Amplification of target sequences

Abstract

The present disclosure relates to methods, kits, and products for amplifying target DNA sequences from genomic DNA. Certain embodiments of the present disclosure provide methods for amplifying target DNA sequences from genomic DNA, the method comprising amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, whereby the target DNA sequences are amplified.

Description

[0001] This application is a divisional application of patent application No. 201780033969.0, filed on March 31, 2017, entitled "Amplification of Target Sequence".

[0002] This application claims priority to Australian Provisional Patent Application No. 2016901197, filed on March 31, 2016, the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to methods, kits, and products for amplifying target DNA sequences from genomic DNA. Background Technology

[0004] The ability to amplify specific target sequences present in genomic DNA is used in various fields, such as for research purposes, recombinant DNA technology, genetic screening, and for forensic analysis.

[0005] Although target-specific amplification (STA) is widely used, it has several drawbacks, especially when the amount of starting genomic material is limited. For example, STSTA often leads to the rapid depletion of limited genomic material and typically allows analysis only of the specifically amplified sequence. As a result, it is often difficult to obtain other important genetic information from the remaining genomic DNA.

[0006] Whole-genome amplification (WGA) is a method that was developed in part to address the problem of having limited genomic DNA available for analysis. WGA enables the amplification of the entire genome using only a very small amount of starting material, thus providing perpetuity for a limited sample that can be used for other desired analyses.

[0007] Various methods have been developed for whole-genome amplification, including degenerate oligonucleotide PCR (DOP-PCR), primer extension pre-amplification (PEP), and multiple substitution amplification (MDA). DOP-PCR and PEP are based on standard PCR techniques, while MDA is a non-PCR amplification technique that utilizes the strand substitution capabilities of some polymerases. The genomic DNA amplified from the whole genome can then be analyzed, for example, by techniques such as comparative genomic hybridization (CGH) analysis, SNP analysis, and sequencing.

[0008] However, while whole-genome amplification (WGA) provides the ability to amplify genomic DNA for analysis, amplifying specific target sequences from the amplified genomic DNA has several drawbacks, such as one or more of the following: allele dropout and / or bias events introduced during WGA, the possibility of uneven sequence coverage, over- or under-representation of specific regions, duplication, and the introduction of errors into the specific target sequence.

[0009] Therefore, there is an urgent need for improved methods and / or products for amplifying specific target sequences from DNA amplified from the whole genome. Summary of the Invention

[0010] This disclosure relates to methods, kits, and products for amplifying target DNA sequences from genomic DNA.

[0011] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, thereby amplifying the target DNA sequence.

[0012] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, thereby amplifying the target DNA sequence.

[0013] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence is also amplified using one or more target DNA sequence-specific primers. This amplifies the target DNA sequence.

[0014] Certain embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising: Whole-genome amplification of the genomic DNA using one or more degenerate oligonucleotide primers; and At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to amplify the target DNA sequence. This amplifies the target DNA sequence.

[0015] Some embodiments of this disclosure provide target DNA sequences amplified by the methods described herein.

[0016] Some embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence amplified from genomic DNA using the methods described herein.

[0017] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0018] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, and evaluating the nucleotide content of the amplified target DNA sequence.

[0019] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0020] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Whole-genome amplification of the genomic DNA was performed using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0021] Certain embodiments of this disclosure provide a method for determining the nucleotide sequence of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and Determine the nucleotide sequence of the amplified target DNA sequence. This determines the nucleotide sequence of the target DNA sequence.

[0022] Certain embodiments of this disclosure provide a method for pre-implantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0023] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising amplifying a target DNA sequence from the embryo with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the embryo with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the embryo.

[0024] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the embryo is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0025] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising: Whole-genome amplification of genomic DNA from the embryo was performed using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers from the embryo to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0026] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Amplifying genomic DNA from an oocyte or its polar body using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0027] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising amplifying a target DNA sequence from the oocyte or its polar body with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the oocyte or polar body with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the oocyte.

[0028] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Genomic DNA from the oocyte or its polar body is amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the oocyte or polar body is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0029] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Whole-genome amplification of genomic DNA from the oocyte or its polar body was performed using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence from the oocyte or polar body is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0030] Some embodiments of this disclosure provide a method for screening sperm, the method comprising: Amplifying genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0031] Certain embodiments of this disclosure provide a method for screening sperm, the method comprising amplifying a target DNA sequence from one or more sperm using one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the sperm.

[0032] Some embodiments of this disclosure provide a method for screening sperm, the method comprising: Amplification of genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, target DNA sequences from the one or more sperm cells are further amplified using one or more target DNA sequence-specific primers to produce amplified target DNA sequences; and The amplified target DNA sequence was evaluated to screen the sperm.

[0033] Some embodiments of this disclosure provide a method for screening sperm, the method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers from the one or more sperm cells to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0034] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Genomic DNA from the fetus is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0035] Certain embodiments of this disclosure provide a method for prenatal genetic screening of a fetus, the method comprising amplifying a target DNA sequence from the fetus with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the fetus with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the fetus.

[0036] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Genomic DNA from the fetus was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the fetus is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0037] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0038] Some embodiments of this disclosure provide kits for performing the methods described herein.

[0039] Certain embodiments of this disclosure provide the use of the amplified target DNA sequence as described herein for preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, screening for polymorphisms, mutations, deletions, insertions, translocations, expansions, inversions, methylation states and / or epigenetic alterations, and / or forensic screening.

[0040] Some embodiments of this disclosure provide a combined product comprising the following components: (i) One or more degenerate oligonucleotide primers used for whole genome amplification; (ii) One or more primers for amplifying the target DNA sequence; and (iii) One or more reagents and / or instructions for amplifying the target DNA sequence during whole-genome amplification.

[0041] Other implementation schemes are described in this article. Attached Figure Description

[0042] Some implementation schemes are illustrated in the following figures. It should be understood that the following description is for the purpose of describing specific implementation schemes only and is not intended to be limiting.

[0043] Figure 1 The images show the amplification products of single cells after whole-genome amplification and gene targeting. Lanes 2 to 5 show single-cell products as a result of whole-genome amplification and gene targeting performed with a final primer concentration of 0.25 μM. Cell amplification in lane 5 failed. Lanes 7 to 10 show single-cell products as a result of whole-genome amplification and gene targeting performed with a final primer concentration of 0.1 μM. Weaker amplification was observed in cells with gene targeting compared to control cells (lanes 12 to 15).

[0044] Figure 2 The images show amplification products from a single cell and 30 pg gDNA after whole-genome amplification. Lanes 2 to 9 showed weaker amplification compared to the control samples in lanes 11 to 18 due to the presence of gene-specific primers at a final concentration of 0.5 μM.

[0045] Figure 3 The image shows electrophoresis patterns of amplicon generated after the second round of PCR following combined whole-genome amplification and seeding of gene-targeted single-cell products into two gene-specific PCRs. Compared to the control (lanes 9-12), the D4S43 PCR gel showed enriched amplification of the target D4S43 marker due to the addition of 0.25 μM gene-specific primers (lanes 2-4). Similarly, compared to the control (lanes 8-11), Y-chromosome PCR showed enriched amplification of the target gene (lanes 1-3 and 4-7) due to the addition of gene-specific primers at final concentrations of 0.25 μM and 0.1 μM. Samples were electrophoresed using the molecular weight standard DMW-100M (Geneworks).

[0046] Figure 4 The image shows electrophoresis patterns of amplicon generated in the second round of PCR after seeding the combined whole-genome amplification and gene-targeted single-cell products into two gene-specific PCRs. Compared to the control (lanes 6-9), the D4S43 PCR gel showed enriched amplification of the target D4S43 marker due to the addition of 0.5 μM gene-specific primers (lanes 2-5). Similarly, compared to the control (lanes 5-8), Y-chromosome PCR showed enriched amplification of the target gene due to the addition of gene-specific primers at a final concentration of 0.5 μM (lanes 1-4). Samples were electrophoresed using the molecular weight standard DMW-100M (Geneworks).

[0047] Figure 5This figure shows the breadth of HBB coverage after combined whole-genome amplification and gene-targeted amplification, compared to whole-genome amplification alone, using next-generation sequencing. After combined whole-genome amplification and gene-targeted amplification, the products were seeded into two gene-specific PCRs. The second-round PCR amplicons were merged with the DNA from the whole-genome amplification with gene-targeted amplification to generate a 1:20 final dilution. Whole-genome PCR products alone (WGA), combined whole-genome amplification and gene-targeted amplification products (WGA + TSE), and the 1:20 dilution merge were sequenced. The figure shows that 100% coverage of the two HBB target sequences (exons 1 and 2, and exon 3) was achieved when the 1:20 dilution of the second-round HBB PCR products was merged back into the sample with combined whole-genome amplification with gene-targeted amplification. The mean is plotted.

[0048] Figure 6 The results, using next-generation sequencing, showed the coverage depth for exons 1 and 2 and exon 3 of HBB after combined whole-genome amplification and gene-targeted amplification, followed by further merging of HBB PCR products, compared to whole-genome amplification and gene-targeted amplification alone. Following combined whole-genome amplification and gene-targeted amplification, the products were seeded into two HBB-specific PCRs. The second-round HBB PCR amplicons were merged with the DNA from the whole-genome amplification with gene-targeted amplification to generate a 1:20 final dilution. Sequencing was performed on the combined whole-genome amplification and gene-targeted amplification products (WGA + TSE) and the 1:20 diluted merged DNA. When sequencing the 1:20 diluted merged DNA, an average HBB sequence coverage depth of >60x was obtained.

[0049] Figure 7 This figure shows the breadth of HLA coverage of the target gene after combined whole-genome amplification and gene-targeted amplification, compared to whole-genome amplification alone, using next-generation sequencing. After combined whole-genome amplification and gene-targeted amplification, the products were seeded into an HLA-specific PCR. This second-round HLA PCR amplicons were merged with DNA from the whole-genome amplification with gene-targeted amplification to generate 1:20 and 1:10 final dilutions. The whole-genome amplification-only PCR product (WGA), the combined whole-genome amplification and gene-targeted amplification product (WGA + TSE), the merged DNA from the 1:20 and 1:10 dilutions, and the second-round HLA PCR amplicons (amplicon) were sequenced. The figure shows that 100% HBB coverage was achieved when the 1:20 and 1:10 dilutions of the second-round HLA PCR product were merged back into the combined whole-genome amplification with gene-targeted amplification, as well as the HLA PCR product alone. Mean values ​​are plotted.

[0050] Figure 8The results show the HLA coverage depth achieved using next-generation sequencing following combined whole-genome amplification and gene-targeted amplification. After combined whole-genome amplification and gene-targeted amplification, the products were seeded into HLA-specific PCR. This second-round HLA PCR amplicons were merged with the DNA from the whole-genome amplification with gene-targeted amplification to generate 1:20 and 1:10 final dilutions. The products of combined whole-genome amplification and gene-targeted amplification (WGA + TSE), the merged DNA from the 1:20 and 1:10 dilutions, and the second-round HLA PCR amplicons (amplicones) were sequenced. When the merged DNA from the 1:20 and 1:10 dilutions, along with the second-round HLA PCR amplicons, was sequenced, an average HLA coverage depth of >60x was obtained. Detailed Implementation

[0051] This disclosure relates to methods, kits, and products for amplifying target DNA sequences from genomic DNA.

[0052] This disclosure is based on the premise that it is possible to amplify specific target DNA sequences during whole-genome amplification.

[0053] Certain embodiments of this disclosure relate to methods and products that have one or more combinations of advantages. For example, some advantages of some embodiments disclosed herein include one or more of the following: reduced likelihood of allele decoupling when amplifying target DNA sequences from DNA amplified from the whole genome; improved ability to analyze target DNA sequences amplified from DNA amplified from the whole genome; reduced bias associated with amplification of target DNA sequences from DNA amplified from the whole genome; reduced number of PCR cycles required for amplifying target DNA sequences from DNA amplified from the whole genome; improved time for amplifying specific target DNA sequences from DNA amplified from the whole genome; reduced use of consumables and / or human resources; reduced costs; and / or provision of commercial options.

[0054] Some embodiments of this disclosure provide methods for amplifying target DNA sequences from genomic DNA.

[0055] The term "amplifying," or variations such as "amplification" and "amplified," refers to the process of copying nucleic acids to produce more copies of all or part of that nucleic acid. For example, nucleic acid amplification can be enzymatically achieved using polymerase chain reaction (PCR), isothermal methods such as multiple displacement amplification using appropriate polymerases, or rollover amplification. Other types of amplification methods are also considered. The methods used to perform amplification are described herein.

[0056] The term "genomic DNA" refers to all or part of the DNA derived from an organism, including chromosomal DNA, cellular DNA, mitochondrial DNA, and viral DNA. This genomic DNA can be isolated, processed, or extracted, for example, from prokaryotic or eukaryotic cell DNA, viral DNA, somatic cell DNA, germ cell DNA, gamete DNA, polar body DNA, embryonic cell DNA, or fetal DNA. The term includes DNA derived from exon DNA, intron DNA, non-coding DNA, RNA-coding DNA, non-repetitive DNA, repetitive DNA, transposon DNA, extrachromosomal DNA, organelle DNA, mitochondrial DNA, chloroplast DNA, viral DNA, plasmid DNA, nuclear DNA, extranuclear DNA, cytoplasmic DNA, cDNA, and cell-free DNA. Other types of DNA are also considered.

[0057] The term "target DNA sequence" refers to a specific region or portion of the DNA to be amplified. For example, the target DNA sequence can be all or part of a gene, a locus, a coding region, one or more exons and / or one or more introns, a SNP, a region of mutation to be evaluated, an allelic region, or all or part of DNA encoding RNA. Other types of target DNA sequences are also considered.

[0058] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, thereby amplifying the target DNA sequence.

[0059] In some embodiments, the amplification includes a polymerase chain reaction (PCR). Methods for employing a PCR are as described herein. Methods for performing the amplification are, for example, described in Fakruddin et al. (2013). J Pharm Bioallied Sci 5(4) It is described in 245-252.

[0060] In some embodiments, the amplification includes an isothermal amplification reaction. Methods for performing isothermal amplification are known in the art, such as loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), helicase-dependent amplification (HDA), and nicking enzyme amplification (NEAR). In some embodiments, the amplification includes a reaction employing multiple substitution amplification. Methods for performing multiple substitution amplification (WGA) are known in the art, for example, Dean et al. (2002). Proc Natl. Acad. Sci USA 99 As described in 5261. In some embodiments, the amplification includes a reaction employing rollover amplification. Other types of amplification are also considered. Methods used for amplification are, for example, described by Walker et al. (1992). Nucleic Acids Res . 20 1691-1696; Walker et al. (1993) PCR Methods Appl . 3 :1-6; Notomi et al. (2000) Nucleic Acids Res .28:e63; Tomita N et al. (2008) Nat. Protoc . 3 :877-882; Lizardi et al. (1998) Nat. Genet . 19 :225-232; Blanco et al. (1989) J. Biol.Chem . 264 :8935-8940; and Dean et al. . (2001) Genome Res .11:1095-1099, and Fakruddin et al. (2013) J Pharm Bioallied Sci 5(4) It is described in :245-252.

[0061] In some embodiments, the target DNA sequence includes all or part of a gene, a locus, a coding region, one or more exons and / or one or more introns, a region to be evaluated for mutations, an allelic region, a region with SNPs or other types of polymorphisms, or all or part of DNA encoding RNA, a region to be evaluated for the presence of mutations, marker DNA, and / or a region used to identify the presence or type of DNA. Other types of target DNA sequences are also considered.

[0062] In some embodiments, the genomic DNA includes DNA from one or more embryonic cells, DNA from an oocyte or its polar body, DNA from sperm, DNA from one or more germ cells, DNA from one or more individual cells, DNA from one or more human or animal cells, DNA from one or more plant cells, DNA from one or more microbial cells, DNA from one or more cells used for disease screening, DNA from one or more cancer cells, DNA for forensic testing, mitochondrial DNA, extrachromosomal DNA, cell-free DNA, DNA from samples comprising one or more cells, DNA from samples comprising cell-free DNA, DNA from cell lysates, DNA from biopsies, DNA from tissue samples, DNA from cell samples, DNA from body fluids, DNA from blood samples, fetal DNA, cell-free DNA, DNA from amniotic fluid, DNA from formalin-fixed samples, DNA from paraffin-embedded samples, and DNA from swabs. Other types of DNA are also considered. Methods for obtaining or using DNA in amplification reactions are known in the art.

[0063] In some embodiments, the genomic DNA comprises DNA from lysed cells. In some embodiments, the genomic DNA comprises unpurified DNA. In some embodiments, the genomic DNA comprises extracted DNA. In some embodiments, the genomic DNA comprises processed DNA. In some embodiments, the genomic DNA comprises purified or partially purified DNA. In some embodiments, the genomic DNA comprises cell-free DNA.

[0064] In some embodiments, the method includes amounts of genomic DNA to be amplified of less than 0.5 pg, less than 1 pg, less than 2.5 pg, less than 5 pg, less than 10 pg, less than 25 pg, less than 50 pg, less than 100 pg, less than 250 pg, less than 500 pg, less than 1 ng, less than 2.5 ng, less than 5 ng, or less than 10 ng. Other amounts are also considered.

[0065] In some embodiments, the method includes at least 0.5 pg, at least 1 pg, at least 2.5 pg, at least 5 pg, at least 10 pg, at least 25 pg, at least 50 pg, at least 100 pg, at least 250 pg, at least 500 pg, at least 1 ng, at least 2.5 ng, at least 5 ng, or at least 10 ng of genomic DNA to be amplified. Other amounts are also considered.

[0066] In some embodiments, the method includes 0.5 pg to 10 ng, 1 pg to 10 ng, 2.5 pg to 10 ng, 5 pg to 10 ng, 10 pg to 10 ng, 25 pg to 10 ng, 50 pg to 10 ng, 100 pg to 10 ng, 250 pg to 10 ng, 500 pg to 10 ng, 1 ng to 10 ng, 2.5 ng to 10 ng, 5 ng to 10 ng, 0.5 pg to 5 ng, 1 pg to 5 ng, 2.5 pg to 5 ng, 5 pg to 5 ng, 10 pg to 5 ng, 25 pg to 5 ng, 50 pg to 5 ng, 100 pg to 5 ng, 250 pg to 5 ng, 500 pg to 5 ng, 1 ng to 5 ng, 2.5 ng to 5 ng, 0. 5pg to 2.5ng, 1pg to 2.5ng, 2.5pg to 2.5ng, 5pg to 2.5ng, 10pg to 2.5ng, 25pg to 2.5ng, 50pg to 2.5ng, 100pg to 2.5ng, 250pg to 2.5ng, 500pg to 2.5ng, 1ng to 2.5ng, 0.5pg to 1ng, 1pg to 1ng, 2.5pg to 1ng, 5pg to 1ng, 10pg to 1ng, 25pg to 1ng, 50pg to 1ng, 100pg to 1ng, 250pg to 1ng, 500pg to 1ng, 0.5pg to 500pg, 1pg to 500pg 2.5pg to 500pg, 5pg to 500pg, 10pg to 500pg, 25pg to 500pg, 50pg to 500pg, 100pg to 500pg, 250pg to 500pg, 0.5pg to 250pg, 1pg to 250pg, 2.5pg to 250pg, 5pg to 250pg, 10pg to 250pg, 25pg to 250pg, 50pg to 250pg, 100pg to 250pg, 0.5pg to 100pg, 1pg to 100pg, 2.5pg to 100pg, 5pg to 100pg, 10pg to 100pg, 25pg to 100pg Genomic DNA to be amplified in amounts of 50 pg to 100 pg, 0.5 pg to 50 pg, 1 pg to 50 pg, 2.5 pg to 50 pg, 5 pg to 50 pg, 10 pg to 50 pg, 25 pg to 50 pg, 0.5 pg to 25 pg, 1 pg to 25 pg, 2.5 pg to 25 pg, 5 pg to 25 pg, 10 pg to 25 pg, 0.5 pg to 10 pg, 1 pg to 10 pg, 2.5 pg to 10 pg, 5 pg to 10 pg, 0.5 pg to 5 ng, 1 pg to 5 ng, 2.5 pg to 5 pg, 0.5 pg to 2.5 pg, 1 pg to 2.5 pg, or 0.5 pg to 1 pg. Other amounts are also considered.

[0067] In some implementations, the method includes 1 pg to 6 ng of genomic DNA to be amplified.

[0068] In some embodiments, the genomic DNA comprises DNA from 1 to 50 cells. In some embodiments, the genomic DNA comprises DNA from a single cell, 1 to 10 cells, 1 to 20 cells, 1 to 50 cells, more than 10 cells, less than 10 cells, more than 50 cells, less than 50 cells, more than 100 cells, or less than 100 cells. DNA from other numbers of cells is also considered.

[0069] Examples of cell types include one or more embryonic cells, oocytes or their polar bodies, sperm, one or more germ cells, one or more individual cells, one or more personal or animal cells, one or more plant cells, one or more cells derived from microorganisms, one or more cells used for screening for diseases, conditions, or states, one or more cancer cells or precancerous cells, cells derived from biopsies, one or more fetal cells, cells derived from tissue samples, cells in / from body fluids, cells in / from blood samples, cells in / from amniotic fluid, cells in / from formalin-fixed samples, cells in / from paraffin-embedded samples, and cells in / from swabs. Other cell types and cells from other sources are also considered. Methods for obtaining cells are known in the art.

[0070] The term "degenerate oligonucleotide primer" refers to an oligonucleotide whose nucleotide sequence is wholly or partially degenerate. Such primers can potentially bind to many sequences. Typically, these oligonucleotides either have a nucleotide sequence portion that has the potential to contain more than one base at a particular position, and / or have a nucleotide sequence portion that contains bases that can form base pairs with one or more other bases. Methods and procedures for designing primers, including degenerate oligonucleotide primers, are known in the art.

[0071] In some implementations, genomic DNA amplification using degenerate oligonucleotide primers results in whole genome amplification (WGA).

[0072] The degenerate oligonucleotide primer may include DNA, RNA, a variant of DNA or RNA, a variant of the sugar-phosphate backbone, and / or a variant of one or more bases, such as modified or methylated bases. Other types of variants are also considered.

[0073] Examples of degenerate bases that can bind to more than one base include 2-aminopurine, 5-methylisodeoxycytosine (Me isodC), 5-nitroindole, inosine deoxy, inosine ribo, and isodeoxyguanosine dG. Other bases are also considered.

[0074] Methods for designing degenerate primers are known in the art, including procedures such as iCODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers), NCBI Primer-BLAST, and HYDEN (Highly Degenerate primers). Methods for generating degenerate oligonucleotide primers are known in the art, including chemical synthesis.

[0075] In some embodiments, the one or more degenerate oligonucleotide primers include a sequence of one or more defined / fixed nucleotides at their 3' end. In some embodiments, the degenerate oligonucleotide primers include a sequence of one or more defined / fixed nucleotides at their 3' end and at their 5' end.

[0076] In some embodiments, the one or more degenerate oligonucleotide primers comprise a nucleotide sequence of 6 to 12 consecutive random nucleotides. For example, degenerate oligonucleotide primers used in WGA are primers having the sequence 5'-CTCGAGNNNNATGTGG-3' (SEQ ID NO. 5) (where N = A, C, G, or T), primers having the sequence 5'-CGACTCGAGNNNNNNATGTGG-3' (SEQ ID NO. 6) (where N = A, C, G, or T), or primers having the sequence 5'-CCGACCGAGNNNNNNATGTGG-3' (SEQ ID NO. 7) (where N = A, C, G, or T). Primers having other sequences are also considered.

[0077] In some embodiments, the degenerate oligonucleotide primer has a size of at least 12 bases, at least 13 bases, at least 14 bases, at least 15 bases, at least 16 bases, at least 17 bases, at least 18 bases, at least 19 bases, at least 20 bases, or at least 25 bases. Other sizes are also considered.

[0078] In some embodiments, the degenerate oligonucleotide primer has a size of less than 25 bases, less than 20 bases, less than 19 bases, less than 18 bases, less than 17 bases, less than 16 bases, less than 15 bases, less than 14 bases, less than 13 bases, or less than 12 bases. Other sizes are also considered.

[0079] In some embodiments, the degenerate oligonucleotide has 12 to 25 bases, 13 to 25 bases, 14 to 25 bases, 15 to 25 bases, 16 to 25 bases, 17 to 25 bases, 18 to 25 bases, 19 to 25 bases, 20 to 25 bases, 21 to 25 bases, 22 to 25 bases, 23 to 25 bases, 24 to 25 bases, 12 to 24 bases, 13 to 24 bases, 14 to 24 bases, 15 to 24 bases, 16 to 24 bases, 17 to 24 bases, 18 to 24 bases, 19 to 24 bases, 20 to 24 bases, 21 to 24 bases, 22 to 24 bases, 23 to 24 bases, 12 to 23 bases, 13 to 23 bases, 14 to 23 bases, 15 to 23 bases, 16 to 23 bases, 17 to 23 bases, 18 to 23 bases, 19 to 23 bases, 20 to 23 bases, 21 to 23 bases, 22 to 23 bases, 12 to 22 bases, 13 to 22 bases, 14 to 22 bases, 15 to 22 bases, 1 6 to 22 bases, 17 to 22 bases, 18 to 22 bases, 19 to 22 bases, 20 to 22 bases, 21 to 22 bases, 12 to 20 bases, 13 to 20 bases, 14 to 20 bases, 15 to 20 bases, 16 to 20 bases, 17 to 20 bases, 18 to 20 bases, 19 to 20 bases, 12 to 19 bases, 13 to 19 bases, 14 to 19 bases, 15 to 19 bases, 16 to 19 bases, 17 to 19 bases, 18 to 19 bases, 12 Sizes of up to 18 bases, 13 to 18 bases, 14 to 18 bases, 15 to 18 bases, 16 to 18 bases, 17 to 18 bases, 12 to 17 bases, 13 to 17 bases, 14 to 17 bases, 15 to 17 bases, 16 to 17 bases, 12 to 16 bases, 13 to 16 bases, 14 to 16 bases, 15 to 16 bases, 12 to 15 bases, 13 to 15 bases, 14 to 15 bases, 12 to 14 bases, 13 to 14 bases, or 12 to 13 bases. Other sizes are also considered.

[0080] The use of degenerate oligonucleotide primers is known in the art, for example in Telenius, H., Carter, NP, Bebb, CE, et al. (1992) "Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer". Genomics 13 It is described in :718.

[0081] In some embodiments, the one or more degenerate oligonucleotide primers comprise a set of primers having the same general nucleotide sequence. In some embodiments, the one or more degenerate oligonucleotide primers comprise nucleotide sequences having the same general formula. In some embodiments, the one or more degenerate primers comprise a set of primers having different general nucleotide sequences. In some embodiments, the one or more degenerate oligonucleotide primers are different.

[0082] In some embodiments, the method includes the concentration of one or more degenerate oligonucleotide primers being at least 0.1 µM, at least 0.25 µM, at least 0.5 µM, at least 1 µM, at least 2.5 µM, or at least 5 µM. Other concentrations are also considered.

[0083] In some embodiments, the method includes the concentration of one or more degenerate oligonucleotide primers being less than 0.1 µM, less than 0.25 µM, less than 0.5 µM, less than 1 µM, less than 2.5 µM, or less than 5 µM. Other concentrations are also considered.

[0084] In some embodiments, the method includes the concentration of one or more degenerate oligonucleotide primers being 0.05 to 5 µM, 0.1 to 5 µM, 0.25 to 5 µM, 0.5 to 5 µM, 1 to 5 µM, 2.5 to 5 µM, 0.05 to 4 µM, 0.1 to 4 µM, 0.25 to 4 µM, 0.5 to 4 µM, 1 to 4 µM, 2.5 to 4 µM, 0.05 to 2.5 µM, 0.1 to 2.5 µM, 0.25 to 2.5 µM, 0.5 to 2.5 µM, 1 to 2.5 µM, 0.05 to 1 µM, 0.1 to 1 µM, 0.25 to 1 µM, 0.5 to 1 µM, 0.05 to 0.5 µM, 0.1 to 0.5 µM, 0.25 to 0.5 µM, 0.05 to 0.25 µM, 0.1 to 0.25 µM, or 0.05 to 0.1 µM. Other concentrations are also considered.

[0085] In some embodiments, the method includes one or more degenerate oligonucleotide primers at a concentration of 0.1 to 4 µM.

[0086] In some embodiments, the one or more target DNA sequence-specific primers have a size of at least 12 bases, at least 13 bases, at least 14 bases, at least 15 bases, at least 16 bases, at least 17 bases, at least 18 bases, at least 19 bases, at least 20 bases, or at least 25 bases. Other sizes are also considered.

[0087] In some embodiments, the one or more target DNA sequence-specific primers have a size of less than 25 bases, less than 20 bases, less than 19 bases, less than 18 bases, less than 17 bases, less than 16 bases, less than 15 bases, less than 14 bases, less than 13 bases, or less than 12 bases. Other sizes are also considered.

[0088] In some embodiments, the one or more target DNA sequence-specific primers have 12 to 25 bases, 13 to 25 bases, 14 to 25 bases, 15 to 25 bases, 16 to 25 bases, 17 to 25 bases, 18 to 25 bases, 19 to 25 bases, 20 to 25 bases, 21 to 25 bases, 22 to 25 bases, 23 to 25 bases, 24 to 25 bases, 12 to 24 bases, 13 to 24 bases, 14 to 24 bases, 15 to 24 bases, 16 to 24 bases, 17 Up to 24 bases, 18 to 24 bases, 19 to 24 bases, 20 to 24 bases, 21 to 24 bases, 22 to 24 bases, 23 to 24 bases, 12 to 23 bases, 13 to 23 bases, 14 to 23 bases, 15 to 23 bases, 16 to 23 bases, 17 to 23 bases, 18 to 23 bases, 19 to 23 bases, 20 to 23 bases, 21 to 23 bases, 22 to 23 bases, 12 to 22 bases, 13 to 22 bases, 14 to 22 bases, 15 to 22 16 to 22 bases, 17 to 22 bases, 18 to 22 bases, 19 to 22 bases, 20 to 22 bases, 21 to 22 bases, 12 to 20 bases, 13 to 20 bases, 14 to 20 bases, 15 to 20 bases, 16 to 20 bases, 17 to 20 bases, 18 to 20 bases, 19 to 20 bases, 12 to 19 bases, 13 to 19 bases, 14 to 19 bases, 15 to 19 bases, 16 to 19 bases, 17 to 19 bases, 18 to 19 bases Sizes of 12 to 18 bases, 13 to 18 bases, 14 to 18 bases, 15 to 18 bases, 16 to 18 bases, 17 to 18 bases, 12 to 17 bases, 13 to 17 bases, 14 to 17 bases, 15 to 17 bases, 16 to 17 bases, 12 to 16 bases, 13 to 16 bases, 14 to 16 bases, 15 to 16 bases, 12 to 15 bases, 13 to 15 bases, 14 to 15 bases, 12 to 14 bases, 13 to 14 bases, or 12 to 13 bases. Other sizes are also considered.

[0089] Methods for designing and generating primers are known in the art. Typically, primers / oligonucleotides are generated from nucleoside phosphoramides using chemical synthesis, but other methods of chemical synthesis are also considered.

[0090] In some embodiments, the one or more target DNA sequence-specific primers include a melting temperature of at least 50°C, at least 55°C, at least 60°C, or at least 65°C. Other melting temperatures are also considered.

[0091] Methods for determining the melting temperature are known in the art. The melting temperature cited herein is provided as a temperature (Tm) under the amplification conditions used. It should be understood that this Tm value depends on several factors, including salt concentration and Mg. 2+ Concentration, and various different types of calculations are known in the art to determine Tm.

[0092] In some embodiments, the one or more target DNA sequence-specific primers include melting temperatures below 50°C, below 55°C, below 60°C, or below 65°C. Other melting temperatures are also considered.

[0093] In some embodiments, the one or more target DNA sequence-specific primers include melting temperatures ranging from 45°C to 65°C, 50°C to 65°C, 55°C to 65°C, 60°C to 65°C, 45°C to 60°C, 50°C to 60°C, 55°C to 60°C, 45°C to 55°C, 50°C to 55°C, or 45°C to 50°C. Other melting temperatures are also considered.

[0094] In some embodiments, the one or more target DNA sequence-specific primers comprise a GC content of at least 18%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65%. Other levels of GC content are also considered. Methods for determining GC content are known in the art.

[0095] In some embodiments, the one or more target DNA sequence-specific primers include GC contents of less than 18%, less than 20%, less than 25%, less than 30%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, or less than 65%. Other levels of GC content are also considered.

[0096] In some embodiments, the one or more target DNA sequence-specific primers include 18% to 65%, 20% to 65%, 25% to 65%, 25% to 65%, 30% to 65%, 35% to 65%, 40% to 65%, 45% to 65%, 50% to 65%, 55% to 65%, 60% to 65%, 18% to 60%, 20% to 60%, 25% to 60%, 25% to 60%, 30% to 60%, 35% to 60%, 40% to 60%, 45% to 60%, 50% to 60%, 55% to 60%, 18% to 55%, 20% to 55%, 25% to 55%, 25% to 55%, 30% to 55%, 35% to 55%, 40 ...25% to 55%, 25% to 55%, 25% to 55%, 25% to 55%, 25% to 55%, 25% to 55%, GC content of 5%, 45% to 55%, 50% to 55%, 18% to 50%, 20% to 50%, 25% to 50%, 30% to 50%, 35% to 50%, 40% to 50%, 45% to 50%, 18% to 45%, 20% to 45%, 25% to 45%, 30% to 45%, 35% to 45%, 40% to 45%, 18% to 40%, 20% to 40%, 25% to 40%, 30% to 40%, 35% to 40%, 18% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 18% to 30%, 20% to 30%, 25% to 30%, 18% to 25%, 20% to 25%, or 18% to 20%. Other GC content levels are also considered.

[0097] In some embodiments, the one or more target DNA sequence-specific primers include a melting temperature of 50°C to 64°C and a GC content of 18% to 65%.

[0098] In some embodiments, the method includes the concentration of one or more target DNA sequence-specific primers being at least 0.05 µM, at least 0.1 µM, at least 0.25 µM, at least 0.5 µM, at least 1 µM, at least 2.5 µM, or at least 5 µM. Other concentrations are also considered.

[0099] In some embodiments, the method includes the use of one or more target DNA sequence-specific primers at concentrations of less than 0.05 µM, less than 0.1 µM, less than 0.25 µM, less than 0.5 µM, less than 1 µM, less than 2.5 µM, or less than 5 µM. Other concentrations are also considered.

[0100] In some embodiments, the method includes using one or more target DNA sequence-specific primers at concentrations of 0.05 to 5 µM, 0.1 to 5 µM, 0.25 to 5 µM, 0.5 to 5 µM, 1 to 5 µM, 2.5 to 5 µM, 0.05 to 4 µM, 0.1 to 4 µM, 0.25 to 4 µM, 0.5 to 4 µM, 1 to 4 µM, 2.5 to 4 µM, and 0.05 to 2.5 µM. 0.1 to 2.5 µM, 0.25 to 2.5 µM, 0.5 to 2.5 µM, 1 to 2.5 µM, 0.05 to 1 µM, 0.1 to 1 µM, 0.25 to 1 µM, 0.5 to 1 µM, 0.05 to 0.5 µM, 0.1 to 0.5 µM, 0.25 to 0.5 µM, 0.05 to 0.25 µM, 0.1 to 0.25 µM, or 0.05 to 0.1 µM. Other concentrations are also considered.

[0101] In some embodiments, the method includes 0.05 to 4 µM of one or more target DNA sequence-specific primers.

[0102] In some embodiments, genomic DNA for amplification is provided. In some embodiments, the genomic DNA for amplification is provided via a DNA-containing cell. In some embodiments, the genomic DNA is released from the cell for amplification.

[0103] In some embodiments, the genomic DNA is substantially unpurified DNA. In some embodiments, the genomic DNA is lysed cellular genomic DNA. In some embodiments, the genomic DNA is derived from lysed cells. In some embodiments, the genomic DNA is substantially unpurified DNA. In some embodiments, the genomic DNA is partially purified or substantially purified.

[0104] In some embodiments, the method includes a cell lysis step. In some embodiments, the method includes a cell lysis step prior to amplification. Methods for lysing cells are known in the art.

[0105] In some embodiments, the method does not include a separate lysis step. In some embodiments, the method includes simultaneously lysing cells and amplifying genomic DNA. In some embodiments, amplification of genomic DNA from cells occurs without a separate lysis step. In some embodiments, genomic DNA from cells is made available for amplification without a separate lysis step.

[0106] In some implementations, genomic DNA from cells is made available for amplification as part of the amplification process.

[0107] In some embodiments, the method includes amplifying genomic DNA with one or more degenerate oligonucleotide primers for at least one or more cycles of amplification with target DNA-specific primers.

[0108] In some embodiments, the method includes amplification with one or more target DNA sequence-specific primers during amplification of genomic DNA with the one or more degenerate oligonucleotide primers.

[0109] In some implementations, the method includes adding one or more degenerate oligonucleotide primers and target DNA sequence-specific primers before amplifying genomic DNA.

[0110] In some embodiments, the method includes simultaneously adding one or more degenerate oligonucleotide primers and target DNA sequence-specific primers.

[0111] In some embodiments, the method includes adding one or more degenerate oligonucleotide primers, followed by adding target DNA sequence-specific primers.

[0112] In some embodiments, the method includes amplifying genomic DNA with one or more degenerate oligonucleotide primers, followed by amplifying the genomic DNA with the one or more degenerate oligonucleotide primers and the one or more target DNA sequence-specific primers.

[0113] In some embodiments, the method includes adding one or more target DNA sequence-specific primers to genomic DNA amplified with the one or more degenerate oligonucleotide primers, followed by amplification with the one or more target DNA sequence-specific primers and the one or more degenerate oligonucleotide primers.

[0114] In some embodiments, the method includes a polymerase chain reaction comprising one or more cycles of low-strictness amplification and one or more further cycles of higher-strictness amplification.

[0115] In some implementations, the method includes amplification with one or more degenerate oligonucleotide primers for one or more cycles of low-strictness amplification.

[0116] In some embodiments, the method includes amplification for one or more cycles of low-strength amplification using one or more degenerate oligonucleotide primers and target DNA sequence-specific primers.

[0117] In some embodiments, the method includes amplification with the one or more degenerate oligonucleotide primers for one or more cycles of low-strength amplification, and amplification with the one or more degenerate oligonucleotide primers and target DNA sequence-specific primers for one or more cycles of higher-strength amplification.

[0118] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles of annealing at a temperature below 35°C.

[0119] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers for one or more cycles of annealing at temperatures below 35°C.

[0120] In some embodiments, the amplification includes a polymerase chain reaction comprising 1 to 8 cycles of annealing at a temperature below 35°C.

[0121] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers for one to eight cycles of annealing at temperatures below 35°C.

[0122] Other temperature and cycling conditions are also considered.

[0123] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles of annealing at a temperature of 20°C to 35°C.

[0124] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers for one or more cycles of annealing at temperatures of 20°C to 35°C.

[0125] In some embodiments, the amplification includes a polymerase chain reaction comprising 1 to 8 cycles of annealing at temperatures of 20°C to 35°C. Other temperature and cycling conditions are also considered.

[0126] In some implementations, the method includes amplification with one or more degenerate oligonucleotide primers for 1 to 8 cycles at annealing temperatures of 20°C to 35°C.

[0127] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles with an annealing temperature above 48°C or 50°C.

[0128] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers and one or more target DNA sequence-specific primers for one or more cycles of annealing at temperatures above 48°C or 50°C.

[0129] In some embodiments, the amplification includes a polymerase chain reaction comprising 5 to 25 cycles with annealing temperatures above 48°C or 50°C. Other temperature and cycling conditions are also considered.

[0130] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers and one or more target DNA sequence-specific primers for 5 to 25 cycles of annealing at temperatures above 48°C or 50°C.

[0131] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles with an annealing temperature of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C.

[0132] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers and one or more target DNA sequence-specific primers for one or more cycles of annealing at one or more temperatures of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C.

[0133] In some embodiments, the amplification includes a polymerase chain reaction comprising 5 to 25 cycles at one of the following annealing temperatures: 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C. Other temperature and cycling conditions are also considered.

[0134] In some embodiments, the method includes amplification with one or more degenerate oligonucleotide primers and one or more target DNA sequence-specific primers for 5 to 25 cycles at one of the annealing temperatures of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C.

[0135] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles with an annealing temperature below 35°C and one or more further cycles with an annealing temperature above 50°C. Other temperature and cycling conditions are also considered.

[0136] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles of annealing at temperatures below 35°C with the one or more degenerate oligonucleotide primers and one or more further cycles of annealing at temperatures above 48°C or 50°C with the one or more degenerate oligonucleotide primers and the one or more target DNA-specific primers. Other temperature and cycling conditions are also considered.

[0137] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles of annealing at a temperature of 20°C to 35°C, and one or more further cycles of annealing at one of the following temperatures: 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C. Other temperatures are also considered.

[0138] In some embodiments, the amplification includes a polymerase chain reaction comprising one or more cycles of annealing at 20°C to 35°C with the one or more degenerate oligonucleotide primers and one or more further cycles of annealing at one of the following temperatures: 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C with the one or more degenerate oligonucleotide primers and the one or more target DNA-specific primers. Other temperatures are also considered.

[0139] In some embodiments, the amplification includes a polymerase chain reaction comprising 1 to 8 cycles of annealing at 20°C to 35°C, and 5 to 25 cycles of annealing at one of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C. Other temperature and cycling conditions are also considered.

[0140] In some embodiments, the amplification includes a polymerase chain reaction comprising 1 to 8 cycles of annealing at 20°C to 35°C with degenerate oligonucleotide primers and 5 to 25 cycles of annealing at one of the following temperatures: 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C with one or more degenerate oligonucleotide primers and one or more target DNA-specific primers.

[0141] Examples of polymerases are described in this article.

[0142] In some embodiments, the method includes using one or more of the following: Taq polymerase (available, for example, from New England Biolabs), Phi29 polymerase (available, for example, from New England Biolabs), Pfu DNA polymerase (available, for example, from Promega), Bst DNA polymerase (available, for example, from New England Biolabs), VentR™ and Deep VentR™ DNA polymerases (available, for example, from New England Biolabs), 9°Nm DNA polymerase (available, for example, from New England Biolabs), Herculase II Fusion DNA polymerase (available, for example, from Agilent Technologies), KAPA HiFi (available, for example, from Kapa Biosystems), KAPA2G Robust (available, for example, from Kapa Biosystems), the Klenow fragment of DNA polymerase I (available, for example, from New England Biolabs), and PhiPRD1 DNA polymerase (Jung et al., (1987)). Proc. Natl. Acad. Sci. USA84:8287), bacteriophage M2 DNA polymerase (Matsumoto et al. (1989) Gene 84:247), T4 DNA polymerase (which can be obtained, for example, from New England Biolabs) and T5 DNA polymerase (Chatterjee et al., 1991). Gene (97:13-19). Other types of polymerases are also considered and are commercially available. Suitable reaction conditions can be selected for a specific polymerase.

[0143] In some embodiments, the method includes using a polymerase having a processability of 1 to 50 nucleotides per second at 72°C. Polymerases with the desired processability are known in the art.

[0144] In some embodiments, the method includes using a template nucleotide having a density of at least 285 × 10⁻⁶ nucleotides per template nucleotide. -6 A polymerase with incorrect fidelity. In some embodiments, the method includes using a polymerase with a fidelity higher than 285 × 10⁻⁶ per template nucleotide. -6 A polymerase with incorrect fidelity. Polymerases with the desired fidelity are known in the art.

[0145] In some implementations, the method includes one or more rounds of further amplification of the amplified target DNA sequence.

[0146] In some embodiments, the further amplification in one or more rounds comprises 10 to 40 cycles. In some embodiments, the further amplification in one or more rounds comprises cycles with an annealing temperature of one of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C. In some embodiments, the further amplification in one or more rounds comprises 10 to 40 cycles with an annealing temperature of one of 48°C to 68°C, 52°C to 65°C, or 52°C to 62°C.

[0147] In some embodiments, the method includes one or more rounds of further amplification of the amplified target DNA sequence using one or more additional target DNA sequence-specific primers.

[0148] In some implementations, the additional target DNA sequence-specific primer is the same as the one or more target DNA sequence primers.

[0149] In some implementations, the additional target DNA sequence-specific primer is different from the one or more target DNA sequence primers.

[0150] In some implementations, the additional target DNA sequence primer is a nested primer.

[0151] Methods for designing and generating such additional primers (including nested primers) are known in the art.

[0152] In some implementations, the method includes amplifying two or more target DNA sequences.

[0153] In some embodiments, the method includes amplifying two or more target DNA sequences from genomic DNA by amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of primers specific to the two or more target DNA sequences.

[0154] In some embodiments, the method includes amplifying a target DNA sequence from genomic DNA by amplifying genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, and subsequently amplifying one or more other target DNA sequences.

[0155] In some embodiments, the method further includes evaluating the nucleotide content of the amplified target DNA sequence. Methods for evaluating nucleotide content are known in the art.

[0156] In some implementations, assessing nucleotide content includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation status, and / or epigenetic changes. Other methods for assessing nucleotide content are also considered.

[0157] In some implementations, evaluating nucleotide content includes sequencing all or part of the amplified target DNA sequence.

[0158] In some embodiments, the method further includes assessing nucleotide content by sequencing all or part of the amplified target DNA sequence, hybridizing the strands of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and one or more high-resolution melting analyses. Other methods are also considered.

[0159] In some implementations, this method can be used for genetic screening, preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, and / or forensic screening. Other uses are also considered.

[0160] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA described herein, which is performed by amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers.

[0161] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, thereby amplifying the target DNA sequence.

[0162] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying genomic DNA, the target DNA sequence is also amplified using one or more target DNA sequence-specific primers. This amplifies the target DNA sequence.

[0163] The amplification method is described herein. In some embodiments, the amplification includes a polymerase chain reaction, as described herein.

[0164] Some embodiments of this disclosure provide methods for amplifying target DNA sequences from genomic DNA using whole-genome amplification, as described herein.

[0165] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA by whole-genome amplification of the genomic DNA using one or more degenerate oligonucleotide primers; and for at least a portion of the whole-genome amplification, the target DNA sequence is further amplified using one or more target DNA sequence-specific primers.

[0166] Some embodiments of this disclosure provide a method for amplifying a target DNA sequence from genomic DNA, the method comprising: Whole-genome amplification of genomic DNA using one or more degenerate oligonucleotide primers; and For at least a portion of the whole genome amplification, the target DNA sequence is further amplified using one or more primers specific to the target DNA sequence. This amplifies the target DNA sequence.

[0167] In some implementations, the whole genome amplification includes amplification using polymerase chain reaction.

[0168] Methods for whole-genome amplification are described in this paper. For example, refer to Arneson N., Hughes S., Houlston R. and Done S. (2007) "PCR-Based Whole Genome Amplification," Chapter 18. PCRThe description of whole-genome amplification is found in (eds. Hughes and Moody). Scion Publishing Ltd., Oxfordshire, UK, 2007.

[0169] Some embodiments of this disclosure provide target DNA sequences amplified by the methods described herein.

[0170] In some embodiments, the target DNA sequence may also be amplified. In some embodiments, the target DNA sequence may also be amplified using the same primers. In some embodiments, the target DNA sequence may also be amplified using one or more other target DNA sequence-specific primers. Methods for amplification using one or more other / additional target DNA sequence-specific primers are described herein.

[0171] Some implementations provide the use of the target DNA sequence amplified as described herein.

[0172] Certain embodiments of this disclosure provide target DNA sequences for use in genetic screening, genetic diagnosis, preimplantation genetic screening, prenatal screening, disease screening, cancer screening, genetic marker screening, screening for polymorphisms, mutations, deletions, insertions, translocations, expansions, inversions, methylation states, and / or epigenetic alterations, and / or forensic screening. Other uses are also contemplated.

[0173] Certain embodiments of this disclosure provide methods for evaluating the nucleotide content of a target DNA sequence amplified from genomic DNA using the methods described herein. Methods for evaluating nucleotide content are described herein.

[0174] Some embodiments of this disclosure provide a method for assessing the nucleotide content of a target DNA sequence by evaluating the nucleotide content of a target DNA sequence amplified by the methods described herein.

[0175] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0176] Certain embodiments of this disclosure provide a method for assessing the nucleotide content of a target DNA sequence in genomic DNA, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, and assessing the nucleotide content of the amplified target DNA sequence in order to assess the nucleotide content of the target DNA sequence.

[0177] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying genomic DNA, the target DNA sequence is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0178] Certain embodiments of this disclosure provide a method for evaluating the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Whole-genome amplification of genomic DNA using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0179] Methods for assessing nucleotide content are described herein. In some embodiments, assessing nucleotide content includes determining all or part of the nucleotide sequence of the amplified target DNA.

[0180] In some implementations, methods for assessing nucleotide content include detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0181] In some implementations, methods for assessing nucleotide content include sequencing all or part of the amplified target DNA sequence, hybridizing the strands of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and one or more high-resolution melting analysis. Other methods are also considered.

[0182] In some implementations, the methods used to assess nucleotide content are used for genetic screening, genetic diagnosis, preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, and / or forensic screening. Other uses are also considered.

[0183] Some embodiments of this disclosure provide a method for determining all or part of a target DNA sequence in genomic DNA by amplifying the target DNA sequence as described herein and determining all or part of the nucleotide sequence of the amplified target DNA sequence.

[0184] Certain embodiments of this disclosure provide a method for determining the nucleotide sequence of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and Determine the nucleotide sequence of the amplified target DNA sequence. This allows us to determine the nucleotide sequence of the target DNA.

[0185] In some embodiments, the method includes determining the nucleotide sequence of substantially all nucleotides in the amplified target DNA sequence. In some embodiments, the method includes determining the nucleotide sequence of one or more nucleotides in the amplified target DNA sequence.

[0186] Methods for determining the nucleotide sequence of DNA are known in the art. Examples include dideoxy chain termination techniques, next-generation sequencing, shotgun sequencing, hybridization with one or more oligonucleotides, real-time PCR, and high-resolution melting analysis. Other methods are also considered.

[0187] In some implementations, the methods described herein are used for genetic screening or diagnosis.

[0188] Some embodiments of this disclosure provide methods for genetic screening or diagnosis by amplifying a target DNA sequence as described herein and evaluating the amplified target DNA sequence for screening or diagnosis.

[0189] Some embodiments of this disclosure provide methods for genetic screening or diagnosis of embryos. Genetic screening or diagnosis of other cells is also considered and described herein.

[0190] Certain embodiments of this disclosure provide a method for genetic screening of embryos, the method comprising: Genomic DNA from the embryo is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the embryo.

[0191] Methods for obtaining one or more cells from an embryo for analysis are known in the art. Methods for amplifying genomic DNA from an embryo using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce amplified target DNA sequences are described herein.

[0192] In some implementations, evaluating the amplified target DNA sequence includes determining the nucleotide content of the amplified target DNA sequence.

[0193] In some implementations, evaluating the amplified target DNA sequence includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0194] In some implementations, the evaluation includes one or more of the following: sequencing of all or part of the amplified target DNA sequence, strand hybridization of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and high-resolution melting analysis. Other methods are also considered.

[0195] In some implementations, the methods described herein are used for embryo screening, such as preimplantation genetic screening.

[0196] Some embodiments of this disclosure provide methods for preimplantation genetic screening of embryos, such as for suitability for implantation, presence of genetic diseases, and / or presence of desired and / or undesirable mutations. Other types of screening are also considered.

[0197] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the embryo.

[0198] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising amplifying a target DNA sequence from the embryo with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the embryo with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the embryo.

[0199] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying genomic DNA, the target DNA sequence from the embryo is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0200] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of embryos, the method comprising: Whole-genome amplification of genomic DNA from the embryo was performed using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence from the embryo is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the embryo.

[0201] In some implementations, the methods described herein are used for oocyte screening, such as genetic screening. For example, oocytes may be screened for their suitability for fertilization, implantation suitability, presence of genetic diseases, their mitochondrial contents or genome, and / or the presence of desired and / or undesirable mutations. Other types of screening are also considered.

[0202] Some embodiments of this disclosure provide a method for genetic screening of oocytes.

[0203] Methods for obtaining oocytes or their polar bodies for analysis are known in the art. Methods for amplifying genomic DNA from oocytes (or their polar bodies) using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce amplified target DNA sequences are described herein.

[0204] In some implementations, evaluating the amplified target DNA sequence includes determining the nucleotide content of the amplified target DNA sequence.

[0205] In some implementations, evaluating the amplified target DNA sequence includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0206] In some implementations, the evaluation includes one or more of the following: sequencing of all or part of the amplified target DNA sequence, strand hybridization of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and high-resolution melting analysis. Other methods are also considered.

[0207] In some implementations, the methods described herein are used for preimplantation genetic screening of oocytes.

[0208] In some implementations, this method is used to screen the fertilization suitability of oocytes.

[0209] Some embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes.

[0210] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Amplifying genomic DNA from an oocyte or its polar body using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the oocyte.

[0211] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising amplifying a target DNA sequence from the oocyte or its polar body with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the oocyte or polar body with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the oocyte.

[0212] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Genomic DNA from the oocyte or its polar body is amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying genomic DNA, the target DNA sequence from the oocyte or polar body is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0213] Certain embodiments of this disclosure provide a method for preimplantation genetic screening of oocytes, the method comprising: Whole-genome amplification of genomic DNA from the oocyte or its polar body using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence from the oocyte or polar body is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0214] The methods used for whole-genome amplification are described in this paper.

[0215] In some implementations, the methods described herein are used for sperm screening, such as genetic screening. For example, sperm may be screened for their suitability for fertilization, the presence of genetic diseases, and / or the presence of desired and / or unwanted mutations. Other types of screening are also considered.

[0216] Some embodiments of this disclosure provide methods for genetic screening of sperm.

[0217] Certain embodiments of this disclosure provide a method for genetic screening of sperm, the method comprising: Amplifying genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0218] Methods for obtaining sperm for analysis are known in the art. Methods for amplifying genomic DNA from sperm using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce amplified target DNA sequences are described herein.

[0219] In some implementations, evaluating the amplified target DNA sequence includes determining the nucleotide content of the amplified target DNA sequence.

[0220] In some implementations, evaluating the amplified target DNA sequence includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0221] In some implementations, the evaluation includes sequencing all or part of the amplified target DNA sequence, hybridizing the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and one or more high-resolution melting analysis.

[0222] Certain embodiments of this disclosure provide a method for genetic screening of sperm, the method comprising amplifying a target DNA sequence from one or more sperm using one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the sperm.

[0223] Certain embodiments of this disclosure provide a method for genetic screening of sperm, the method comprising: Amplification of genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplified genomic DNA, the target DNA sequence from the one or more sperm cells is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0224] Some embodiments of this disclosure provide a method for screening sperm, the method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence from the one or more sperm cells is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0225] In some implementations, the methods described herein are used to screen sperm for fertilization suitability.

[0226] In some implementations, the methods described herein are used for fetal screening, such as genetic screening. For example, fetal oocytes may be screened for the presence of genetic diseases, genetic markers, chromosomal content, and / or desired and / or unwanted mutations. Other types of screening are also considered.

[0227] Some embodiments of this disclosure provide methods for screening fetuses.

[0228] Some embodiments of this disclosure provide methods for prenatal screening of the fetus.

[0229] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Genomic DNA from the fetus is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0230] Methods for obtaining genomic DNA from a fetus are known in the art, including amniocentesis, chorionic villus sampling, and obtaining cell-free DNA. Methods for amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce amplified target DNA sequences are described herein.

[0231] In some implementations, evaluating the amplified target DNA sequence includes determining the nucleotide content of the amplified target DNA sequence.

[0232] In some implementations, evaluating the amplified target DNA sequence includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0233] In some implementations, the evaluation includes sequencing all or part of the amplified target DNA sequence, hybridizing the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and one or more high-resolution melting analysis.

[0234] In some implementations, the methods described herein are used to screen for genetic diseases in the fetus.

[0235] Certain embodiments of this disclosure provide a method for prenatal genetic screening of a fetus, the method comprising amplifying a target DNA sequence from the fetus with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the fetus with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the fetus.

[0236] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Genomic DNA from the fetus was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying genomic DNA, the target DNA sequence from the fetus is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0237] Some embodiments of this disclosure provide a method for prenatal screening of the fetus, the method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; For at least a portion of the whole genome amplification, the target DNA sequence from the fetus is further amplified using one or more target DNA sequence-specific primers to produce the amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0238] Some embodiments of this disclosure provide kits for performing the methods described herein.

[0239] In some embodiments, the kit includes one or more components described herein, such as buffers, reagents, enzymes, solutions, dNTPs, controls, and / or instructions for use.

[0240] For example, a kit for amplifying a target DNA sequence may contain one or more degenerate oligonucleotide primers for whole-genome amplification, one or more primers for amplifying the target DNA sequence, and optionally one or more reagents and / or instructions for amplifying the target DNA sequence during whole-genome amplification.

[0241] Some embodiments of this disclosure provide a combined product comprising one or more components described herein.

[0242] In some implementations, combined products are used to perform the methods described herein.

[0243] Some embodiments of this disclosure provide a combined product comprising the following components: (i) One or more degenerate oligonucleotide primers used for whole genome amplification; (ii) One or more primers for amplifying the target DNA sequence; and (iii) One or more reagents and / or instructions for use in amplifying the target DNA sequence during whole-genome amplification.

[0244] In some embodiments, the combined product may optionally also include one or more components described herein, such as buffers, reagents, enzymes, solutions, dNTPs, controls, and / or instructions for use.

[0245] Standard techniques and equipment can be used for recombinant DNA technology, oligonucleotide synthesis, molecular biology, and enzymatic reactions. The aforementioned techniques and operations can generally be performed according to methods known in the art and / or commercially available, and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) and Ausubel et al., Current Protocols in Molecular Biology (2003) John Wiley & Sons, which are incorporated herein by reference.

[0246] This disclosure also describes the following aspects. These items form part of the disclosure of this specification and are used to further illustrate alternative embodiments of this disclosure. Unless the context expressly specifies otherwise, any technical feature described in the following items may be combined with any embodiment, aspect, feature, step, component, or use described in other parts of this specification.

[0247] 1. A method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, thereby amplifying the target DNA sequence.

[0248] 2. The method of item 1, wherein the amplification comprises a polymerase chain reaction.

[0249] 3. The method of claim 1 or 2, wherein the method comprises amplifying the genomic DNA with the one or more degenerate oligonucleotide primers, and subsequently amplifying the genomic DNA with the one or more degenerate oligonucleotide primers and the one or more target DNA sequence-specific primers.

[0250] 4. The method of claim 3, wherein the method comprises adding the one or more target DNA sequence-specific primers to genomic DNA amplified with the one or more degenerate oligonucleotide primers, and subsequently amplifying with the one or more target DNA sequence-specific primers and the one or more degenerate oligonucleotide primers.

[0251] 5. The method of any one of claims 1 to 4, wherein the one or more degenerate oligonucleotide primers comprise nucleotide sequences of the same general formula.

[0252] 6. The method of any one of claims 1 to 5, wherein the one or more degenerate oligonucleotide primers comprise a nucleotide sequence of 6 to 12 consecutive random nucleotides.

[0253] 7. The method of any one of claims 2 to 6, wherein the method comprises a polymerase chain reaction, the polymerase chain reaction comprising one or more cycles of low-strength amplification and one or more cycles of further higher-strength amplification.

[0254] 8. The method of claim 7, wherein the method comprises amplifying with the one or more degenerate oligonucleotide primers for the one or more cycles of low-strength amplification, and amplifying with the one or more degenerate oligonucleotide primers and the target DNA sequence-specific primers for the one or more cycles of higher-strength amplification.

[0255] 9. The method of any one of claims 1 to 8, wherein the method comprises a polymerase chain reaction, the polymerase chain reaction comprising one or more cycles of annealing at a temperature below 35°C and one or more further cycles of annealing at a temperature above 50°C.

[0256] 10. The method of any one of claims 1 to 9, wherein the method comprises a polymerase chain reaction, the polymerase chain reaction comprising one or more cycles of annealing at a temperature of 20°C to 35°C for 1 to 8 cycles and one or more further cycles of annealing at a temperature of 52°C to 65°C for 5 to 25 cycles.

[0257] 11. The method of any one of claims 1 to 10, wherein the one or more target DNA sequence-specific primers comprise a melting temperature of 50°C to 64°C and a GC content of 18% to 65%.

[0258] 12. The method of any one of claims 1 to 11, wherein the method comprises an amount of 1 pg to 6 ng of genomic DNA to be amplified.

[0259] 13. The method of any one of claims 1 to 12, wherein the method comprises one or more degenerate oligonucleotide primers at a concentration of 0.1 to 4 µM.

[0260] 14. The method of any one of claims 1 to 13, wherein the method comprises one or more target DNA-specific primers at a concentration of 0.05 to 4 µM.

[0261] 15. The method of any one of claims 1 to 14, wherein the method comprises further amplifying the amplified target DNA sequence by one or more rounds using additional target DNA sequence-specific primers.

[0262] 16. The method of claim 15, wherein the additional target DNA sequence-specific primer is different from the one or more target DNA sequence primers.

[0263] 17. The method of claim 16, wherein the additional target DNA sequence primer is a nested primer.

[0264] 18. The method of any one of claims 1 to 17, wherein the method comprises amplifying two or more target DNA sequences.

[0265] 19. The method of any one of claims 1 to 18, wherein the genomic DNA comprises DNA from one or more embryonic cells, DNA from an oocyte or its polar body, DNA from a sperm, DNA from one or more germ cells, DNA from one or more individual cells, DNA from one or more human or animal cells, DNA from one or more plant cells, DNA from one or more microbial cells, DNA from one or more cells used for disease screening, DNA from one or more cancer cells, DNA for forensic testing, mitochondrial DNA, extrachromosomal DNA, cell-free DNA, DNA from a sample comprising one or more cells, DNA from a sample comprising cell-free DNA, DNA from cell lysates, DNA from a biopsy, DNA from a tissue sample, DNA from a cell sample, DNA from a body fluid, DNA from a blood sample, DNA from amniotic fluid, DNA from a formalin-fixed sample, DNA from a paraffin-embedded sample, and DNA from a swab.

[0266] 20. The method of any one of claims 1 to 19, wherein the genomic DNA comprises DNA from 1 to 50 cells.

[0267] 21. The method of any one of claims 1 to 20, wherein the genomic DNA comprises DNA derived from a single cell.

[0268] 22. The method of any one of claims 1 to 21, wherein the method further comprises evaluating the nucleotide content of the amplified target DNA sequence.

[0269] 23. The method of claim 22, wherein the assessment of nucleotide content includes detecting the presence of polymorphisms, mutations, deletions, insertions, translocations, methylation states, and / or epigenetic changes.

[0270] 24. The method of any one of claims 22 or 23, wherein the method comprises one or more of sequencing all or part of the amplified target DNA sequence, hybridizing the strand of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and high-resolution melting analysis.

[0271] 25. A method for amplifying a target DNA sequence from a genome, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, thereby amplifying the target DNA sequence.

[0272] 26. A method for amplifying a target DNA sequence from genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence is also amplified using one or more target DNA sequence-specific primers. This amplifies the target DNA sequence.

[0273] 27. The method of claim 25 or 26, wherein the amplification comprises a polymerase chain reaction.

[0274] 28. A method for amplifying a target DNA sequence from genomic DNA, the method comprising: Whole-genome amplification of the genomic DNA using one or more degenerate oligonucleotide primers; and At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to amplify the target DNA sequence. This amplifies the target DNA sequence.

[0275] 29. The method of claim 28, wherein the whole genome amplification comprises amplification using polymerase chain reaction.

[0276] 30. The method of any one of claims 1 to 29, wherein the method is used for preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, and / or forensic screening.

[0277] 31. The target DNA sequence amplified by any of the methods in items 1 to 29.

[0278] 32. A method for evaluating the nucleotide content of a target DNA sequence amplified from genomic DNA, the method comprising evaluating the nucleotide content of the target DNA sequence of item 31.

[0279] 33. A method for assessing the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0280] 34. A method for assessing the nucleotide content of a target DNA sequence in genomic DNA, the method comprising amplifying the target DNA sequence with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA with one or more degenerate oligonucleotide primers, and assessing the nucleotide content of the amplified target DNA sequence in order to assess the nucleotide content of the target DNA sequence.

[0281] 35. A method for assessing the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Amplifying genomic DNA using one or more degenerate oligonucleotide primers; and For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0282] 36. A method for assessing the nucleotide content of a target DNA sequence in genomic DNA, the method comprising: Whole-genome amplification of the genomic DNA was performed using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The nucleotide content of the amplified target DNA sequence is evaluated in order to assess the nucleotide content of the target DNA sequence.

[0283] 37. The method of any one of claims 32 to 36, wherein the assessment of nucleotide content comprises determining the nucleotide content of the amplified target DNA.

[0284] 38. The method of any one of claims 32 to 37, wherein the method includes detecting the presence of polymorphism, mutation, deletion, insertion, translocation, methylation status, and / or epigenetic changes.

[0285] 39. The method of any one of claims 32 to 38, wherein the method comprises one or more of sequencing all or part of the amplified target DNA sequence, hybridizing the strand of the amplified target DNA sequence to one or more oligonucleotides, real-time PCR, and high-resolution melting analysis.

[0286] 40. The method of any one of claims 32 to 39, wherein the method is used for preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, and / or forensic screening.

[0287] 41. A method for determining the nucleotide sequence of a target DNA sequence in genomic DNA, the method comprising: Amplifying the genomic DNA with one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and Determine the nucleotide sequence of the amplified target DNA sequence. This determines the nucleotide sequence of the target DNA sequence.

[0288] 42. A method for preimplantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0289] 43. A method for preimplantation genetic screening of embryos, the method comprising amplifying a target DNA sequence from the embryo with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the embryo with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the embryo.

[0290] 44. A method for preimplantation genetic screening of embryos, the method comprising: Genomic DNA from the embryo was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the embryo is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0291] 45. A method for preimplantation genetic screening of embryos, the method comprising: Whole-genome amplification of genomic DNA from the embryo was performed using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers from the embryo to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the embryos.

[0292] 46. ​​A method for preimplantation genetic screening of oocytes, the method comprising: Amplifying genomic DNA from an oocyte or its polar body using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0293] 47. A method for preimplantation genetic screening of oocytes, the method comprising amplifying a target DNA sequence from the oocyte or its polar body with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the oocyte or polar body with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the oocyte.

[0294] 48. A method for preimplantation genetic screening of oocytes, the method comprising: Genomic DNA from the oocyte or its polar body is amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the oocyte or polar body is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0295] 49. A method for preimplantation genetic screening of oocytes, the method comprising: Whole-genome amplification of genomic DNA from the oocyte or its polar body was performed using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers from the oocyte or polar body to produce the amplified target DNA sequence; and The amplified target DNA sequence is evaluated to screen the oocytes.

[0296] 50. The method of any one of claims 46 to 50, wherein the method is used to screen the fertilization suitability of the oocyte.

[0297] 51. A method for screening sperm, the method comprising: Amplifying genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0298] 52. A method for screening sperm, the method comprising amplifying a target DNA sequence from the one or more sperm with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the one or more sperm with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the sperm.

[0299] 53. A method for screening sperm, the method comprising: Amplification of genomic DNA from one or more sperm using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, target DNA sequences from the one or more sperm cells are further amplified using one or more target DNA sequence-specific primers to produce amplified target DNA sequences; and The amplified target DNA sequence was evaluated to screen the sperm.

[0300] 54. A method for screening sperm, the method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers from the one or more sperm cells to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the sperm.

[0301] 55. The method of any one of claims 51 to 54, wherein the method is used to screen the fertilization suitability of the sperm.

[0302] 56. A method for prenatal screening of the fetus, said method comprising: Genomic DNA from the fetus is amplified using one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0303] 57. A method for prenatal screening of a fetus, the method comprising amplifying a target DNA sequence from the fetus with one or more target DNA sequence-specific primers for at least one or more cycles of amplifying genomic DNA from the fetus with one or more degenerate oligonucleotide primers to produce an amplified target DNA sequence, and evaluating the amplified target DNA sequence to screen the fetus.

[0304] 58. A method for prenatal screening of the fetus, said method comprising: Genomic DNA from the fetus was amplified using one or more degenerate oligonucleotide primers; For at least one or more cycles of amplifying the genomic DNA, the target DNA sequence from the fetus is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0305] 59. A method for prenatal screening of the fetus, said method comprising: Whole-genome amplification of genomic DNA from one or more sperm cells using one or more degenerate oligonucleotide primers; At least a portion of the whole genome amplification is further amplified using one or more target DNA sequence-specific primers to produce an amplified target DNA sequence; and The amplified target DNA sequence was evaluated to screen the fetus.

[0306] 60. The method of any one of claims 56 to 59, wherein the method is used to screen the fetus for genetic diseases.

[0307] 61. A kit for performing any one of the methods in items 1 to 30 or 32 to 60.

[0308] 62. The target DNA sequence of item 31 may be used for preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, disease screening, cancer screening, genetic marker screening, screening for polymorphisms, mutations, deletions, insertions, translocations, expansions, inversions, methylation status, and / or epigenetic alterations, and / or forensic screening.

[0309] 63. A combined product, said product comprising the following components: (i) One or more degenerate oligonucleotide primers used for whole genome amplification; (ii) One or more primers for amplifying the target DNA sequence; and (iii) One or more reagents and / or instructions for amplifying the target DNA sequence during whole-genome amplification.

[0310] The present disclosure is further described through the following embodiments. It should be understood that the following description is for the purpose of describing specific embodiments only and is not intended to limit the above description.

[0311] Example Example 1 - Whole Genome Amplification and Gene Targeting Procedure The following procedures are used for the whole-genome amplification (WGA) and gene targeting studies described in this article.

[0312] 1. Materials and Methods First round of whole-genome amplification and gene targeting procedures: (i) Add 1 μl of cell lysin (thermosin; EmbryoCellect) to 6.5 μl of PCR-grade H2O. TM The kit (RHS; number RHS1001) is called cell lysin Dil 1.

[0313] (ii) Generate a cell lysis mixture by mixing the reagents in Table 1: Table 1

[0314] (iii) Add 3 µl of cell lysis mixture to the cell or DNA tube. Tap the solution to the bottom of the tube or briefly centrifuge. Do not vortex.

[0315] Therefore, a single cell typically contains about 6 pg of DNA.

[0316] (iv) Incubate the samples in a thermal cycler using the parameters in Table 2: Table 2

[0317] (v) Prepare the mastermix in the required quantity of samples as given in Table 3, using the listed commercial reagents: Table 3

[0318] (vi) Other suitable WGA DNA polymerases are described in this paper. For example, Cat. # 600675, KAPAHiFi; Kapa Biosystems Cat # KK2101, KAPA2G Robust PCR kit; Kapa Biosystems Cat. # KK5004, Herculase II Fusion DNA polymerase (Cat # 600675); and AmpliTaq DNA polymerase ThermoFisher Scientific Cat. # N8080171.

[0319] (vii) Aliquot 22 µL of the master mixture into PCR tubes containing genomic DNA or lysed cells.

[0320] (viii) Run the sample according to the procedure in Table 4: Table 4

[0321] (ix) Maintaining the temperature at 4°C allows for the addition of gene-specific primers to the PCR.

[0322] (x) Dilute the gene-specific primers to the required working stock concentrations (10 μM, 5 μM, 2 μM) using PCR-grade H2O.

[0323] (xi) During the 4°C holding period, while the samples are on ice: add 1.4 μL of forward gene-specific primer and 1.4 μL of reverse gene-specific primer to each tube. Briefly centrifuge the sample tubes and return them to the thermal cycler.

[0324] (xii) Run the sample according to the procedure shown in Table 5: Table 5

[0325] (xiii) The amplification products were observed by running at 100 v for 30 minutes on a 1% agarose gel.

[0326] Second round of gene-specific PCR procedure: (i) Dilute the PCR products generated in the first round of PCR with PCR-grade water by 1 / 50 to a concentration of approximately 1 ng / μL.

[0327] (ii) Prepare the main mixture given in Table 6: Table 6

[0328] (iii) Use the KAPA2G Robust kit with dNTPS (Kapa Biosystems, USA, MA; product number KK5004 or KK5005) to specifically amplify the target DNA.

[0329] (iv) Distribute the master mixture (24 µL) equally into each PCR tube.

[0330] (v) Add 1 μL of template DNA diluted 1 / 50.

[0331] (vi) Run the PCR program for the samples as shown in Table 7: Table 7

[0332] (vii) The amplification products were observed by running at 100 v for 30 minutes on a 2% agarose gel.

[0333] Table 8. Gene-specific primers used:

[0334] (viii) Final master mixture concentration (total volume 27.8 μL) for each forward and reverse primer in 25 μL solution + 2.8 μL additive: (ix) 10μM working stock solution: 0.5035μM; 5μM working stock solution: 0.2518μM; 2μM working stock solution: 0.1007μM.

[0335] If necessary, the DNA sequence of the amplified product can be determined.

[0336] One or more of the above reagents may be included in a kit or combination product, which may also include instructions for use.

[0337] As described above, amplification of target DNA sequences has various uses, such as preimplantation genetic screening, preimplantation genetic diagnosis, prenatal screening, genetic screening, disease screening, cancer screening, genetic marker screening, and / or forensic screening.

[0338] Example 2 - Validation of target gene enrichment in WGA PCR using single cells To conduct research to validate the procedures used for enriching target genes.

[0339] Two sets of gene-specific primers (P) (0.5 μM, 0.25 μM, or 0.1 μM concentrations) were added to the WGA (multiplex PCR); Figure 1 and Figure 2The first round of whole-genome amplification and gene targeting was performed using single cells manually sorted from commercially available cell lines (Coriell; 47,XY,+15 or 48,XXY,+21) as described in Example 1.

[0340] As observed, the amplification process resulted in whole-genome amplification of genomic DNA and specific amplification of the target gene, with amplification of the target gene observed at the selected primer concentrations tested. It was noted that WGA amplification was slightly weaker in the presence of the gene-specific primer (P) than with control WGA. These results confirm the ability to amplify target genes from single cells using overlapping WGA and gene-specific amplification.

[0341] The data also confirms that primer concentrations of at least 0.1 µM can be used to amplify target genes from single cells.

[0342] To evaluate whole-genome amplification, WGA samples were hybridized using an EmbryoCellect™ array according to the EmbryoCellect™ TDS protocol to confirm the karyotype of individual cells. Correct aneuploidy results were obtained for all individual cells tested (Table 9).

[0343] Table 9: Single-cell samples after whole-genome amplification combined with DNA gene-targeted amplification EmbryoCellect™ array results

[0344] The presence of the target gene was then tested in the WGA product using gene-specific PCR. Gene enrichment was observed when the correct size band was produced compared to the missing band in the control. Figure 3 and Figure 4 ).

[0345] These results confirm that the target sequence can be amplified during whole-genome amplification.

[0346] The ability to amplify the target sequence during whole-genome amplification offers several advantages. For example, the ability to amplify the target sequence provides one or more of the following advantages: (i) reduced allele decoupling when using whole-genome amplification, (ii) improved ability to analyze the target DNA sequence amplified from whole-genome amplified DNA, (iii) reduced bias associated with the amplification of the target DNA sequence amplified from whole-genome amplified DNA, (iv) reduced number of PCR cycles required to amplify the target DNA sequence from genomic DNA, (v) improved time for amplifying specific target DNA sequences from whole-genome amplified DNA, and (vi) reduced use of consumables and / or human resources and / or lower costs.

[0347] Example 3 - Target Sequence Enrichment (TSE) of HBB Gene To further confirm that the procedure can effectively amplify other target sequences, and to evaluate the use of the procedure in genetic screening and / or diagnosis, such as for preimplantation genetic diagnosis and preimplantation genetic screening, an analysis was performed on the enrichment of target sequences of the HBB gene encoding β-globin using the procedure.

[0348] Multicellular samples (5-cells) were used for combined whole-genome amplification and HBB gene-targeted amplification using two primer sets. The PCR products were then seeded into two HBB-specific PCRs; major exons 1 and 2 were amplified, and exon 3 was amplified. The second-round HBB PCR amplicons were combined with the DNA from the whole-genome amplification with HBB gene-targeted amplification to generate a 1:20 final dilution. Samples containing only WGA PCR products, combined whole-genome amplification and HBB gene-targeted amplification products, and the 1:20 diluted combined DNA were sequenced in a 40-sample multiplex MiSeq run; read length 2 × 75 bp. Next-generation sequencing data were bioinformatically aligned to hg19. The breadth and depth of HBB coverage were calculated for each sample and compared. An average coverage depth >60x was obtained when sequencing the 1:20 diluted combined DNA.

[0349] Figure 5 This figure shows the breadth of HBB coverage after combined whole-genome amplification and gene-targeted amplification, compared to whole-genome amplification alone, using next-generation sequencing. After combined whole-genome amplification and gene-targeted amplification, the products were seeded into two gene-specific HBB PCRs. The second-round PCR amplicons were merged with the DNA from the whole-genome amplification with gene-targeted amplification to generate a 1:20 final dilution. The combined whole-genome amplification and gene-targeted amplification products (WGA + TSE) and the 1:20 dilution merge were sequenced. Next-generation sequencing data were bioinformatically aligned to hg19, and coverage was determined for each sample at a depth of 1×. The figure shows that 100% HBB coverage was achieved when the 1:20 dilution of the second-round HBB PCR gene-specific products was merged back into the combined whole-genome amplification with gene-targeted amplification. Mean values ​​are plotted.

[0350] Figure 6 Analysis of HBB gene coverage depth is shown. An average HBB coverage depth of >60× was obtained when sequencing the pooled DNA at a 1:20 dilution. This is a suitable depth for genetic analysis used for SNP determination.

[0351] Combined whole-genome amplification and sequence-specific amplification can be used to achieve 100% coverage of the target DNA sequence and increase coverage depth, making it suitable for genetic analysis for SNP detection.

[0352] Furthermore, we have demonstrated successful multiplex amplification with up to four primer sets in related experiments.

[0353] Example 4 - Target sequence enrichment for genes in major histocompatibility complex, class I, and class A To further evaluate the effectiveness of this protocol in amplifying other target sequences, and to further evaluate its use in genetic screening and / or diagnosis, such as preimplantation genetic diagnosis and preimplantation genetic screening, an analysis was conducted on the enrichment of target sequences encoding major histocompatibility complex, class I and A proteins using this protocol.

[0354] Multicellular samples (5-cells) were used for combined whole-genome amplification and HLA gene-targeted amplification. The PCR product was then seeded into a gene-specific HLA PCR. This second-round HLA PCR amplicons were merged with the DNA from the whole-genome amplification with HLA gene-targeted amplification to generate 1:20 and 1:10 final dilutions. Samples containing only WGA PCR products, combined whole-genome amplification and HLA gene-targeted amplification products, the merged DNA from the 1:20 and 1:10 dilutions, and the second-round HLA PCR amplicons were sequenced in a 40-sample multiplex MiSeq run; read length 2 × 75 bp. Next-generation sequencing data were bioinformatically aligned to hg19. HLA breadth and depth were calculated for each sample and compared. An average HLA coverage depth >60 × 10⁻⁶ was obtained when sequencing the merged DNA from the 1:20 and 1:10 dilutions and the second-round PCR amplicons.

[0355] Figure 7 This study demonstrates the breadth of HLA coverage achieved using next-generation sequencing after combined whole-genome amplification and gene-targeted amplification, compared to whole-genome amplification alone. Following combined whole-genome amplification and gene-targeted amplification, the products were seeded into an HLA-specific PCR. This second-round PCR amplicons were merged with the DNA from the whole-genome amplification with gene-targeted amplification to generate 1:20 and 1:10 final dilutions. Sequencing was performed on the PCR products from WGA alone, the products from combined whole-genome amplification and gene-targeted amplification, the merged DNA from the 1:20 and 1:10 dilutions, and the second-round PCR amplicons. Next-generation sequencing data were bioinformatically aligned to hg19, and exon 2 and exon 3 coverage was determined for each sample at a depth of 1×. An average coverage depth of >60× was obtained when sequencing the merged DNA from the 1:20 and 1:10 dilutions, as well as the second-round PCR amplicons.

[0356] Figure 8 The analysis shows the coverage depth of HLA genes.

[0357] When the merged DNA from the 1:20 and 1:10 dilutions was sequenced, along with the second round of HLA PCR amplicon sequencing, an average coverage depth of >60× was obtained. This is a suitable depth for genetic analysis used for SNP detection.

[0358] Example 5 - PGS of Embryos One use of the target gene enrichment protocol described in this article is for preimplantation genetic screening before embryo transfer into the uterus.

[0359] This screening requires the removal of one to several cells from an embryo via biopsy. Methods for removing cells from an embryo are known in the art. These cells are then placed in a tube, and because the biopsy material contains a limited amount of DNA, this DNA needs to be amplified to generate sufficient DNA for downstream techniques.

[0360] This targeted sequence enrichment protocol can then be used to identify chromosome complements in embryos. Furthermore, the targeted sequence enrichment protocol can be used for preimplantation genetic screening of specific genes and / or other target loci.

[0361] Example 6 - General Procedures, Reagent Kits, and Products The general procedures, components, and instructions for use with various kits for targeted sequence enrichment are as follows: 1. Reagent kit contents Kits for targeted sequence enrichment may contain the following components: PCR grade H2O Cell Lysis Enzyme Cell Lysis Buffer polymerase PCR buffer DOP primers 2. Overview of the Procedure Whole-genome amplification (WGA) based on degenerate oligonucleotide primer PCR (DOP-PCR) generates representative amplifications from total DNA or their DNA equivalents from single cells. This DOP-PCR-based WGA can reproducibly amplify total DNA from single cells, producing micrograms of amplified DNA in less than 3 hours. This procedure has been successfully used for both cellular and purified genomic DNA input.

[0362] This procedure involves the following general steps: (i) Sample collection Place the sample into a PCR tube and add <2 μl of buffer. Mark the sample location on the tube. (ii) Pyrolysis Add 3 μl of lysis solution above the sample. Gently tap the PCR tube to allow the lysis solution to swirl on the sample. Incubate for 15 minutes according to the pyrolysis procedure. It should be understood that in some cases the cell lysis step can be omitted, for example, to allow the procedure to be used on cell-free DNA, or by using cells directly into the amplification procedure without a separate lysis step before amplification.

[0363] (iii) Genome-wide amplification and targeted sequence enrichment Add 22 μl of PCR master mixture to the lysed sample PCR was performed using the WGA PCR program and sequence-specific primers. WGA was evaluated by agarose gel electrophoresis. 3. Input Instructions (i) Cell count The procedures described herein are suitable for single cells or their DNA equivalents, as well as small numbers of cells (e.g., <10 cells).

[0364] (ii) Cell collection methods Flow cytometry sorting, dilution, and micromanipulation are collection methods compatible with this procedure. Single cells should be transferred to PCR tubes with the minimum amount of transfer buffer (<2 μl). The position of cells within the tube should be marked with dots on the outside of the tube using a permanent marker to facilitate cell localization during the lysis step.

[0365] (iii) DNA dilution It is recommended to dilute the DNA to a final concentration of 30 pg / μl in 10 mM Tris-HCl (pH 8.0) (without EDTA).

[0366] (iv) Compatible buffer solution Recommended cell transfer buffers include 10 mM Tris-HCl (pH 8.0) (EDTA-free) and PBS (Mg-free). 2 + Ca2+ And without BSA).

[0367] 4. Procedure; DOP-PCR whole genome amplification (i) Cell lysis In this optional step, cells are lysed and DNA becomes soluble by adding a cell lysis mixture and briefly incubating in a PCR thermal cycler.

[0368] Consumables: 0.2 mL or 0.5 mL sterile PCR tubes PCR grade H2O Cell Lysis Enzyme Cell Lysis Buffer Cell or DNA sample preparation: Remove PCR-grade H2O and cell lysis buffer from storage and thaw to room temperature.

[0369] Remove the cell lysin from the storage area and store it in a 4°C cold block.

[0370] Mix the reagents thoroughly, then briefly centrifuge to collect the contents at the bottom of the tube.

[0371] Calculate the required reagent volume for the Cell Lysis Mix.

[0372] process: Cell Lysis Enzyme Dilution 1 was prepared by combining the following reagents in a 4°C cold block:

[0373] Mix thoroughly, then briefly centrifuge. Cell lysis mixtures for the required number of reactions were prepared by combining the following reagents in a 4°C cold block.

[0374]

[0375] Steps for lysing single-cell / multi-cell samples: (a) Add 3 μl of cell lysis mixture above the cell sample in the PCR tube. Ensure the lysis mixture rolls over the marked sample location on the tube by gently tapping the tube on the lab bench. Do not mix or vortex.

[0376] (b) If it is necessary to collect the contents at the bottom of the tube, briefly centrifuge in a microcentrifuge.

[0377] (c) Repeat with other samples.

[0378] Steps for preparing template-free control (NTC): (a) Add 3 μl of cell lysis mixture to a sterile PCR tube labeled with NTC.

[0379] (b) Add 1 μl of PCR-grade H2O to the tube labeled with NTC.

[0380] Steps for DNA sample preparation prior to WGA PCR (if required): (a) Add 3 μl of cell lysis mixture to the required number of sterile empty PCR tubes.

[0381] (b) Add 1 μl of 30 pg / μl DNA sample to each tube containing the cell lysis mixture. Proceed to the next step.

[0382] Incubate all samples and NTC in a thermal cycler programmed as follows:

[0383] Place all samples inside a cold block.

[0384] (ii) Genome-wide amplification and targeted sequence enrichment In this step, a master mixture is generated and added to the lysed sample prior to whole-genome amplification (WGA) using degenerate oligonucleotide primer PCR (DOP-PCR). This generates a representative amplification of total DNA from cells or their DNA equivalents.

[0385] Consumables: 1.5mL tube PCR grade H2O WGA PCR buffer DOP primers WGA polymerase Lysed samples and NTCs (from cell lysis) Sequence-specific primers It is possible to use a variety of different DOP primers. Examples of DOP primers include those whose portion of the nucleotide sequence allows for the possibility of having more than one base at a specific position, and DOP primers that can form base pairs with one or more other bases.

[0386] preparation: Dilute the sequence-specific primers to the required concentration in the PCR-grade H2O provided by the user.

[0387] Remove PCR-grade H2O, WGA PCR buffer, and primers from storage and thaw to room temperature.

[0388] Remove the WGA polymerase from the storage area and store it in a 4°C cold block.

[0389] Mix the reagents thoroughly, then briefly centrifuge to collect the contents at the bottom of the tube.

[0390] Calculate the reagent volume required for the WGA master mixture.

[0391] process: Prepare the WGA master mixture for the required amount of reaction by combining the following reagents in the order listed in a 1.5 mL tube.

[0392]

[0393] Mix thoroughly, then briefly spin in a microcentrifuge.

[0394] Transfer 22 μl of the PCR master mixture to individual tubes containing the lysed template (sample or NTC from the cell lysis mixture). To prevent any DNA from being extracted from the sample, do not insert the pipette tip into the lysed sample mixture. Do not mix or vortex the PCR tube. Briefly centrifuge or rotate the tube in a microcentrifuge to collect the contents at the bottom of the tube.

[0395] Transfer 22 μl of the PCR master mixture to each tube containing the DNA template (in the cell lysis mixture). To prevent any DNA from being extracted from the sample, do not insert the pipette tip into the lysed sample mixture. Mix thoroughly, then briefly centrifuge to collect the contents at the bottom of the tube.

[0396] Incubate all samples and NTC in a thermal cycler programmed as follows:

[0397] (iii) Steps for diluting sequence-specific primers before WGA with TSE PCR process: It is recommended to use a 30pg gDNA template to optimize each primer set used in WGA PCR.

[0398] Dilute the sequence-specific primers to the required working stock concentration using PCR-grade H2O.

[0399] As a guideline, primer concentrations were titrated using working stock concentrations of 2 μM, 5 μM, and 10 μM. Sequence enrichment was confirmed using semi-quantitative PCR (a low number of PCR cycles, where the target band is just visible on the gel; do not run PCR to the plateau phase) or alternative methods to determine the optimal working stock primer concentrations.

[0400] As an example, nested PCR targeting the target region can be used to confirm that target sequence enrichment has occurred.

[0401] Running the WGA product on an agarose gel ensures that the PCR amplicons are of typical size and quantity for DOPlify™ WGA, and also provides confirmation that the optimal primer concentration has been used.

[0402] During the hold step of the PCR procedure, transfer 1.4 μl of the 5' (forward) primer and 1.4 μl of the 3' (reverse) primer to each tube containing the DNA template in the WGA mixture. The total volume of all primers added to the multiplexed set of samples should not exceed 2.8 μl. To prevent any sample DNA from being extracted, do not insert the pipette tip into the sample mixture. Instead, pipette the primers to the side of the PCR tube, just above the level of the master sample mixture.

[0403] Briefly centrifuge to collect the contents at the bottom of the tube.

[0404] Continue the WGA PCR procedure.

[0405] After PCR is completed, the DNA can be stored for a short period at 4°C or for a long period at -20°C.

[0406] The quality of WGA DNA can be assessed, for example, by gel electrophoresis. WGA DNA products should appear as a diffuse product, ranging from 200 bp to 2000 bp. NTC should appear clean and contain primer dimers.

[0407] Failed WGA amplification is indicated by the presence of primer dimers but no larger amplification products.

[0408] Poor WGA amplification is indicated by low-intensity smears, or by PCR products that are significantly larger or smaller than the expected size range (observed on other samples on the same agarose gel).

[0409] Enrichment of the target sequence was confirmed using sequence-specific PCR and DNA amplified from the whole locus as a template. Due to the addition of gene-specific primers to the WGA PCR, agarose gel electrophoresis showed enriched amplification of the target sequence compared to the control.

[0410] Although this disclosure has been described with reference to specific embodiments, it should be understood that this disclosure may be embodied in many other forms. It should also be understood that this disclosure described herein allows for alterations and modifications different from those specifically described. It should be understood that this disclosure includes all such alterations and modifications. This disclosure also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more steps or features.

[0411] It should also be noted that, when used in this text, the singular forms of “a,” “an,” and “the” include the plural aspect, unless the context otherwise indicates.

[0412] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” shall be understood to imply coverage of the said element or integer or group of elements or integers, but not to exclude any other element or integer or group of elements or integers.

[0413] Any reference to prior art in this specification is not, and should not be construed as, an admission or implication in any way that such prior art constitutes part of the common general knowledge of any country.

[0414] The subject headings included herein are for reader convenience only and should not be used to limit the subject matter found throughout the disclosure or claims. These subject headings should not be used to interpret the scope or limit of the claims.

[0415] The description provided herein relates to several embodiments that may share common features and characteristics. It should be understood that one or more features of one embodiment may be combined with one or more features of other embodiments. Furthermore, individual features or combinations of features of these embodiments may constitute additional embodiments.

[0416] All methods described herein may be performed in a suitable order unless otherwise stated herein or obviously contradicted by the context. Unless otherwise stated, any and all examples or exemplary language provided herein (e.g., “for example”) are intended only to better illustrate the exemplary embodiments and are not intended to limit the scope of the claimed invention. No language in the specification should be construed as indicating that any non-claim element is necessary.

[0417] Future patent applications may be filed based on this application, for example by claiming priority from this application, by claiming divisional status, and / or by claiming continuation status. It should be understood that the appended claims are provided by way of example only and are not intended to limit the scope of protection that may be claimed in any such future application. Nor should the claims be considered to limit the understanding of this disclosure (or exclude other understandings). Features may be added to or omitted from the example claims in the future.

Claims

1. A method for amplifying a target DNA sequence from genomic DNA, the method comprising amplifying the genomic DNA using a polymerase chain reaction with one or more degenerate oligonucleotide primers, and subsequently amplifying the amplified genomic DNA with the one or more degenerate oligonucleotide primers in the presence of one or more target DNA sequence-specific primers, wherein the amplification with the one or more degenerate oligonucleotide primers comprises amplifying the one or more degenerate oligonucleotide primers for 1 to 8 cycles at an annealing temperature of 20°C to 35°C, and the subsequent amplification comprises amplifying the target DNA sequence for 5 to 25 cycles at an annealing temperature of 52°C to 65°C.

2. The method of claim 1, wherein the one or more degenerate oligonucleotide primers comprise a nucleotide sequence of 6 to 12 consecutive random nucleotides.

3. The method according to claim 1 or 2, wherein the method comprises one or more degenerate oligonucleotide primers at a concentration of 0.1 to 4 µM.

4. The method according to any one of claims 1 to 3, wherein the method comprises one or more target DNA sequence-specific primers at a concentration of 0.05 to 4 µM.

5. The method according to any one of claims 1 to 4, wherein the one or more target DNA sequence-specific primers comprise a melting temperature of 50°C to 64°C and a GC content of 18% to 65%.

6. The method according to any one of claims 1 to 5, wherein the method comprises an amount of 1 pg to 6 ng of genomic DNA to be amplified.

7. The method according to any one of claims 1 to 6, wherein the method comprises further amplifying the amplified target DNA sequence by one or more rounds using additional target DNA sequence-specific primers.

8. The method according to any one of claims 1 to 7, wherein the genomic DNA comprises DNA from one or more embryonic cells, DNA from an oocyte or its polar body, DNA from a sperm, DNA from one or more germ cells, DNA from one or more individual cells, DNA from one or more human or animal cells, DNA from one or more plant cells, DNA from one or more microbial cells, DNA from one or more cells used for disease screening, DNA from one or more cancer cells, DNA for forensic testing, mitochondrial DNA, extrachromosomal DNA, cell-free DNA, DNA from a sample comprising one or more cells, DNA from a sample comprising cell-free DNA, DNA from cell lysates, DNA from a biopsy, DNA from a tissue sample, DNA from a cell sample, DNA from a body fluid, DNA from a blood sample, DNA from amniotic fluid, DNA from a formalin-fixed sample, DNA from a paraffin-embedded sample, and DNA from a swab.

9. The method according to any one of claims 1 to 8, wherein the genomic DNA comprises DNA from a single cell.

10. A kit for use in performing the method of any one of claims 1 to 9, the kit comprising one or more degenerate oligonucleotide primers, one or more primers for amplifying a target DNA sequence, and optionally one or more reagents and / or instructions for amplifying the target DNA sequence during whole-genome amplification.

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