyzed, for example, by next-generation sequencing (NGS). Nucleic acids can be analyzed using any suitable sequencing method. Exemplary NGS methods include the Roche 454 sequencer, Life Technologies SOLiD system, Life Technologies Ion Torrent, BGI / MGI system, Genapsys system, and Illumina systems such as the Illumina Genome Analyzer II, Illumina MiSeq, Illumina HiSeq, Illumina NextSeq, and Illumina NovaSeq instruments.

[0021] The oligonucleotide primer system provided herein may include a first primer set. The first primer set may include a plurality of first forward primers. The first primer set may also include a plurality of first reverse primers. As used herein, the terms "forward primer" and "reverse primer" can be used interchangeably with the terms "+ strand primer" and "- strand primer", respectively, unless otherwise specified in the context. Each of the plurality of first forward primers and each of the plurality of first reverse primers may include a 3' end sequence that is complementary to a repetitive sequence in a nucleic acid whose quality is determined using the primer system provided herein. Each of the plurality of first forward primers and each of the plurality of first reverse primers may also include a 5' end common sequence that is not present in the nucleic acid whose quality is determined. Thus, the primers included in the first primer set may be hybrid primers. As used herein, the term "hybrid primer" means a primer having at least two sequences that are complementary to at least two sequences within a nucleic acid molecule that may not be contiguous or adjacent to each other, or having at least two sequences having at least one sequence that is not initially present within the nucleic acid molecule. As an example, when a hybrid primer includes a 3' end sequence complementary to a nucleic acid sequence and a 5' end sequence non-complementary to the nucleic acid, a nucleic acid molecule having a sequence complementary to the 5' end sequence of the hybrid primer can be generated, for example, by polymerase chain reaction (PCR), thereby generating a nucleic acid molecule complementary to both the 3' end and the 5' end sequences of the hybrid primer.

[0022] As used herein, the terms "complementary" and "complementarity" refer to the ability of polynucleotides to form base pairs with each other. Base pairs are typically formed by hydrogen bonds between nucleotides in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick-like fashion (e.g., A-T, A-U, C-G) or in any other fashion that allows for the formation of a duplex. As will be understood by those skilled in the art, when using RNA rather than DNA, uracil rather than thymine is the base that is considered to be complementary to adenine.

[0023] Exact or perfect complementarity or 100% complementarity refers to a situation where each nucleotide of one polynucleotide strand can hydrogen bond with a nucleotide of an antiparallel polynucleotide strand. Less than exact complementarity refers to a situation where only some, rather than all, of the nucleotides of the two strands can hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. As another example, if 18 out of 20 nucleotides on each strand can hydrogen bond with each other, the polynucleotide strands exhibit 90% complementarity. "Substantial complementarity" refers to a polynucleotide strand that exhibits 75% or greater complementarity, excluding regions of the polynucleotide strand that are selected to be non-complementary, such as overhangs. Thus, complementarity does not take into account overhangs that are selected to be non-similar or non-complementary to nucleotides on the antiparallel strand, unless specifically stated otherwise in the context. In some embodiments, a 3' end sequence having complementarity to a repetitive nucleic acid sequence exhibits exact or perfect complementarity to the repetitive nucleic acid sequence. In some embodiments, a 3' end sequence having complementarity to a repetitive nucleic acid sequence exhibits substantial complementarity to the repetitive nucleic acid sequence.

[0024] As used herein, “5'-terminus common sequence” means that some or all forward primers contain the same or substantially the same sequence at the 5' end of the primer, and some or all reverse primers contain the same or substantially the same sequence at the 5' end of the primer. For example, the 5'-terminus common sequences of the first and second forward primers (described below) may contain the same sequence, and may also contain both the further nucleotides 5', 3', or 5' and 3' of the 5'-terminus common sequence. As another example, the 5'-terminus common sequences of the first and second reverse primers (described below) may contain the same sequence, and may also contain both the further nucleotides 5', 3', or 5' and 3' of the 5'-terminus common sequence. The 5'-terminus common sequences of the forward primers may be the same or different. For example, a forward primer may contain two or more different 5'-terminus common sequences shared between the forward primers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The 5' common sequences of the reverse primers may be the same or different. For example, a reverse primer may contain two or more 5' common sequences shared between reverse primers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different 5' common sequences. The 5' common sequences of the forward primer and reverse primer may be the same or different. Any sequence not present in the nucleic acid being amplified can be a 5' common sequence.

[0025] As used herein, “3' end sequence” when referring to a primer sequence means the 3' end sequence of each variable first forward primer and each first reverse primer. The 3' end sequences of the first forward primers and first reverse primers may be of any length containing about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 34, or about 35 nucleotides. Any number of different 3' end sequences may be included in the first forward primers and first reverse primers of the first primer set provided herein. For example, the first forward primer and the first reverse primer may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 different 3' terminal sequences.

[0026] Any combination of a different number of 3' sequences may be included in the first forward primer and the first reverse primer. For example, the first forward primer may have four different 3' terminal sequences. The first forward primer may include sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. In some embodiments, the first reverse primer includes three different 3' terminal sequences. In some embodiments, the first reverse primer includes sequences of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof. Thus, the first primer set of the primer system provided herein may include an unequal number of first forward primers and first reverse primers. As an example, the first forward primer may include four different 3' terminal sequences and a common 5' terminal sequence, and the first reverse primer may include three different 3' terminal sequences and a common 5' terminal sequence. The first primer set of the primer system provided herein may also include an equal number of first forward primers and first reverse primers. As an example, the first forward primer and the first reverse primer may contain the same number of different 3'-terminal sequences and common 5'-terminal sequences. In some embodiments, the first primer set contains about 1 to 20 first forward primers. In some embodiments, the first primer set contains about 1 to 20 first reverse primers. The first primer set may contain any number of first forward primers and any number of first reverse primers.

[0027] The 5' common sequence may be of any length, containing approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, or 35 nucleotides. The 5' common sequences of the first forward primer and the first reverse primer may contain the same or substantially the same sequences. The 5' common sequences of the first forward primer and the first reverse primer may contain any sequences not present in nucleic acids analyzed for quality using the primer system provided herein. In some embodiments, the 5' common sequence of the first forward primer contains the sequence of SEQ ID NO: 1. In some embodiments, the 5' common sequence of the first reverse primer contains the sequence of SEQ ID NO: 2.

[0028] The 3' terminal sequences of the first forward primer and the first reverse primer included in the primer system for determining nucleic acid quality provided herein may have a first melting temperature. The 5' terminal common sequences of the first forward primer and the first reverse primer included in the primer system for determining nucleic acid quality provided herein may have a second melting temperature. The second melting temperature may be higher than the first melting temperature. In some embodiments, the second melting temperature is about 5°C to 25°C higher than the first melting temperature. The first melting temperature may be about 45°C to 70°C. The first melting temperature may be about 45°C to 50°C, about 45°C to 55°C, about 45°C to 60°C, about 45°C to 65°C, or about 45°C to 70°C. In some embodiments, for example, the first melting temperature is approximately 70°C to 65°C, approximately 70°C to 60°C, approximately 70°C to 55°C, approximately 70°C to 50°C, and approximately 70°C to 45°C. In some embodiments, the first melting temperature is approximately 50°C to 53°C. In some embodiments, the first melting temperature is approximately 50.7°C to 52.5°C. Generally, the melting temperatures of the 3' terminal sequences of the first forward primer and the first reverse primer are 0.1°C, 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, and any number or range in between.

[0029] The second melting temperature may be around 60°C to 85°C. In some embodiments, for example, the second melting temperature is approximately 60°C to 85°C, approximately 60°C to 80°C, approximately 60°C to 75°C, approximately 60°C to 70°C, and approximately 60°C to 65°C. In some embodiments, the second melting temperature is approximately 80°C to 85°C, approximately 75°C to 85°C, approximately 70°C to 85°C, approximately 65°C to 85°C, and approximately 60°C to 85°C. In some embodiments, the second melting temperature is approximately 70°C to 75°C. In some embodiments, the second melting temperature is approximately 70°C to 73°C. In some embodiments, the second melting temperature is approximately 70°C to 72°C. In some embodiments, the second melting temperature is approximately 70°C to 71°C. In some embodiments, the second melting temperature is approximately 70°C to 70.9°C.

[0030] The first forward primer and first reverse primer of the first primer set provided herein may include a 3' terminal sequence complementary to a repetitive sequence in a nucleic acid whose quality is determined using the primer system provided herein. The repetitive nucleic acid sequence may include a retrotransposon. Retrotransposons, also called class I transposers or RNA intermediate-mediated transposons, are genetic elements that can copy and paste themselves to different genomic locations by converting RNA to DNA through reverse transcription via an RNA transposition intermediate.

[0031] Retrotransposons are classified into LTR and non-LTR retrotransposons. LTR retrotransposons are larger than 5 kilobases in size. Each end of an LTR retrotransposon contains a long chain of repeating DNA called a long-terminal repeat (LTR). Exemplary LTR retrotransposons include Ty1-copia-like (Pseudoviridae), Ty3-gypsy-like (Metaviridae), and the BEL-Pao-like group of retrotransposons. Millions of copies per haploid nucleus of Ty1-copia-like and Ty3-gypsy-like retrotransposons can be found in the genomes of animals, fungi, protists, and plants, while BEL-Pao-like factors can only be found in animals.

[0032] Non-LTR retrotransposons include long-chain scattered repeat sequences (LINEs) and short-chain scattered repeat sequences (SINEs). LINE transcripts contain an RNA polymerase II promoter that allows the LINE to be copied after insertion into a genomic site. LINE transcripts are translocation intermediates that move from the nucleus to the cytoplasm for translation by reverse transcriptase. Reverse transcriptase produces a DNA copy of the LINE RNA that can be incorporated into the new site in the genome. Each LINE is approximately 7,000 base pairs (bp) long, and the human genome contains an estimated 100,000–4,000 full-length LINE-1 factors. Many LINEs are not transcribed or translated due to the accumulation of mutations. The five main groups of LINEs are L1, RTE, R2, I, and Jockey. Human LINEs include LINE-1 / L1 as well as remnants of L2 and L3. The human genome contains approximately 850,000 LINE factors, including approximately 516,000 copies of L1 factors, approximately 315,000 copies of L2 factors, and approximately 37,000 copies of L3 factors. LINE-1 / L1 factors are widely found in mammals and remain active in the human genome. Further LINE factors include Tad, CRE, Deceiver, and Inkcap-like factors.

[0033] SINE factors include non-autonomous, non-coding transmutation factors (TEs) of approximately 100–700 bp. Three types of SINE factors are identified: CORE-SINE, V-SINE, and AmnSINE. SINE factors are transcribed by RNA polymerase III, and the transcription region contains a promoter element. SINE factors do not code for proteins and are likely to use proteins coded by LINE for reverse transcription and integration into the genome. An exemplary SINE is the Alu factor, a short, scattered repeat sequence of approximately 300 nucleotides, which can be found in humans and other species. The Alu factor is the most common SINE in humans, with over 1,000,000 copies across the entire human genome. Further exemplary SINEs include the canine SINE_Cf repeat, as well as the plant Au-SINE and Angio-SINE.

[0034] The 3' terminal sequences of the first forward primer and the first reverse primer of the first primer set may be complementary to the sequence of any retrotransposon derived from any organism. For example, the 3' terminal sequences of the first forward primer and the first reverse primer can be designed based on a nucleic acid source whose quality is determined using the primer system provided herein. Thus, the 3' terminal sequence may be complementary to a retrotransposon found in the organism from which the quality-determined nucleic acid was obtained. The nucleic acid may be of human origin. In some embodiments, the retrotransposon is an L1 retrotransposon.

[0035] The 3' terminal sequences of the first forward primer and the first reverse primer can be designed to complement the zigzag sequences along both strands of the retrotransposon. The zigzag sequences along both strands of the retrotransposon do not need to overlap. In some embodiments, each first forward primer generates an amplicon with each first reverse primer, and each first reverse primer generates an amplicon with each first forward primer. In this way, a large number of amplicons can be generated along a size range including approximately 50bp-200bp, approximately 50bp-300bp, approximately 50bp-400bp, approximately 50bp-500bp, approximately 50bp-600bp, approximately 50bp-700bp, approximately 50bp-800bp, approximately 50bp-900bp, approximately 50bp-1,000bp, approximately 50bp-1,500bp, approximately 50bp-2,000bp, approximately 50bp-2,500bp, approximately 50bp-3,000bp, approximately 50bp-3,500bp, approximately 50bp-4,000bp, approximately 50bp-4,500bp, and approximately 50bp-5,000bp. In some embodiments, the amplicons are in the range of approximately 100-2,000bp.

[0036] A primer system for determining the quality of nucleic acids in a sample may include a second primer set. The second primer set may include multiple second forward primers and multiple second reverse primers. Each second forward primer and each second reverse primer may include a common 5' terminal sequence.

[0037] The 5' common sequence contained in the first forward primer and the first reverse primer may also be contained in the second forward primer and the second reverse primer. For example, each forward primer may contain the same or substantially the same sequence at the 5' end of the primer, and each reverse primer may contain the same or substantially the same sequence at the 5' end of the primer. As another example, the 5' common sequence of the first and second forward primers may contain the same sequence, and may also contain both the further nucleotides 5', 3', or 5' and 3' of the 5' common sequence. As yet another example, the 5' common sequence of the first and second reverse primers may contain the same sequence, and may also contain both the further nucleotides 5', 3', or 5' and 3' of the 5' common sequence. The 5' common sequences of the forward primers may be the same or different. The 5' common sequences of the reverse primers may be the same or different. The 5' common sequences of the forward primers and reverse primers may be the same or different.

[0038] In some embodiments, the second forward primer and the second reverse primer contain only the 5' common sequence present in the first forward primer and the first reverse primer, and do not contain any other sequences or nucleotides. In some embodiments, the second forward primer and the second reverse primer may contain other sequences or nucleotides in addition to the 5' common sequence present in the first forward primer and the first reverse primer. For example, the second forward primer and the second reverse primer may contain nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more upstream or at 5' of the 5' common sequence. As another example, the second forward primer and the second reverse primer may also contain nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more downstream or at 3' of the 5' common sequence. Further nucleotides downstream of the 5' common sequence or at the 3' end may include the 5' nucleotides present in the 3' end sequences of the first forward primer and the first reverse primer, but not all of the nucleotides present in the 3' end sequences of the first forward primer and the first reverse primer. Thus, further nucleotides included downstream of the 5' common sequence or at the 3' end of the 5' common sequence of the second forward primer and the second reverse primer can extend to the 3' end sequences of the first forward primer and the first reverse primer. In some embodiments, the 3' end sequences of the second forward primer and the second reverse primer contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 fewer 3' nucleotides than the 3' end sequences of the first forward primer and the first reverse primer.Therefore, the 3' terminal sequences of the first forward primer and the first reverse primer may contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 more 3' nucleotides than the 3' terminal sequences contained in the second forward primer and the second reverse primer.

[0039] The second primer set may include any combination of a second forward primer and a second reverse primer, including a second forward primer containing a 5' common sequence, a second forward primer containing a 5' common sequence and any number of additional nucleotides, a second reverse primer containing a 5' common sequence, and a second reverse primer containing a 5' common sequence and any number of additional nucleotides. Thus, the second primer set may include an equal number of second forward primers and second reverse primers. The second primer set may also include an unequal number of second forward primers and second reverse primers. In some embodiments, the second primer set includes about 1 to 20 second forward primers. In some embodiments, the second primer set includes about 1 to 20 second reverse primers. The second primer set may include any number of second forward primers and any number of second reverse primers.

[0040] The second forward primer and the second reverse primer may have a higher melting temperature than the first melting temperature of the 3' terminal sequence of the first forward primer and the first reverse primer. The melting temperature of the second forward primer and the second reverse primer may be similar to, or correspond to, the second melting temperature of the 5' terminal common sequence of the first forward primer and the first reverse primer. For example, if the second forward primer and the second reverse primer do not contain any sequences or nucleotides in addition to the 5' terminal common sequence, the melting temperature of the second forward primer and the second reverse primer may correspond to the second melting temperature of the 5' terminal common sequence contained in the first forward primer and the first reverse primer. As another example, if the second forward primer and the second reverse primer contain any sequences or nucleotides in addition to the 5' terminal common sequence, the melting temperature of the second forward primer and the second reverse primer may be higher than the second melting temperature of the 5' terminal common sequence contained in the first forward primer and the first reverse primer. The melting temperatures of the second forward primer and the second reverse primer are approximately 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, 10.0°C, 10.5°C, and 11°C, respectively. It can be higher at 0.0℃, 11.5℃, 12.0℃, 12.5℃, 13.0℃, 13.5℃, 14.0℃, 14.5℃, 15.0℃, 15.5℃, 16.0℃, 16.5℃, 17.0℃, 17.5℃, 18.0℃, 18.5℃, 19.0℃, 19.5℃, 20.0℃, 20.5℃, 21.0℃, 21.5℃, 22.0℃, 22.5℃, 23.0℃, 23.5℃, 24.0℃, 24.5℃, 25.0℃, and any number or range in between.

[0041] In some embodiments, the second forward primer and second reverse primer of the primer set provided herein include a tag or label. The tag or label can be used, for example, to detect amplicons produced by PCR. Any type of tag or label, including color tags or labels, can be used. Exemplary color tags or labels include fluorophores. Any fluorophores can be used, including, for example, fluorescent lanthanide complexes including those of europium and terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red (for example, available from Invitrogen), and others described in the 11th Edition of the Molecular Probes Handbook by Richard P. Haugland, which is explicitly incorporated herein in whole by reference.Other fluorophores include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (GE Healthcare), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red(registered trademark)-5-dUTP, Cascade Blue(registered trademark)-7-dUTP, BODIPY(registered trademark)FL-14-dUTP, BODIPY(registered trademark)R-14-dUTP, BODIPY(registered trademark)TR-14-dUTP, rhodamine green(trademark)-5-dUTP, Oregon green(registered trademark)488-5-dUTP, Texas Red(registered trademark)-12-dUTP, BODIPY(registered trademark)630 / 650-14-dUTP, BODIPY(registered trademark)650 / 665-14-dUTP, and Alexa. Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-1 4-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, Texas Red®-5-UTP, Cascade Blue®-7-UTP, BODIPY® FL-14-UTP, BODIPY® TMR-14-UTP, BODIPY® TR-14-UTP, Rhodamine Green®-5-UTP, Alexa Fluor® 488-5-UTP, and Alexa Fluor® 546-1 4-UTP (Invitrogen), Alexa Fluor® 350, Alexa Fluor(registered trademark) 532, Fluor(registered trademark) 546, Alexa Fluor(registered trademark) 568, Alexa Fluor(registered trademark) 594, Fluor(registered trademark) 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY. 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lisamin Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6 Examples include G, Rhodamine Green, Rhodamine Red, as well as Cy2, Cy3.5, Cy5.5, and Cy7 (GE Healthcare).

[0042] In some embodiments, methods for determining the quality of nucleic acids in a sample are provided herein.

[0043] A method for determining nucleic acid quality in a sample provided herein may include the step of preparing a polymerase chain reaction (PCR) mixture. The PCR mixture may include a first primer set comprising a plurality of first forward primers and a plurality of first reverse primers. Each first forward primer and each first reverse primer may include a 3' terminal sequence complementary to a repeat sequence present in the nucleic acid and having a first melting temperature. Each first forward primer and each first reverse primer may also include a 5' terminal common sequence not present in the nucleic acid and having a second melting temperature. The second melting temperature may be higher than the first melting temperature. In some embodiments, the first forward primer and the first reverse primer are included in the PCR reaction mixture at low or final limiting concentrations, e.g., about 0.05 μM, about 0.04 μM, about 0.03 μM, about 0.02 μM, about 0.01 μM, about 0.009 μM, about 0.008 μM, about 0.007 μM, about 0.006 μM, about 0.005 μM, about 0.004 μM, about 0.003 μM, about 0.002 μM, about 0.001 μM, and any number or range in between. In some embodiments, the final concentrations of the first forward primer and the first reverse primer are the same or similar. As used herein, “similar primer concentrations” means primer concentrations that differ by no more than twofold.

[0044] The PCR mixture may also include a second primer set. The second primer set may include a plurality of second forward primers and a plurality of second reverse primers. Each second forward primer and each second reverse primer may include a common 5' terminal sequence. In some embodiments, the second forward primers and second reverse primers are included in the PCR reaction mixture at final concentrations corresponding to large molar excesses, such as 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, and any number or range in between. In some embodiments, the second forward primers and second reverse primers are included in the PCR reaction mixture at final concentrations of 0.1 μM to 0.5 μM. In some embodiments, the final concentrations of the second forward primer and the second reverse primer are the same or similar.

[0045] In some embodiments, the final concentrations of the second forward primer and the second reverse primer in the PCR reaction mixture are higher than the final concentrations of the first forward primer and the first reverse primer in the PCR reaction mixture. The final concentrations of the second forward primer and the second reverse primer in the PCR reaction mixture corresponding to a large molar excess may be higher than the final concentrations of the first forward primer and the first reverse primer corresponding to a low or limiting concentration. In some embodiments, the final concentrations of the second forward primer and the second reverse primer in the PCR reaction mixture are at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 50,000 times, at least 100,000 times, and any number or range in between.

[0046] Any set of primers included in the primer system provided herein may be included in the PCR mixture of the method for determining nucleic acid quality provided herein.

[0047] The PCR mixture of the method for determining nucleic acid quality in a sample provided herein may include a first primer set. The first primer set may include a plurality of first forward primers. The first primer set may also include a plurality of first reverse primers. Each of the plurality of first forward primers and first reverse primers may include a 3' terminal sequence complementary to the repetitive sequence in the nucleic acid whose quality is to be determined using the method provided herein. Each of the plurality of first forward primers and first reverse primers may also include a 5' common terminal sequence that is not present in the nucleic acid whose quality is to be determined. Thus, the primers included in the first primer set may be hybrid primers as described above. The 3' terminal sequence complementary to the repetitive nucleic acid sequence may exhibit exact or complete complementarity to the repetitive sequence in the nucleic acid. The 3' terminal sequence complementary to the repetitive nucleic acid sequence may also exhibit substantial complementarity to the repetitive sequence in the nucleic acid.

[0048] Each first forward primer and each first reverse primer may contain the same 5' common end sequence. The 3' end sequences of each first forward primer and each first reverse primer may vary. The 3' end sequences of the first forward primers and first reverse primers may be of any length, containing about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 34, or about 35 nucleotides. The first forward primers and first reverse primers of the first primer set contained in the PCR mixture of the method provided herein may contain any number of different 3' end sequences. For example, the first forward primer and the first reverse primer may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 different 3' terminal sequences.

[0049] Any combination of a different number of 3' sequences may be included in the first forward primer and first reverse primer of the PCR mixture prepared in the method provided herein. For example, the first forward primer may have four different 3' terminal sequences. In some embodiments, the first forward primer includes sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. In some embodiments, the first reverse primer includes three different 3' terminal sequences. In some embodiments, the first reverse primer includes sequences of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof. Thus, the first primer set of the PCR mixture prepared in the method provided herein may include an unequal number of first forward primers and first reverse primers. As an example, the first forward primer may include four different 3' terminal sequences and a common 5' terminal sequence, and the first reverse primer may include three different 3' terminal sequences and a common 5' terminal sequence. The first primer set of a PCR mixture prepared by the method provided herein may also include an equal number of first forward primers and first reverse primers. For example, the first forward primers and first reverse primers may include the same number of different 3'-terminal sequences and common 5'-terminal sequences. In some embodiments, the first primer set includes about 1 to 20 first forward primers. In some embodiments, the first primer set includes about 1 to 20 first reverse primers. The first primer set may include any number of first forward primers and any number of first reverse primers.

[0050] The 5' common sequence may be of any length, containing approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, or 35 nucleotides. The 5' common sequences of the first forward primer and the first reverse primer may contain the same or substantially the same sequences. The 5' common sequences of the first forward primer and the first reverse primer may contain any sequences not present in nucleic acids analyzed for quality using the methods provided herein. In some embodiments, the 5' common sequence of the first forward primer contains the sequence of SEQ ID NO: 1. In some embodiments, the 5' common sequence of the first reverse primer contains the sequence of SEQ ID NO: 2.

[0051] The 3' end sequences of the first forward primer and the first reverse primer contained in the PCR mixture of the method for determining nucleic acid quality provided herein may have a first melting temperature. The 5' end common sequences of the first forward primer and the first reverse primer contained in the PCR mixture of the method for determining nucleic acid quality may have a second melting temperature. The second melting temperature may be higher than the first melting temperature. In some embodiments, the second melting temperature is about 5°C to 25°C higher than the first melting temperature. The first melting temperature may be about 45°C to 70°C. In some embodiments, the first melting temperature is about 45°C to 50°C, about 45°C to 55°C, about 45°C to 60°C, about 45°C to 65°C, or about 45°C to 70°C. In some embodiments, for example, the first melting temperature is approximately 70°C to 65°C, approximately 70°C to 60°C, approximately 70°C to 55°C, approximately 70°C to 50°C, and approximately 70°C to 45°C. In some embodiments, the first melting temperature is approximately 50°C to 53°C. In some embodiments, the first melting temperature is approximately 50.7°C to 52.5°C. In some embodiments, the first melting temperature is approximately 50.7°C to 52.5°C. Generally, the melting temperatures of the 3' terminal sequences of the first forward primer and the first reverse primer are 0.1°C, 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, and any number or range in between.

[0052] The second melting temperature may be around 60°C to 85°C. In some embodiments, for example, the second melting temperature is approximately 60°C to 85°C, approximately 60°C to 80°C, approximately 60°C to 75°C, approximately 60°C to 70°C, and approximately 60°C to 65°C. In some embodiments, the second melting temperature is approximately 80°C to 85°C, approximately 75°C to 85°C, approximately 70°C to 85°C, approximately 65°C to 85°C, and approximately 60°C to 85°C. In some embodiments, the second melting temperature is approximately 70°C to 75°C. In some embodiments, the second melting temperature is approximately 70°C to 73°C. In some embodiments, the second melting temperature is approximately 70°C to 72°C. In some embodiments, the second melting temperature is approximately 70°C to 71°C. In some embodiments, the second melting temperature is approximately 70°C to 70.9°C.

[0053] The first forward primer and first reverse primer of the first primer set contained in the PCR mixture may include a 3' terminal sequence complementary to a repetitive sequence in the nucleic acid whose quality is determined using the method for determining nucleic acid quality in the sample provided herein. The repetitive nucleic acid sequence may include a retrotransposon. The 3' terminal sequence may be complementary to any retrotransposon, including LTR and non-LTR retrotransposons. Examples of retrotransposons include Ty1-copia-like (Pseudoviridae), Ty3-gypsy-like (Metaviridae), and any group of retrotransposons including the BEL-Pao-like group, L1, L2, L3, RTE, R2, I, and Jockey, and Tad, CRE, Deceiver, and Inkcap-like factors, as well as SINEs, such as CORE-SINE, V-SINE, and AmnSINE.

[0054] The 3' terminal sequences of the first forward primer and the first reverse primer of the first primer set of a PCR mixture prepared by the method provided herein may be complementary to the sequence of any retrotransposon derived from any organism. For example, the 3' terminal sequences of the first forward primer and the first reverse primer can be designed based on a nucleic acid source whose quality is determined using the method provided herein. Thus, the 3' terminal sequence may be complementary to a retrotransposon found in the organism from which the quality-determined nucleic acid was obtained. The nucleic acid may be of human origin. In some embodiments, the retrotransposon is an L1 retrotransposon.

[0055] The 3' terminal sequences of the first forward primer and the first reverse primer can be designed to complement the zigzag sequences along both strands of the retrotransposon. The zigzag sequences along both strands of the retrotransposon do not need to overlap. In some embodiments, each first forward primer can generate an amplicon with each first reverse primer, and each first reverse primer can generate an amplicon with each first forward primer. In this way, a large number of amplicons can be generated along a size range including approximately 50bp-200bp, approximately 50bp-300bp, approximately 50bp-400bp, approximately 50bp-500bp, approximately 50bp-600bp, approximately 50bp-700bp, approximately 50bp-800bp, approximately 50bp-900bp, approximately 50bp-1,000bp, approximately 50bp-1,500bp, approximately 50bp-2,000bp, approximately 50bp-2,500bp, approximately 50bp-3,000bp, approximately 50bp-3,500bp, approximately 50bp-4,000bp, approximately 50bp-4,500bp, and approximately 50bp-5,000bp. In some embodiments, the amplicons are in the range of approximately 100-2,000bp.

[0056] PCR mixtures prepared by the method for determining the quality of nucleic acids in a sample provided herein may include a second primer set. The second primer set may include a plurality of second forward primers and a plurality of second reverse primers. Each second forward primer and each second reverse primer may include a common 5' terminal sequence.

[0057] The 5' common sequence contained in the first forward primer and the first reverse primer may also be contained in the second forward primer and the second reverse primer. In some embodiments, the second forward primer and the second reverse primer contain only the 5' common sequence contained in the first forward primer and the first reverse primer, and do not contain any other sequences or nucleotides. In some embodiments, the second forward primer and the second reverse primer may contain other sequences or nucleotides in addition to the 5' common sequence contained in the first forward primer and the first reverse primer. For example, the second forward primer and the second reverse primer may contain nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more upstream of the 5' common sequence or at 5'. As another example, the second forward primer and the second reverse primer may also contain nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more downstream of the 5' common sequence or at 3'. Further sequences or nucleotides downstream of the 5' common sequence or at the 3' end may include the 5' nucleotides present in the 3' end sequences of the first forward primer and the first reverse primer, but not all of the nucleotides present in the 3' end sequences of the first forward primer and the first reverse primer. Thus, further nucleotides included downstream of the 5' common sequence or at the 3' end of the second forward primer and the second reverse primer can extend to the 3' end sequences included in the first forward primer and the first reverse primer. In some embodiments, the 3' end sequences included in the second forward primer and the second reverse primer contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 fewer 3' nucleotides than the 3' end sequences included in the first forward primer and the first reverse primer.Therefore, in some embodiments, the 3' terminal sequences of the first forward primer and the first reverse primer contain at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, and at least 20 more 3' nucleotides than the 3' terminal sequences contained in the second forward primer and the second reverse primer.

[0058] Any combination of a second forward primer and a second reverse primer, including a second forward primer containing a 5' common sequence, a second forward primer containing a 5' common sequence and any number of further nucleotides, a second reverse primer containing a 5' common sequence, and a second reverse primer containing a 5' common sequence and any number of further nucleotides, as well as a second reverse primer containing a 5' common sequence and any number of further nucleotides, can be included in the second primer set of the PCR mixture prepared in the nucleic acid quality determination method provided herein. Thus, the second primer set may contain an equal number of second forward primers and second reverse primers. The second primer set may also contain an unequal number of second forward primers and second reverse primers. In some embodiments, the second primer set contains about 1 to 20 second forward primers. In some embodiments, the second primer set contains about 1 to 20 second reverse primers. The second primer set may contain any number of second forward primers and any number of second reverse primers.

[0059] The second forward primer and the second reverse primer may have a higher melting temperature than the first melting temperature of the 3' terminal sequence of the first forward primer and the first reverse primer. The melting temperature of the second forward primer and the second reverse primer may be similar to, or correspond to, the second melting temperature of the 5' terminal common sequence of the first forward primer and the first reverse primer. For example, if the second forward primer and the second reverse primer do not contain any sequences other than the 5' terminal common sequence, the melting temperature of the second forward primer and the second reverse primer may correspond to the second melting temperature of the 5' terminal common sequence contained in the first forward primer and the first reverse primer. As another example, if the second forward primer and the second reverse primer contain nucleotides in addition to the 5' terminal common sequence, the melting temperature of the second forward primer and the second reverse primer may be higher than the second melting temperature of the 5' terminal common sequence contained in the first forward primer and the first reverse primer. The melting temperatures of the second forward primer and the second reverse primer are approximately 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, 10.0°C, 10.5°C, and 11°C, respectively. It can be higher at 0.0℃, 11.5℃, 12.0℃, 12.5℃, 13.0℃, 13.5℃, 14.0℃, 14.5℃, 15.0℃, 15.5℃, 16.0℃, 16.5℃, 17.0℃, 17.5℃, 18.0℃, 18.5℃, 19.0℃, 19.5℃, 20.0℃, 20.5℃, 21.0℃, 21.5℃, 22.0℃, 22.5℃, 23.0℃, 23.5℃, 24.0℃, 24.5℃, 25.0℃, and any number or range in between.

[0060] In some embodiments, the second forward primer and second reverse primer of the primer set used in the methods provided herein include a tag or label. The tag or label can be used, for example, to detect amplicons produced by PCR. Any type of tag or label, including color tags or labels, can be used. Exemplary color tags or labels include fluorophores. Any fluorophores can be used, including, for example, fluorescent lanthanide complexes including those of europium and terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red (for example, available from Invitrogen), and others described in the 11th Edition of the Molecular Probes Handbook by Richard P. Haugland, which is explicitly incorporated herein in whole by reference. Other fluorophores include, for example, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (GE Healthcare), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, and Texas Red(registered trademark)-5-dUTP, Cascade Blue(registered trademark)-7-dUTP, BODIPY(registered trademark)FL-14-dUTP, BODIPY(registered trademark)R-14-dUTP, BODIPY(registered trademark)TR-14-dUTP, Rhodamine Green(trademark)-5-dUTP, Oregon Green(registered trademark)488-5-dUTP, Texas Red(registered trademark)-12-dUTP, BODIPY(registered trademark)630 / 650-14-dUTP, BODIPY(registered trademark)650 / 665-14-dUTP, Alexa Fluor(registered trademark)488-5-dUTP, Alexa Fluor(registered trademark)532-5-dUTP, Alexa Fluor(registered trademark)568-5-dUTP, Alexa Fluor(registered trademark)594-5-dUTP, Alexa Fluor(registered trademark)546-1 4-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, Texas Red®-5-UTP, Cascade Blue®-7-UTP, BODIPY®-FL-14-UTP, BODIPY®-TMR-14-UTP, BODIPY®-TR-14-UTP, Rhodamine Green®-5-UTP, Alexa Fluor®-488-5-UTP, and Alexa Fluor®-546-1 4-UTP (Invitrogen), Alexa Fluor® 350, Alexa Fluor® 532, Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Fluor® 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Examples include Yellow, Dansyl, Lisamin Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6 G, Rhodamine Green, Rhodamine Red, as well as Cy2, Cy3.5, Cy5.5, and Cy7 (GE Healthcare).

[0061] A method for determining the quality of nucleic acids in a sample provided herein may include the step of performing a first polymerase chain reaction (PCR) on the sample. In some embodiments, the first extension step of each cycle of the first PCR is at a temperature approximately the first melting temperature of the 3' terminal sequences of the first forward primer and first reverse primer contained in the PCR reaction mixture provided herein. In some embodiments, the first extension step is at a temperature from about 5°C below the first melting temperature to 5°C above the first melting temperature. In some embodiments, the first extension step is at a temperature at least 5°C below the second melting temperature. In some embodiments, the first extension step is at a temperature from about 5°C below the first melting temperature to 5°C above the first melting temperature and at least 5°C below the second melting temperature. In some embodiments, the first extension step is at about 45°C to 70°C. In some embodiments, the first elongation step is approximately 45°C to 50°C, approximately 45°C to 55°C, approximately 45°C to 60°C, approximately 45°C to 65°C, and approximately 45°C to 70°C. In some embodiments, for example, the first elongation step is approximately 70°C to 65°C, approximately 70°C to 60°C, approximately 70°C to 55°C, approximately 70°C to 50°C, and approximately 70°C to 45°C. In some embodiments, the first elongation step is approximately 50°C to 53°C. In some embodiments, the first elongation step is approximately 50.7°C to 52.5°C. In some embodiments, the first elongation step is approximately 50°C.

[0062] The first stretching step can be any suitable length of time. For example, the first stretching step can be about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1 minute 10 seconds, about 1 minute 20 seconds, about 1 minute 30 seconds, about 1 minute 40 seconds, about 1 minute 50 seconds, about 2 minutes, about 2 minutes 10 seconds, about 2 minutes 20 seconds, about 2 minutes 30 seconds, about 2 minutes 40 seconds, about 2 minutes 50 seconds, about 3 minutes, and any number or range in between. In some embodiments, the first stretching step is about 2 minutes.

[0063] The first PCR can consist of any appropriate number of cycles. Generally, the number of cycles in the first PCR is less than 10, but more cycles are also possible. The first PCR may consist of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles. In some embodiments, the first PCR consists of approximately 3 to 5 cycles.

[0064] A method for determining the quality of nucleic acids in a sample provided herein may include the step of performing a second polymerase chain reaction (PCR) on the sample. In some embodiments, the second extension step of each cycle in the second PCR is approximately the second melting temperature of the 5' common sequence of the second forward primer and second reverse primer contained in the PCR reaction mixture provided herein. In some embodiments, the second extension step is at a temperature from about 5°C lower than the second melting temperature to 5°C higher than the second melting temperature. In some embodiments, the second extension step is at a temperature at least 5°C higher than the first melting temperature. In some embodiments, the second extension step is at a temperature from about 5°C lower than the second melting temperature to 5°C higher than the second melting temperature and at least 5°C higher than the first melting temperature. In some embodiments, the second extension step is approximately 60°C to 85°C. In some embodiments, for example, the second elongation step is approximately 60°C to 85°C, approximately 60°C to 80°C, approximately 60°C to 75°C, approximately 60°C to 70°C, and approximately 60°C to 65°C. In some embodiments, the second elongation step is approximately 80°C to 85°C, approximately 75°C to 85°C, approximately 70°C to 85°C, approximately 65°C to 85°C, and approximately 60°C to 85°C. In some embodiments, the second elongation step is approximately 70°C to 75°C. In some embodiments, the second elongation step is approximately 70°C to 73°C. In some embodiments, the second elongation step is approximately 70°C to 72°C. In some embodiments, the second elongation step is approximately 70°C to 71°C. In some embodiments, the second elongation step is approximately 70°C to 70.9°C. In some embodiments, the second elongation step is approximately 70°C.

[0065] The second extension step can be any appropriate length of time. For example, the second extension step could be about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 1 minute 10 seconds, about 1 minute 20 seconds, about 1 minute 30 seconds, about 1 minute 40 seconds, about 1 minute 50 seconds, about 2 minutes, about 2 minutes 10 seconds, about 2 minutes 20 seconds, about 2 minutes 30 seconds, about 2 minutes 40 seconds, about 2 minutes 50 seconds, about 3 minutes, and any number or range in between. In some embodiments, the second extension step is about 1 minute.

[0066] The second PCR can include any appropriate number of cycles. Generally, the number of cycles in the second PCR exceeds 10, but it can also be performed with fewer cycles. The second PCR may consist of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 cycles, or more. In some embodiments, the second PCR may consist of approximately 10 to 35 cycles. In some embodiments, the second PCR involves approximately 15–25 cycles.

[0067] A method for determining the quality of nucleic acids in a sample provided herein may include a step of determining the size range of the amplicon. As used herein, "amplicon" means nucleic acids that are the product of an amplification or replication event, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), or gene duplication. When referring to the product of an amplification reaction such as PCR, the terms "amplicon" and "PCR product" can be used interchangeably unless otherwise specified in the context.

[0068] The first forward primer and the first reverse primer can be designed to complement the zigzag and / or non-overlapping sequences along both strands of the retrotransposon at their 3' ends. Since each first forward primer can generally generate an amplicon with each first reverse primer, and each first reverse primer can generally generate an amplicon with each first forward reverse primer, the method provided herein can be used to generate amplicons of various sizes individually, including approximately 50–200 bp, approximately 50–300 bp, approximately 50–400 bp, approximately 50–500 bp, approximately 50–600 bp, approximately 50–700 bp, approximately 50–800 bp, approximately 50–900 bp, approximately 50–1,000 bp, approximately 50–1,500 bp, approximately 50–2,000 bp, approximately 50–2,500 bp, approximately 50–3,000 bp, approximately 50–3,500 bp, approximately 50–4,000 bp, approximately 50–4,500 bp, and approximately 50–5,000 bp. In some embodiments, the amplicon is in the range of approximately 100 to 2,000 bp. Any size range of amplicons produced by the methods provided herein can be used for nucleic acid quality analysis.

[0069] A method for determining nucleic acid quality provided herein may include a step of determining the amplicon intensity ratio. Amplicon size and amplicon intensity ratio can be determined using any suitable method, including Agilent 4200 TapeStation system, Agilent 2200 TapeStation system, Agilent 2100 Bioanalyzer system, Agilent DNA ScreenTape Analysis, Agilent D1000 and High Sensitivity D1000 Screen Tape Assays, Lab901 TapeStation, and Shimadzu MCE-202 MultiNA. In some embodiments, the presence of a predicted amplicon size correlates with nucleic acid quality. In some embodiments, the presence of a predicted amplicon size correlates with nucleic acid size. In some embodiments, the presence of a predicted amplicon intensity correlates with nucleic acid quality. In some embodiments, the presence of a predicted amplicon intensity correlates with nucleic acid quality.

[0070] Where used herein, the singular “a,” “a,” and “the” include multiple references unless otherwise explicitly indicated by the context. Thus, for example, a reference to “method” includes one or more methods and / or steps of the kind described herein, which would be obvious to those skilled in the art by reading this disclosure, etc.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0072] As used herein when referring to measurable values ​​such as quantity or duration, “about” means to include a variation of ±20%, ±10%, ±5%, or even ±1% from the specified value, such variation being appropriate for carrying out the disclosed composition or method.

[0073] As used herein, the term “nucleic acid” refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analog. DNA or RNA molecules may be double-stranded or single-stranded and may be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), mRNA, tRNA, rRNA, siRNA, microRNA (miRNA or miR), and hnRNA. Exemplary nucleic acid analogs include peptide nucleic acids, morpholino and locked nucleic acids, glycol nucleic acids, and threose nucleic acids. As used herein, the terms “nucleic acid molecule” and “nucleic acid” mean, for example, nucleic acid molecules and nucleic acid fragments, as well as any full-length or unfragmented nucleic acid molecules and nucleic acids.

[0074] As used herein, the term “nucleotide” includes individual units of both ribonucleic acid and deoxyribonucleic acid, as well as nucleosides and nucleotide analogs, and modified nucleotides such as labeled nucleotides. Furthermore, “nucleotide” includes analog structures that do not exist in nature, such as those in which sugars, phosphates, and / or base units are absent or replaced by other chemical structures. Thus, the term “nucleotide” encompasses individual peptide nucleic acid (PNA) (Nielsen et al., Bioconjug. Chem. 1994; 5(l): 3-7) units and locked nucleic acid (LNA) (Braasch and Corey, Chem. Biol. 2001; 8(1): 1-7) units, as well as other similar units.

[0075] As used herein, the terms “sample” and “biological sample” refer to any sample suitable for use with the compositions and methods provided herein. Samples used with the compositions and methods may be tissue samples or bodily fluids derived from the subject, or from tissue obtained by a biopsy procedure (e.g., needle biopsy) or surgical procedure. Biological samples for the methods may be bodily fluids such as cerebrospinal fluid (CSF), blood, serum, plasma, urine, saliva, tears, and ascites. Bodily fluid samples may be collected by any suitable method known to those skilled in the art.

[0076] As used herein, the term “Subject” refers to any individual or patient on which the methods disclosed herein are performed. The term “Subject” may also include any individual or patient that is a source of nucleic acids for use with the compositions and methods provided herein. The term “Subject” may be used interchangeably with the terms “Individual” or “Patient.” While the subject may be human, as will be understood to those skilled in the art, the subject may also be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), livestock such as cats, dogs, rabbits, cattle, horses, goats, sheep and pigs, primates (including monkeys, chimpanzees, orangutans and gorillas), reptiles, birds, amphibians, bony fish, cartilaginous fish and invertebrates, are included in the definition of the subject. The subject may also be a plant or a microorganism. [Examples]

[0077] Example 1 This example illustrates the design of a novel assay that provides a wider range of DNA sizes for nucleic acid quality analysis from a single amplification reaction, can be used with smaller amounts of DNA, and is less susceptible to the effects of DNA alterations at individual sites.

[0078] Current methods for determining DNA quality are insufficient to effectively assess, for example, whether a sample should be advanced for library preparation for next-generation sequencing (NGS). Having a functional assay that predicts the success of whether a sample can be successfully converted would be extremely useful. Quantitative index-of-function (QFI) assays examine a small number of DNA segments for amplification, but their usefulness is limited due to the limited number and size of fragments. Therefore, to improve the predictability of the assay, we developed an assay that amplifies more genomic fragments across a more appropriate size range.

[0079] Novel assays for determining DNA quality rely on the amplification of human genome repetitive sequences using a small number of primers capable of amplifying hundreds of different sites on the genome. These primers can amplify regions relevant to NGS, resulting in a more accurate understanding of DNA quality.

[0080] Primers were designed based on the highly repeatable 5' end sequence of the L1 retrotransposon. Zigzag sequences along both strands were selected to have a uniform melting temperature and produce fragment sizes ranging from 107 to 833 bp. Each of the seven selected primers should generate over 68 different amplicons with each of the other primers on the opposite strand. This results in fragments with many sizes in the 100–2,000 bp region. Figure 1 provides an overview of the DNA primers and assay method.

[0081] The L1 binding primers are fused to a common sequence not present in human DNA, with one sequence added to the +-strand primer and a second sequence fused to the --strand primer. The bases adjacent to the fusion site were selected to minimize overlap with the L1 sequence. The fusion primers are then added to the amplification mixture at a low concentration and used to amplify the genomic DNA at the Tm of the L1 sequence for a very small number of cycles (e.g., 3-5 cycles, the number of cycles can be optimized). Figure 2 shows a representative LINE sequence from the initial PCR, along with the expected amplicon length and the expected number of amplicons.

[0082] After a few cycles, the temperature is increased to the Tm of the 5' end fusion sequence, and then the reaction is amplified for many more cycles (e.g., 15–25 cycles, the number of cycles can be optimized) with a large molar excess of fusion primers. This generates a wide range of DNA fragments that can be separated by standard DNA sizing equipment (e.g., TapeStation, DNA sequencer, or other methods for separating DNA by size). The intensity ratio at a specific size or size range is used to correlate with DNA size and quality. The primers and primer components are shown in Figure 3. The predicted amplicon size distribution is shown in Figure 4.

[0083] Several modifications to the assay are possible. For example, a common primer can be extended to the L1 sequence by just one or a few bases and labeled with a different colored tag, which may result in better resolution of specific fragments. Different primers can also be used for possible performance improvements.

[0084] The advantages of this method stem from the use of repetitive DNA as the starting material. The presence of 100 to 1000 times more copies of DNA per genome allows for the detection of much smaller amounts of DNA and the use of very small amounts of starting material. Since many DNA fragments have slightly different sizes, a wider range of sizes can be examined compared to standard QFI assays. Furthermore, because regions from the entire genome are examined, changes in a single region of the genome have no substantial impact on the assay.

[0085] In summary, we designed a novel assay for determining nucleic acid quality based on the analysis of repeating nucleic acid sequences. This novel assay is useful for determining nucleic acid quality in applications such as library preparation and next-generation sequencing (NGS), as well as for any other applications where nucleic acid quality is critical. [ka] [Table 1]

[0086] Any references and citations made through this disclosure to other documents, such as patents, patent applications, patent publications, journals, books, articles, and web content, are incorporated herein by reference in their entirety for any purpose.

[0087] Although the present invention has been described with reference to the above examples, it will be understood that modifications and variations are enshrined within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims. In certain embodiments, for example, the following items are provided: (Item 1) A system for determining the quality of nucleic acids in a sample, (a) A first primer set comprising a plurality of first forward primers and a plurality of first reverse primers, wherein each first forward primer and each first reverse primer is (i) a 3' terminal sequence that is complementary to the repeat sequence in the nucleic acid and has a first melting temperature, (ii) A common 5' terminal sequence that is not present in the nucleic acid and has a second melting temperature, the second melting temperature of which is higher than the first melting temperature. A first primer set including, (b) A second primer set comprising a plurality of second forward primers and a plurality of second reverse primers, wherein each second forward primer and each second reverse primer comprises a common 5' terminal sequence. A system that includes this. (Item 2) The primer system described in item 1, wherein the second melting temperature is approximately 5°C to 25°C higher than the first melting temperature. (Item 3) The primer system described in item 1, wherein the first melting temperature is approximately 45°C to 70°C. (Item 4) The primer system described in item 1, wherein the second melting temperature is approximately 60°C to 85°C. (Item 5) The primer system according to item 1, wherein the first primer set comprises an equal number of first forward primers and first reverse primers. (Item 6) The primer system according to item 1, wherein the first primer set comprises an unequal number of first forward primers and first reverse primers. (Item 7) The primer system according to item 1, wherein the second primer set comprises an equal number of second forward primers and second reverse primers. (Item 8) The primer system according to item 1, wherein the second primer set comprises an unequal number of second forward primers and second reverse primers. (Item 9) The primer system according to item 1, wherein the first primer set comprises about 1 to 20 first forward primers and about 1 to 20 first reverse primers. (Item 10) The primer system according to item 1, wherein the second primer set comprises about 1 to 20 second forward primers and about 1 to 20 second reverse primers. (Item 11) A system of primers according to item 1, wherein the repeating nucleic acid sequence includes a retrotransposon. (Item 12) The primer system described in item 11, wherein the retrotransposon is an L1 retrotransposon. (Item 13) The primer system according to item 1, wherein the common 5' terminal sequence of each first forward primer contains the sequence of sequence number 1. (Item 14) The primer system according to item 1, wherein the common 5' terminal sequence of each first reverse primer contains the sequence of sequence number 2. (Item 15) The primer system according to item 1, wherein the 3' terminal sequence of each first forward primer contains the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. (Item 16) The primer system according to item 1, wherein the 3' terminal sequence of each first reverse primer contains the sequence of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof. (Item 17) A method for determining the quality of nucleic acids in a sample, comprising the step of preparing a polymerase chain reaction (PCR) mixture, (i) A first primer set comprising a plurality of first forward primers and a plurality of first reverse primers, wherein each first forward primer and each first reverse primer is (i) a 3' terminal sequence that is complementary to the repeat sequence in the nucleic acid and has a first melting temperature, (ii) A common 5' terminal sequence that is not present in the nucleic acid and has a second melting temperature, the second melting temperature of which is higher than the first melting temperature. A first primer set including, ii) A second primer set comprising a plurality of second forward primers and a plurality of second reverse primers, wherein each second forward primer and each second reverse primer contains a common 5' terminal sequence. The steps include preparing a polymerase chain reaction (PCR) mixture containing, (a) A step of performing a first polymerase chain reaction (PCR) on the sample, wherein the first extension step of each cycle of the first PCR is at a temperature approximately the first melting temperature, (b) A step of performing a second polymerase chain reaction (PCR) on the sample, wherein the second extension step of each cycle in the second PCR is at a temperature approximately the second melting temperature, (c) A step of determining the size range of the amplicon and Methods that include... (Item 18) The method according to item 17, wherein the second melting temperature is approximately 5°C to 25°C higher than the first melting temperature. (Item 19) The method according to item 17, wherein the first melting temperature is approximately 45°C to 70°C. (Item 20) The method according to item 17, wherein the second melting temperature is approximately 60°C to 85°C. (Item 21) The method according to item 17, wherein the first extension step is at a temperature between approximately 5°C lower than the first melting temperature and 5°C higher than the first melting temperature, and at least 5°C lower than the second melting temperature. (Item 22) The method according to item 17, wherein the second extension step is at a temperature between approximately 5°C lower than the second melting temperature and 5.0°C higher than the second melting temperature, and at least 5°C higher than the first melting temperature. (Item 23) The method according to item 17, wherein the first extension step is approximately 2 minutes. (Item 24) The method according to item 17, wherein the second extension step is approximately 1 minute. (Item 25) The method according to item 17, wherein the first primer set comprises an equal number of first forward primers and first reverse primers. (Item 26) The method according to item 17, wherein the first primer set comprises an unequal number of first forward primers and first reverse primers. (Item 27) The method according to item 17, wherein the second primer set comprises an equal number of second forward primers and second reverse primers. (Item 28) The method according to item 17, wherein the second primer set comprises an unequal number of second forward primers and second reverse primers. (Item 29) The method according to item 17, wherein the first primer set comprises about 1 to 20 first forward primers and about 1 to 20 first reverse primers. (Item 30) The method according to item 17, wherein the second primer set comprises about 1 to 20 second forward primers and about 1 to 20 second reverse primers. (Item 31) The method according to item 17, wherein the final concentrations of the second forward primer and the second reverse primer are higher than the final concentrations of the first forward primer and the first reverse primer. (Item 32) The method according to item 31, wherein the final concentration of the first forward primer is the same as or similar to the final concentration of the first reverse primer. (Item 33) The method according to item 31, wherein the final concentration of the second forward primer is the same as or similar to the final concentration of the second reverse primer. (Item 34) The method according to item 17, wherein the repeating nucleic acid sequence includes a retrotransposon. (Item 35) The method according to item 34, wherein the retrotransposon is an L1 retrotransposon. (Item 36) The method described in item 17, wherein the first PCR comprises approximately 3 to 5 cycles. (Item 37) The method described in item 17, wherein the second PCR comprises approximately 10 to 35 cycles. (Item 38) The method according to item 17, further comprising the step of determining the intensity ratio of the amplicon. (Item 39) The method described in item 17, which correlates the presence of predicted amplicon size with nucleic acid quality. (Item 40) The method described in item 17, which correlates the presence of a predicted amplicon size with nucleic acid size. (Item 41) The method described in item 17, which correlates the presence of a predicted amplicon intensity ratio with nucleic acid quality. (Item 42) The method described in item 17, which correlates the presence of a predicted amplicon intensity ratio with nucleic acid size. (Item 43) The method according to item 17, wherein the common 5' terminal sequence of each first forward primer contains the sequence of SEQ ID NO: 1. (Item 44) The method according to item 17, wherein the common 5' terminal sequence of each first reverse primer includes the sequence of SEQ ID NO: 2. (Item 45) The method according to item 17, wherein the 3' terminal sequence of each first forward primer includes the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof. (Item 46) The method according to item 17, wherein the 3' terminal sequence of each first reverse primer comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or any combination thereof.

Claims

[Claim 1] The invention described herein.

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