Compositions and methods for determining nucleic acid quality

Abstract

Provided herein is a composition and method for determining the quality of nucleic acid in a sample, comprising a first primer set and a second primer set.Further provided herein is a composition and method for determining the quality of nucleic acid in a sample, comprising a primer set for amplifying repetitive nucleic acid sequences.The present invention generally relates to determining the quality of nucleic acid in a sample, and more particularly relates to determining the quality of nucleic acid before library preparation and sequencing.

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 March 31, 2020. The disclosure of that prior application is considered part of the disclosure of this application and is incorporated by reference into the disclosure of this application.

[0002] Incorporating a sequence listing The data in the attached Sequence Listing is incorporated into this application by reference. The attached Sequence Listing text file, filename PGDX3150-1 WO_SL.txt, was created on March 15, 2021, and is 3,116 bytes. The file can be accessed using Microsoft Word on a computer using the Windows® OS.

[0003] Background of the Invention FIELD OF THE INVENTION The present invention relates generally to determining the quality of nucleic acids in a sample, and more particularly to determining the quality of nucleic acids prior to library preparation and sequencing. [Background technology]

[0004] Background information Determining the quality of nucleic acid samples, such as DNA, prior to extensive library preparation and sequencing is extremely useful to avoid wasting time and money on samples that are too degraded to be successfully analyzed. Current assays are based on DNA length or amplifibility. None of the assays are sufficiently robust and predictive.

[0005] In principle, amplifiability is closest to functional assessment of nucleic acids, but it is not always informative because it is performed on a small number of specific fragments. Therefore, there is a need for assays that provide robust and predictive information based on a wider range of nucleic acid sizes for analysis. Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention relates to compositions and methods for determining nucleic acid quality using amplification of repetitive 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, each of which 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 consensus sequence absent from the nucleic acid and having a second melting temperature 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, each of which comprises a 5'-terminal consensus sequence. 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 second melting temperature is about 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 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 repetitive nucleic acid sequence comprises a retrotransposon. In one embodiment, the retrotransposon is an L1 retrotransposon. In one embodiment, the 5' terminal consensus sequence of each first forward primer comprises the sequence of SEQ ID NO: 1. In one embodiment, the 5' terminal consensus sequence of each first reverse primer comprises the sequence of SEQ ID NO: 2.In one embodiment, the 3' terminal sequence of each first forward primer comprises 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 comprises 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 provides 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 of the first forward primers and each of the first reverse primers comprising: (i) a 3' terminal sequence that is complementary to a repetitive sequence in the nucleic acid and has a first melting temperature; and (ii) a 5' terminal consensus sequence that is not present in the nucleic acid and has 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. and a second primer set, each of the second forward primers and each of the second reverse primers comprising a 5'-terminal consensus sequence; (a) 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 about a first melting temperature; (b) performing a second polymerase chain reaction (PCR) on the sample, wherein the second extension step of each cycle of the second PCR is at a temperature about a second melting temperature; and (c) determining the size range of the amplicon. 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 first extension step is about 2 minutes. In one embodiment, the second extension step is about 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 repetitive nucleic acid sequence comprises a retrotransposon. In one embodiment, the retrotransposon is an L1 retrotransposon.In one embodiment, the second PCR comprises about 10 to 35 cycles.

[0009] In yet another aspect, the method further comprises determining an intensity ratio of the amplicons. In one aspect, the presence of the predicted amplicon size correlates with nucleic acid quality. In one aspect, the 5'-terminal consensus sequence of each first forward primer comprises the sequence of SEQ ID NO: 1. In one aspect, the 5'-terminal consensus sequence of each first reverse primer comprises the sequence of SEQ ID NO: 2. In one aspect, the 3'-terminal sequence of each first forward primer comprises 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 aspect, 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. [Brief explanation of the drawings]

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

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

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

[0013] [Figure 4] Figure 4 shows the predicted amplicon size distribution. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention The present invention is based on the discovery that amplification of repetitive sequences in the human genome 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, useful, for example, for library preparation and next-generation sequencing (NGS).

[0015] Provided herein is a system of oligonucleotide primers for determining nucleic acid quality. Provided herein is a system of oligonucleotide primers for determining nucleic acid quality, and can include a first oligonucleotide primer set. Provided herein is a system of oligonucleotide primers for determining nucleic acid quality, and can also include a second oligonucleotide primer set.

[0016] The quality of any nucleic acid can be determined using the oligonucleotide primer system provided herein. The nucleic acid can be derived from any sample or any type of sample. For example, the sample can be blood, saliva, plasma, serum, urine, or other biological fluids. Further exemplary biological fluids include serous fluid, lymphatic fluid, 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 single cell. For example, fresh or preserved samples can be used, including preserved frozen samples, formalin-fixed paraffin-embedded (FFPE) samples, and samples preserved by any other method.

[0017] The sample may be derived from a normal, healthy subject. The sample may also be derived from a subject with a disease or disorder. The quality of nucleic acids in a sample from a subject with any disease or disorder can be determined using the primer system provided herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the sample is a fluid sample from a subject with cancer. In some embodiments, the sample is a tissue or cell sample from a healthy subject or a subject with or suspected of having cancer. The cancer sample may be a sample from a solid tumor or a liquid tumor. The cancer may be renal cancer, kidney 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, rhabdomyosarcoma, etc. 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 for determining nucleic acid quality provided herein. Before determining nucleic acid quality, nucleic acids can 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 nucleic acid extraction, isolation, or purification.

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

[0020] The quality of any kind of nucleic acid can be determined using the primer system provided herein, including, for example, DNA, RNA, and nucleic acid fragments.DNA sources include, for example, 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 primer system provided herein can be used to determine DNA quality.After determining nucleic acid quality, the nucleic acid can be used, for example, to prepare a nucleic acid library.In some embodiments, the library is a genomic library.The nucleic acid library can be prepared by, for example, by end repair, A-tailing, and adapter ligation, by binding a set or subset of oligonucleotides, which may include one or more barcodes for identification, to nucleic acid molecules.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, the Life Technologies SOLiD system, the Life Technologies Ion Torrent, the BGI / MGI system, the 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" may be used interchangeably with the terms "+strand primer" and "-strand primer," respectively, unless the context clearly indicates otherwise. Each first forward primer and each first reverse primer of the plurality of first forward primers and the plurality of first reverse primers may include a 3'-terminal sequence complementary to a repeat sequence in the nucleic acid whose quality is to be determined using the primer system provided herein. Each first forward primer and each first reverse primer of the plurality of first forward primers and the plurality of first reverse primers may also include a 5'-terminal common sequence 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 used herein, the term "hybrid primer" refers to a primer having at least two sequences that are complementary to at least two sequences within a nucleic acid molecule that are not contiguous or adjacent to each other within the nucleic acid molecule, or that have at least one of the at least two sequences that are not initially present within the nucleic acid molecule. As an example, if a hybrid primer comprises a 3'-terminal sequence that is complementary to a nucleic acid sequence and a 5'-terminal sequence that is not complementary to the nucleic acid, a nucleic acid molecule having a sequence complementary to the 5'-terminal sequence of the hybrid primer can be generated, for example, by polymerase chain reaction (PCR), thereby generating a nucleic acid molecule that is complementary to both the 3'-terminal and 5'-terminal sequences of the hybrid primer.

[0022] As used herein, the terms "complementary" and "complementarity" refer to the ability of polynucleotides to base pair with one another. Base pairs are typically formed by hydrogen bonds between nucleotides in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., AT, AU, CG) or in any other manner that allows for the formation of a duplex. As one of skill in the art will understand, when using RNA rather than DNA, uracil, rather than thymine, is the base considered complementary to adenosine.

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

[0024] As used herein, "5'-end 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'-end common sequence (described below) of a first forward primer and a second forward primer may contain the same sequence, or may also contain additional nucleotides 5', 3', or both 5' and 3' of the 5'-end common sequence. As another example, the 5'-end common sequence (described below) of a first reverse primer and a second reverse primer may contain the same sequence, or may also contain additional nucleotides 5', 3', or both 5' and 3' of the 5'-end common sequence. The 5'-end common sequences of forward primers may be the same or different. For example, forward primers may contain two or more 5'-end common sequences shared between forward primers, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different 5'-end common sequences. The 5'-end common sequence of the reverse primer can be the same or different. For example, the reverse primer can contain two or more 5'-end common sequences shared between the reverse primers, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different 5'-end common sequences. The 5'-end common sequences of the forward primer and the reverse primer can be the same or different. Any sequence that does not exist in the nucleic acid to be amplified can be the 5'-end common sequence.

[0025] As used herein, the "3' terminal sequence" when referring to a primer sequence refers to the 3' terminal sequence of each first forward primer and each first reverse primer, which may vary. The 3' terminal sequences of the first forward primer and the first reverse primer can be any length, including 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' terminal sequences can be included in the first forward primer and the first reverse primer of the first primer set provided herein. For example, the first forward primer and the first reverse primer can comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 different 3' end sequences.

[0026] Any combination of different numbers of 3' sequences can be included in the first forward primer and the first reverse primer. For example, the first forward primer can have four different 3' end sequences. The first forward primer can include 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' end sequences. In some embodiments, the first reverse primer includes 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 can include unequal numbers of first forward primers and first reverse primers. For example, the first forward primer can include four different 3' end sequences and a 5' end consensus sequence, and the first reverse primer can include three different 3' end sequences and a 5' end consensus sequence. The first primer set of the primer system provided herein can also include equal numbers 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 5'-terminal common 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'-terminal consensus sequence can be any length, including 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 5'-terminal consensus sequences of the first forward primer and the first reverse primer can comprise the same or substantially the same sequence. The 5'-terminal consensus sequences of the first forward primer and the first reverse primer can comprise any sequence not present in the nucleic acid analyzed for quality using the primer system provided herein. In some embodiments, the 5'-terminal consensus sequence of the first forward primer comprises the sequence of SEQ ID NO: 1. In some embodiments, the 5'-terminal consensus sequence of the first reverse primer comprises 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 about 70°C to 65°C, about 70°C to 60°C, about 70°C to 55°C, about 70°C to 50°C, or about 70°C to 45°C. In some embodiments, the first melting temperature is about 50°C to 53°C. In some embodiments, the first melting temperature is about 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 within 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 therebetween from each other.

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

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

[0031] Types of retrotransposons include LTR and non-LTR retrotransposons. LTR retrotransposons are larger than 5 kilobases in size. LTR retrotransposons contain a long chain of repetitive DNA called long terminal repeat (LTR) at each end of the retrotransposon. 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 the Ty1-copia-like and Ty3-gypsy-like groups of retrotransposons can be found in the genomes of animals, fungi, protists, and plants, while BEL-Pao-like elements can only be found in animals.

[0032] Non-LTR retrotransposons include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). LINE transcripts contain an RNA polymerase II promoter, which allows them to be copied after insertion into a genomic site. LINE transcripts are translocation intermediates that are transported from the nucleus to the cytoplasm for translation by reverse transcriptase. Reverse transcriptase generates a DNA copy of the LINE RNA that can be integrated into a new site in the genome. Each LINE is approximately 7,000 base pairs (bp) long, and there are an estimated 100,000 to 4,000 full-length LINE-1 elements in the human genome. Many LINEs are not transcribed or translated due to the accumulation of mutations. Five major groups of LINEs include the L1, RTE, R2, I, and Jockey groups. Human LINEs include remnants of LINE-1 / L1 and L2 and L3. The human genome contains approximately 850,000 LINE elements, including approximately 516,000 copies of L1 elements, approximately 315,000 copies of L2 elements, and approximately 37,000 copies of L3 elements. LINE-1 / L1 elements are widely found in mammals and are still active in the human genome. Additional LINE elements include Tad, CRE, Deceiver, and Inkcap-like elements.

[0033] SINE elements include non-autonomous, non-coding transposable elements (TEs) of approximately 100 to 700 bp. Three types of SINE elements are available: CORE-SINEs, V-SINEs, and AmnSINEs. SINE elements are transcribed by RNA polymerase III, and the transcribed region contains a promoter element. SINE elements do not encode proteins; instead, they likely use proteins encoded by LINEs for reverse transcription and integration into the genome. Exemplary SINEs include Alu elements, which are short interspersed repeats of approximately 300 nucleotides and can be found in humans and other species. Alu elements are the most common SINEs in humans, with over 1,000,000 copies present throughout the human genome. Further exemplary SINEs include canine SINE_Cf repeats and plant Au-SINEs and Angio-SINEs.

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

[0035] The 3'-end sequences of the first forward primer and the first reverse primer can be designed to be complementary to the zigzag sequences along both strands of the retrotransposon.The zigzag sequences along both strands of the retrotransposon may not 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 manner, multiple amplicons can be generated along a range of sizes, including about 50 bp to 200 bp, about 50 bp to 300 bp, about 50 bp to 400 bp, about 50 bp to 500 bp, about 50 bp to 600 bp, about 50 bp to 700 bp, about 50 bp to 800 bp, about 50 bp to 900 bp, about 50 bp to 1,000 bp, about 50 bp to 1,500 bp, about 50 bp to 2,000 bp, about 50 bp to 2,500 bp, about 50 bp to 3,000 bp, about 50 bp to 3,500 bp, about 50 bp to 4,000 bp, about 50 bp to 4,500 bp, and about 50 bp to 5,000 bp. In some embodiments, the amplicons are in the range of about 100 to 2,000 bp.

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

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

[0038] In some embodiments, the second forward primer and the second reverse primer contain only the 5'-terminal 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'-terminal 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional nucleotides upstream or 5' of the 5'-terminal common sequence. As another example, the second forward primer and the second reverse primer may also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional nucleotides downstream or 3' of the 5'-terminal common sequence. The additional nucleotides downstream or 3' of the 5'-terminal consensus sequence may include 5' nucleotides present in the 3'-terminal sequences of the first forward primer and the first reverse primer, but not all of the nucleotides present in the 3'-terminal sequences of the first forward primer and the first reverse primer. Thus, the additional nucleotides downstream or 3' of the 5'-terminal consensus sequence of the second forward primer and the second reverse primer can extend into the 3'-terminal sequences of the first forward primer and the first reverse primer. In some embodiments, the 3'-terminal 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, or at least 20 fewer 3' nucleotides than the 3'-terminal sequences of the first forward primer and the first reverse primer.Thus, 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, or 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 can include any combination of second forward primers and second reverse primers, including a second forward primer containing a 5'-terminal common sequence, a second forward primer containing a 5'-terminal common sequence and any number of additional nucleotides, a second reverse primer containing a 5'-terminal common sequence, and a second reverse primer containing a 5'-terminal common sequence and any number of additional nucleotides. Thus, the second primer set can include an equal number of second forward primers and second reverse primers. The second primer set can also include unequal numbers of second forward primers and second reverse primers. In some embodiments, the second primer set includes approximately 1 to 20 second forward primers. In some embodiments, the second primer set includes approximately 1 to 20 second reverse primers. The second primer set can include any number of second forward primers and any number of second reverse primers.

[0040] The second forward primer and the second reverse primer can have a melting temperature higher than the first melting temperature of the 3'-end sequence of the first forward primer and the first reverse primer.The melting temperature of the second forward primer and the second reverse primer can be similar to or correspond to the second melting temperature of the 5'-end 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 a sequence or nucleotide in addition to the 5'-end common sequence, the melting temperature of the second forward primer and the second reverse primer can correspond to the second melting temperature of the 5'-end 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 a sequence or nucleotide in addition to the 5'-end common sequence, the melting temperature of the second forward primer and the second reverse primer can be higher than the second melting temperature of the 5'-end 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 about 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, 11.0°C, 11.5°C, 12.0°C, 12.5°C, 13.0°C, 13.5°C, 14.0°C, 14.5°C, 15.0°C, 15.5°C, 16.0°C, 16.5°C, 17.0°C, 17.5°C, 18.0°C, 18.5°C, 19.0°C, 19.5°C, 20.0°C, 20.5°C, 21.0°C, 21.5°C, 22.0°C, 22.5°C, 23.0°C, 23.5°C, 24.0°C, 24.5°C, 25.0°C, 25.5°C, 26.0°C, 26.5°C, 27.0°C, 27.5°C, 28.0°C, 28.5°C, 29.0°C, 29.5°C, 30.0°C, 30.5°C, 31.0°C, 31.5°C, 32.0°C, 3 0.0°C, 11.5°C, 12.0°C, 12.5°C, 13.0°C, 13.5°C, 14.0°C, 14.5°C, 15.0°C, 15.5°C, 16.0°C, 16.5°C, 17.0°C, 17.5°C, 18.0°C, 18.5°C, 19.0°C, 19.5°C, 20.0°C, 20.5°C, 21.0°C, 21.5°C, 22.0°C, 22.5°C, 23.0°C, 23.5°C, 24.0°C, 24.5°C, 25.0°C, and any number or range therebetween.

[0041] In some embodiments, the second forward primer and the second reverse primer of the primer set provided herein comprise a tag or label. The tag or label can be used, for example, to detect amplicons generated by PCR. Any type of tag or label can be used, including color tags or labels. Exemplary color tags or labels include fluorophores. Any fluorophore 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 (available, for example, from Invitrogen), and others described in the 11th Edition of the Molecular Probes Handbook by Richard P. Haugland, the entire contents of which are expressly incorporated herein 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®-5-dUTP, Cascade Blue®-7-dUTP, BODIPY® FL-14-dUTP, BODIPY® R-14-dUTP, BODIPY® TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green® 488-5-dUTP, Texas Red®-12-dUTP, BODIPY® 630 / 650-14-dUTP, BODIPY® 650 / 665-14-dUTP, and Alexa Fluor 488. Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-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 Yellow, Dansyl, Lissamine 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).

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

[0043] The method for determining nucleic acid quality in a sample provided herein may include preparing a polymerase chain reaction (PCR) mixture. The PCR mixture may include a first primer set including a plurality of first forward primers and a plurality of first reverse primers. Each of the first forward primers and each of the first reverse primers may include a 3'-end sequence that is complementary to a repeat sequence present in the nucleic acid and has a first melting temperature. Each of the first forward primers and each of the first reverse primers may also include a 5'-end consensus sequence that is not present in the nucleic acid and has 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 PCR reaction mixture at low concentration or final limit concentration, for example, 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 therebetween.In some embodiments, the final concentration of the first forward primer and the first reverse primer is the same or similar.As used herein, "similar primer concentration" refers to primer concentration that is not more than 2-fold different.

[0044] The PCR mixture may also 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 5'-terminal consensus sequence. In some embodiments, the second forward primer and second reverse primer are included in the PCR reaction mixture at a final concentration corresponding to a large molar excess, 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 therebetween. In some embodiments, the second forward primer and second reverse primer are included in the PCR reaction mixture at a final concentration between 0.1 μM and 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 concentration of the second forward primer and the second reverse primer in the PCR reaction mixture is higher than the final concentration of the first forward primer and the first reverse primer in the PCR reaction mixture. The final concentration of the second forward primer and the second reverse primer in the PCR reaction mixture corresponding to a large molar excess can be higher than the final concentration of the first forward primer and the first reverse primer corresponding to a low or limiting concentration. In some embodiments, the final concentration of the second forward primer and the second reverse primer in the PCR reaction mixture is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, and any number or range therebetween.

[0046] Any primer set included in the primer system provided herein can be included in the PCR mixture of the methods 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 first forward primers and each of the first reverse primers of the plurality of first forward primers and first reverse primers may include a 3'-terminal sequence complementary to a repetitive sequence in the nucleic acid whose quality is to be determined using the method provided herein. Each of the first forward primers and each of the first reverse primers of the plurality of first forward primers and first reverse primers may also include a 5'-terminal consensus sequence 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 can contain the same 5' common terminal sequence. The 3' terminal sequence of each first forward primer and each first reverse primer can vary. The 3' terminal sequence of the first forward primer and the first reverse primer can be any length, including 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 primer and the first reverse primer of the first primer set contained in the PCR mixture of the method provided herein can contain any number of different 3' terminal sequences. For example, the first forward primer and the first reverse primer can comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 different 3' end sequences.

[0049] Any combination of different numbers of 3' sequences can be included in the first forward primer and the first reverse primer of the PCR mixture prepared in the methods provided herein. For example, the first forward primer can have four different 3' end sequences. In some embodiments, the first forward primer comprises 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 comprises three different 3' end sequences. In some embodiments, the first reverse primer comprises 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 methods provided herein can comprise unequal numbers of first forward primers and first reverse primers. For example, the first forward primer can comprise four different 3' end sequences and a 5' end consensus sequence, and the first reverse primer can comprise three different 3' end sequences and a 5' end consensus sequence. The first primer set of the PCR mixture prepared by the methods provided herein can also contain an equal number of first forward primers and first reverse primers. As an example, the first forward primers and first reverse primers can contain the same number of different 3'-terminal sequences and 5'-terminal common 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 can contain any number of first forward primers and any number of first reverse primers.

[0050] The 5' terminal consensus sequence can be any length, including 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 5' terminal consensus sequences of the first forward primer and the first reverse primer can comprise the same or substantially the same sequence. The 5' terminal consensus sequences of the first forward primer and the first reverse primer can comprise any sequence not present in the nucleic acid analyzed for quality using the methods provided herein. In some embodiments, the 5' terminal consensus sequence of the first forward primer comprises the sequence of SEQ ID NO: 1. In some embodiments, the 5' terminal consensus sequence of the first reverse primer comprises the sequence of SEQ ID NO: 2.

[0051] The 3'-terminal sequences of the first forward primer and the first reverse primer included in the PCR mixture of the method for determining nucleic acid quality provided herein may have a first melting temperature. The 5'-terminal consensus sequences of the first forward primer and the first reverse primer included 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 about 70°C to 65°C, about 70°C to 60°C, about 70°C to 55°C, about 70°C to 50°C, or about 70°C to 45°C. In some embodiments, the first melting temperature is about 50°C to 53°C. In some embodiments, the first melting temperature is about 50.7°C to 52.5°C. In some embodiments, the first melting temperature is about 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 within 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 therebetween from each other.

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

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

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

[0055] The 3'-end sequences of the first forward primer and the first reverse primer can be designed to be complementary to the zigzag sequences along both strands of the retrotransposon.The zigzag sequences along both strands of the retrotransposon may not 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 manner, multiple amplicons can be generated along a range of sizes, including about 50 bp to 200 bp, about 50 bp to 300 bp, about 50 bp to 400 bp, about 50 bp to 500 bp, about 50 bp to 600 bp, about 50 bp to 700 bp, about 50 bp to 800 bp, about 50 bp to 900 bp, about 50 bp to 1,000 bp, about 50 bp to 1,500 bp, about 50 bp to 2,000 bp, about 50 bp to 2,500 bp, about 50 bp to 3,000 bp, about 50 bp to 3,500 bp, about 50 bp to 4,000 bp, about 50 bp to 4,500 bp, and about 50 bp to 5,000 bp. In some embodiments, the amplicons are in the range of about 100 to 2,000 bp.

[0056] The PCR mixture prepared by the method for determining the quality of nucleic acid in a sample provided herein can include a second primer set.The second primer set can include a plurality of second forward primers and a plurality of second reverse primers.Each second forward primer and each second reverse primer can include a 5'-end common sequence.

[0057] The 5'-terminal common sequence contained in the first forward primer and the first reverse primer can 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'-terminal 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 can contain other sequences or nucleotides in addition to the 5'-terminal common sequence contained in the first forward primer and the first reverse primer. For example, the second forward primer and the second reverse primer can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional nucleotides upstream or 5' of the 5'-terminal common sequence. As another example, the second forward primer and the second reverse primer can also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional nucleotides downstream or 3' of the 5'-terminal common sequence. The additional sequence or nucleotides downstream or 3' of the 5'-terminal consensus sequence may include 5' nucleotides present in the 3'-terminal sequences of the first forward primer and the first reverse primer, but not all of the nucleotides present in the 3'-terminal sequences of the first forward primer and the first reverse primer. Thus, the additional nucleotides present downstream or 3' of the 5'-terminal consensus sequence of the second forward primer and the second reverse primer can extend into the 3'-terminal sequences of the first forward primer and the first reverse primer. In some embodiments, the 3'-terminal 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, or at least 20 fewer 3' nucleotides than the 3'-terminal sequences of the first forward primer and the first reverse primer.Thus, 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, or 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 second forward primers and second reverse primers can be included in the second primer set of the PCR mixture prepared in the methods for determining nucleic acid quality provided herein, including a second forward primer containing a 5'-terminal consensus sequence, a second forward primer containing a 5'-terminal consensus sequence and any number of additional nucleotides, a second reverse primer containing a 5'-terminal consensus sequence and any number of additional nucleotides, and a second reverse primer containing a 5'-terminal consensus sequence and any number of additional nucleotides. Thus, the second primer set can contain an equal number of second forward primers and second reverse primers. The second primer set can also contain unequal numbers 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 can contain any number of second forward primers and any number of second reverse primers.

[0059] The second forward primer and the second reverse primer can have a melting temperature higher than the first melting temperature of the 3'-end sequence of the first forward primer and the first reverse primer.The melting temperature of the second forward primer and the second reverse primer can be similar to or correspond to the second melting temperature of the 5'-end 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 sequence other than the 5'-end common sequence, the melting temperature of the second forward primer and the second reverse primer can correspond to the second melting temperature of the 5'-end 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'-end common sequence, the melting temperature of the second forward primer and the second reverse primer can be higher than the second melting temperature of the 5'-end 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 about 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, 11.0°C, 11.5°C, 12.0°C, 12.5°C, 13.0°C, 13.5°C, 14.0°C, 14.5°C, 15.0°C, 15.5°C, 16.0°C, 16.5°C, 17.0°C, 17.5°C, 18.0°C, 18.5°C, 19.0°C, 19.5°C, 20.0°C, 20.5°C, 21.0°C, 21.5°C, 22.0°C, 22.5°C, 23.0°C, 23.5°C, 24.0°C, 24.5°C, 25.0°C, 25.5°C, 26.0°C, 26.5°C, 27.0°C, 27.5°C, 28.0°C, 28.5°C, 29.0°C, 29.5°C, 30.0°C, 30.5°C, 31.0°C, 31.5°C, 32.0°C, 3 0.0°C, 11.5°C, 12.0°C, 12.5°C, 13.0°C, 13.5°C, 14.0°C, 14.5°C, 15.0°C, 15.5°C, 16.0°C, 16.5°C, 17.0°C, 17.5°C, 18.0°C, 18.5°C, 19.0°C, 19.5°C, 20.0°C, 20.5°C, 21.0°C, 21.5°C, 22.0°C, 22.5°C, 23.0°C, 23.5°C, 24.0°C, 24.5°C, 25.0°C, and any number or range therebetween.

[0060] In some embodiments, the second forward primer and the second reverse primer of the primer set used in the methods provided herein comprise a tag or label. The tag or label can be used, for example, to detect amplicons generated by PCR. Any type of tag or label can be used, including color tags or labels. Exemplary color tags or labels include fluorophores. Any fluorophore 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 (available, for example, from Invitrogen), and others described in the 11th Edition of the Molecular Probes Handbook by Richard P. Haugland, the entire contents of which are expressly incorporated herein 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®-5-dUTP, Cascade Blue®-7-dUTP, BODIPY® FL-14-dUTP, BODIPY® R-14-dUTP, BODIPY® TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green® 488-5-dUTP, Texas Red®-12-dUTP, BODIPY® 630 / 650-14-dUTP, BODIPY® 650 / 665-14-dUTP, and Alexa Fluor 488. Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-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 Yellow, Dansyl, Lissamine 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] The methods for determining the quality of nucleic acids in a sample provided herein may include 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 performed at a temperature approximately equal to the first melting temperature of the 3'-terminal sequences of the first forward primer and the first reverse primer contained in the PCR reaction mixture provided herein. In some embodiments, the first extension step is performed at a temperature ranging from about 5°C lower than the first melting temperature to 5°C higher than the first melting temperature. In some embodiments, the first extension step is performed at a temperature at least 5°C lower than the second melting temperature. In some embodiments, the first extension step is performed at a temperature ranging from about 5°C lower than the first melting temperature to 5°C higher than the first melting temperature and at least 5°C lower than the second melting temperature. In some embodiments, the first extension step is performed at a temperature between about 45°C and 70°C. In some embodiments, the first extension step 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 extension step is about 70°C to 65°C, about 70°C to 60°C, about 70°C to 55°C, about 70°C to 50°C, or about 70°C to 45°C. In some embodiments, the first extension step is about 50°C to 53°C. In some embodiments, the first extension step is about 50.7°C to 52.5°C. In some embodiments, the first extension step is about 50°C.

[0062] The first extension step can be any suitable length of time. For example, the first extension 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 therebetween. In some embodiments, the first extension step is about 2 minutes.

[0063] The first PCR may include any suitable number of cycles. Generally, the number of cycles in the first PCR is less than 10, but a greater number of cycles may be performed. The first PCR may be about 1 cycle, about 2 cycles, about 3 cycles, about 4 cycles, about 5 cycles, about 6 cycles, about 7 cycles, about 8 cycles, about 9 cycles, or about 10 cycles. In some embodiments, the first PCR includes about 3 to 5 cycles.

[0064] The methods for determining the quality of nucleic acids in a sample provided herein may include 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 at about the second melting temperature of the 5'-end consensus sequences of the second forward primer and the second reverse primer included in the PCR reaction mixture provided herein. In some embodiments, the second extension step is performed at a temperature ranging 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 performed at a temperature at least 5°C higher than the first melting temperature. In some embodiments, the second extension step is performed at a temperature ranging 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 performed at a temperature ranging 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 performed at about 60°C to 85°C. In some embodiments, for example, the second extension step is about 60°C to 85°C, about 60°C to 80°C, about 60°C to 75°C, about 60°C to 70°C, or about 60°C to 65°C. In some embodiments, the second extension step is about 80°C to 85°C, about 75°C to 85°C, about 70°C to 85°C, about 65°C to 85°C, or about 60°C to 85°C. In some embodiments, the second extension step is about 70°C to 75°C. In some embodiments, the second extension step is about 70°C to 73°C. In some embodiments, the second extension step is about 70°C to 72°C. In some embodiments, the second extension step is about 70°C to 71°C. In some embodiments, the second extension step is about 70°C to 70.9°C. In some embodiments, the second extension step is about 70°C.

[0065] The second extension step can be any suitable length of time. For example, the second extension 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 therebetween. In some embodiments, the second extension step is about 1 minute.

[0066] The second PCR can include any suitable number of cycles. Generally, the second PCR includes more than 10 cycles, although it can also include fewer cycles. The second PCR can be about 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, or more cycles. In some embodiments, the second PCR comprises about 10 to 35 cycles. In some embodiments, the second PCR comprises about 15 to 25 cycles.

[0067] The method for determining the quality of nucleic acids in a sample provided herein may include determining the size range of amplicons. As used herein, "amplicon" refers to a nucleic acid that is 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 the context clearly indicates otherwise.

[0068] The first forward primer and the first reverse primer can be designed to have complementarity at their 3' ends to zigzag and / or non-overlapping sequences along both strands of the retrotransposon. Because 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 methods provided herein can be used to independently generate amplicons of various sizes, including about 50-200 bp, about 50-300 bp, about 50-400 bp, about 50-500 bp, about 50-600 bp, about 50-700 bp, about 50-800 bp, about 50-900 bp, about 50-1,000 bp, about 50-1,500 bp, about 50-2,000 bp, about 50-2,500 bp, about 50-3,000 bp, about 50-3,500 bp, about 50-4,000 bp, about 50-4,500 bp, and about 50-5,000 bp. In some embodiments, the amplicons range from about 100 to 2,000 bp. Any size range of amplicons generated by the methods provided herein can be used to analyze nucleic acid quality.

[0069] The methods for determining nucleic acid quality provided herein may include determining amplicon intensity ratios. Amplicon size and amplicon intensity ratios can be determined using any suitable method, including an Agilent 4200 TapeStation system, an Agilent 2200 TapeStation system, an Agilent 2100 Bioanalyzer system, an Agilent DNA ScreenTape Analysis, an Agilent D1000 and High Sensitivity D1000 Screen Tape Assays, a Lab901 TapeStation, and a 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] As used herein, the singular forms "a," "a," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0072] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass a variation of ±20%, or ±10%, or ±5%, or even ±1% from the specified value, where such variation is appropriate for practicing the disclosed compositions or disclosed methods.

[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 can be double-stranded or single-stranded and can 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" are meant to include, for example, nucleic acid molecules and fragments of nucleic acids, as well as any full-length or unfragmented nucleic acid molecules and nucleic acids.

[0074] As used herein, the term "nucleotide" includes both individual units of ribonucleic acid and deoxyribonucleic acid, as well as nucleoside and nucleotide analogs, and modified nucleotides such as labeled nucleotides. Furthermore, "nucleotide" includes non-naturally occurring analog structures, such as those in which the sugar, phosphate, 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(1): 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 in the compositions and methods provided herein. The sample used with the present compositions and methods can be obtained from a tissue sample or body fluid from a subject, or from tissue obtained by a biopsy procedure (e.g., needle biopsy) or surgical procedure. The biological sample of the present method can be a sample of body fluid, such as cerebrospinal fluid (CSF), blood, serum, plasma, urine, saliva, tears, and ascites. Body fluid samples can 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 whom the methods disclosed herein are performed. The term "subject" can also include any individual or patient who is a source of nucleic acid for use with the compositions and methods provided herein. The term "subject" can be used interchangeably with the terms "individual" or "patient." A subject may be a human, but as will be understood by those skilled in the art, a subject may also be an animal. Thus, other animals, including mammals, such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, etc., primates (including monkeys, chimpanzees, orangutans, and gorillas), reptiles, birds, amphibians, bony fish, cartilaginous fish, and invertebrates, are included within the definition of a subject. A subject may also be a plant or a microorganism. [Example]

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

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

[0079] A novel assay for determining DNA quality relies on the amplification of repetitive sequences in the human genome using a small number of primers capable of amplifying hundreds of different sites across the genome. These primers can amplify regions relevant to NGS, thereby providing a more accurate picture of DNA quality.

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

[0081] The L1-binding primer is fused to a common sequence not present in human DNA, with one sequence attached to the +strand primer and the second sequence attached to the -strand primer. The bases adjacent to the fusion point are selected to minimize overlap with the L1 sequence. The fusion primer is then added to the amplification mixture at a low concentration and used to amplify genomic DNA at the Tm of the L1 sequence in 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 predicted number of amplicons.

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

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

[0084] The advantages of this method stem from the use of repetitive DNA as the starting material. The presence of 100-1000 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. Because many DNA fragments have slightly different sizes, a larger range of sizes is examined compared to standard QFI assays. Furthermore, because regions from the entire genome are examined, changes in a single region of the genome do not substantially affect the assay.

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

[0086] Any and all references and citations made throughout this disclosure to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc., are hereby incorporated by reference in their entirety for all purposes.

[0087] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims. In particular embodiments, for example, the following items are provided: (Item 1) 1. 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 of the first forward primers and each of the first reverse primers is (i) a 3' terminal sequence having complementarity to a repeat sequence in the nucleic acid and having a first melting temperature; (ii) a 5'-end consensus sequence that is not present in the nucleic acid and has a second melting temperature, the second melting temperature being higher than the first melting temperature; a first primer set comprising: (b) a second primer set comprising a plurality of second forward primers and a plurality of second reverse primers, each of the second forward primers and each of the second reverse primers comprising a 5' terminal consensus sequence; Including, the system. (Item 2) 2. The primer system according to Item 1, wherein the second melting temperature is about 5°C to 25°C higher than the first melting temperature. (Item 3) 2. The primer system according to Item 1, wherein the first melting temperature is about 45°C to 70°C. (Item 4) 2. The primer system according to Item 1, wherein the second melting temperature is about 60°C to 85°C. (Item 5) 2. The system of primers according to item 1, wherein the first primer set comprises an equal number of first forward primers and first reverse primers. (Item 6) 2. The system of primers according to item 1, wherein the first primer set comprises an unequal number of first forward primers and first reverse primers. (Item 7) 2. The system of primers according to item 1, wherein the second primer set comprises an equal number of second forward primers and second reverse primers. (Item 8) 2. The system of primers of item 1, wherein the second primer set comprises an unequal number of second forward primers and second reverse primers. (Item 9) 2. 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) 2. 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) 2. The primer system of item 1, wherein the repetitive nucleic acid sequence comprises a retrotransposon. (Item 12) Item 12. The primer system according to Item 11, wherein the retrotransposon is an L1 retrotransposon. (Item 13) 2. The primer system of item 1, wherein the 5'-end common sequence of each first forward primer comprises the sequence of SEQ ID NO: 1. (Item 14) 2. The primer system of item 1, wherein the 5'-end common sequence of each first reverse primer comprises the sequence of SEQ ID NO: 2. (Item 15) 2. The primer system of item 1, wherein the 3' terminal sequence of each first forward primer comprises 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) 2. The primer system of item 1, 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. (Item 17) 1. A method for determining the quality of nucleic acids 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, wherein each of the first forward primers and each of the first reverse primers is (i) a 3' terminal sequence having complementarity to a repeat sequence in the nucleic acid and having a first melting temperature; (ii) a 5'-end consensus sequence that is not present in the nucleic acid and has a second melting temperature, the second melting temperature being higher than the first melting temperature; a first primer set comprising: ii) a second primer set comprising a plurality of second forward primers and a plurality of second reverse primers, each of the second forward primers and each of the second reverse primers comprising a 5' terminal consensus sequence; and preparing a polymerase chain reaction (PCR) mixture comprising: (a) performing a first polymerase chain reaction (PCR) on the sample, wherein a first extension step of each cycle of the first PCR is at a temperature about the first melting temperature; (b) performing a second polymerase chain reaction (PCR) on the sample, wherein a second extension step of each cycle in the second PCR is at a temperature about the second melting temperature; (c) determining the size range of the amplicons; A method comprising: (Item 18) Item 18. The method according to item 17, wherein the second melting temperature is about 5°C to 25°C higher than the first melting temperature. (Item 19) Item 18. The method according to item 17, wherein the first melting temperature is about 45°C to 70°C. (Item 20) Item 18. The method according to item 17, wherein the second melting temperature is about 60°C to 85°C. (Item 21) 18. The method of claim 17, wherein 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. (Item 22) 18. The method of claim 17, wherein the second extension step is at a temperature from about 5°C below the second melting temperature to 5.0°C above the second melting temperature and at least 5°C above the first melting temperature. (Item 23) 18. The method of claim 17, wherein the first extension step is for about 2 minutes. (Item 24) 18. The method of claim 17, wherein the second extension step is for about 1 minute. (Item 25) 18. The method of claim 17, wherein the first primer set comprises an equal number of first forward primers and first reverse primers. (Item 26) 18. The method of claim 17, wherein the first primer set comprises an unequal number of first forward primers and first reverse primers. (Item 27) 18. The method of claim 17, wherein the second primer set comprises an equal number of second forward primers and second reverse primers. (Item 28) 18. The method of claim 17, wherein the second primer set comprises an unequal number of second forward primers and second reverse primers. (Item 29) Item 18. The method of 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) Item 18. The method of 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) 18. The method of item 17, wherein the final concentration of the second forward primer and the second reverse primer is higher than the final concentration of the first forward primer and the first reverse primer. (Item 32) 32. The method of claim 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) 32. The method of claim 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) 18. The method of item 17, wherein the repetitive nucleic acid sequence comprises a retrotransposon. (Item 35) 35. The method of item 34, wherein the retrotransposon is an L1 retrotransposon. (Item 36) Item 18. The method of item 17, wherein the first PCR comprises about 3 to 5 cycles. (Item 37) 18. The method of item 17, wherein the second PCR comprises about 10 to 35 cycles. (Item 38) 18. The method of item 17, further comprising determining an intensity ratio of the amplicons. (Item 39) 18. The method of item 17, wherein the presence of the predicted amplicon size correlates with the quality of the nucleic acid. (Item 40) 18. The method of item 17, wherein the presence of the predicted amplicon size correlates with nucleic acid size. (Item 41) 18. The method of item 17, wherein the presence of the predicted amplicon intensity ratio correlates with nucleic acid quality. (Item 42) 18. The method of item 17, wherein the presence of the predicted amplicon intensity ratio correlates with nucleic acid size. (Item 43) 18. The method of item 17, wherein the 5'-end consensus sequence of each first forward primer comprises the sequence of SEQ ID NO: 1. (Item 44) 18. The method of item 17, wherein the 5'-end consensus sequence of each first reverse primer comprises the sequence of SEQ ID NO: 2. (Item 45) 18. The method of item 17, wherein the 3' end sequence of each first forward primer comprises 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) 18. The method of 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

1. 1. A method for determining the quality of nucleic acids in a sample, comprising: (a) preparing a polymerase chain reaction (PCR) mixture, (i) a first primer set comprising a plurality of different first forward primer sequences and a plurality of different first reverse primer sequences, wherein each first forward primer sequence and each first reverse primer sequence is (i) a 3' terminal sequence having complementarity to a repeat sequence in the nucleic acid and having a first melting temperature; (ii) a 5'-end consensus sequence that is not present in the nucleic acid and has a second melting temperature, the second melting temperature being higher than the first melting temperature; a first primer set comprising: ii) a second primer set comprising a plurality of different second forward primer sequences and a plurality of different second reverse primer sequences, each of the second forward primer sequences and each of the second reverse primer sequences comprising a 5' terminal consensus sequence that is not present in the nucleic acid; and preparing a polymerase chain reaction (PCR) mixture comprising: (b) performing a first polymerase chain reaction (PCR) on the sample, wherein a first extension step of each cycle of the first PCR is at a temperature about the first melting temperature; (c) performing a second polymerase chain reaction (PCR) on the sample, wherein a second extension step of each cycle in the second PCR is at a temperature about the second melting temperature; (d) determining the size range of the amplicons; A method comprising:

2. 10. The method of claim 1, wherein the second melting temperature is about 5°C to 25°C higher than the first melting temperature.

3. 10. The method of claim 1, wherein the first melting temperature is between about 45°C and 70°C.

4. 10. The method of claim 1, wherein the second melting temperature is between about 60°C and 85°C.

5. 2. The method of claim 1, wherein 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.

6. 2. The method of claim 1, wherein the second extension step is at a temperature from about 5°C below the second melting temperature to 5.0°C above the second melting temperature and at least 5°C above the first melting temperature.

7. 10. The method of claim 1, wherein the first extension step is for about 2 minutes.

8. 10. The method of claim 1, wherein the second extension step is for about 1 minute.

9. 2. The method of claim 1, wherein the first primer set comprises an equal number of different first forward primer sequences and different first reverse primer sequences.

10. 2. The method of claim 1, wherein the first primer set comprises an unequal number of different first forward primer sequences and different first reverse primer sequences.

11. 2. The method of claim 1, wherein the second primer set comprises an equal number of different second forward primer sequences and different second reverse primer sequences.

12. 2. The method of claim 1, wherein the second primer set comprises an unequal number of different second forward primer sequences and different second reverse primer sequences.

13. 2. The method of claim 1, wherein the first primer set comprises about 1 to 20 different first forward primer sequences and about 1 to 20 different first reverse primer sequences.

14. 2. The method of claim 1, wherein the second primer set comprises about 1 to 20 different second forward primer sequences and about 1 to 20 different second reverse primer sequences.

15. 2. The method of claim 1, wherein the final concentration of the second forward primer and the second reverse primer is higher than the final concentration of the first forward primer and the first reverse primer.

16. 16. The method of claim 15, wherein the final concentration of the first forward primer is the same as or similar to the final concentration of the first reverse primer.

17. 16. The method of claim 15, wherein the final concentration of the second forward primer is the same as or similar to the final concentration of the second reverse primer.

18. 2. The method of claim 1, wherein the repetitive nucleic acid sequence comprises a retrotransposon.

19. 19. The method of claim 18, wherein the retrotransposon is an L1 retrotransposon.

20. 2. The method of claim 1, wherein the first PCR comprises about 3 to 5 cycles.

21. 2. The method of claim 1, wherein the second PCR comprises about 10 to 35 cycles.

22. The method of claim 1, further comprising determining an intensity ratio of the amplicons.

23. The method of claim 1, wherein the presence of the predicted amplicon size correlates with the quality of the nucleic acid.

24. The method of claim 1, wherein the presence of the predicted amplicon size correlates with nucleic acid size.

25. The method of claim 1 , wherein the presence of the predicted amplicon intensity ratio correlates with nucleic acid quality.

26. The method of claim 1, wherein the presence of the predicted amplicon intensity ratio correlates with nucleic acid size.

27. 2. The method of claim 1, wherein the 5' terminal consensus sequence of each first forward primer comprises the sequence of SEQ ID NO:

1.

28. 2. The method of claim 1, wherein the 5' terminal consensus sequence of each first reverse primer comprises the sequence of SEQ ID NO:

2.

29. 2. The method of claim 1, wherein the 3' terminal sequence of each first forward primer comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, or any combination thereof.

30. 2. The method of claim 1, 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.

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