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how to amplify dna

A technology for pre-amplification and amplification of products, applied in the field of DNA amplification, which can solve the problems of poor sequencing results, cumbersome operations, and long time-consuming

Active Publication Date: 2020-06-05
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The library preparation process of the CG platform is relatively complicated. Fragmented DNA needs enzyme digestion and two circularization processes after end repair, which is cumbersome and time-consuming.
[0006] When the products amplified by the current mainstream amplification methods are used in the above-mentioned sequencing technologies, either additional library construction is required, or the sequencing effect is not good

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Example 1: Preliminary verification of amplification effects using different linear amplification primer mixtures

[0187] a) Validation using standard genomic DNA samples

[0188] The standard genomic DNA is the genomic DNA of human cells extracted in advance. Dilute the standard genomic DNA with nuclease-free water to a DNA solution of 50 pg / μl, take 1 μl of the above solution (as a source of genomic DNA) and add it to a PCR tube, and add it to each experimental group as shown in Table 1. The primer mix and other relevant reagents, obtain the first reaction mixture (which contains Na + , Mg 2+ , Cl - , Tris-Cl, TritonX-100, dNTPs, Vent polymerase and primer mix).

[0189] linear amplification

[0190] For each primer combination / mixture, two experimental groups were used to conduct parallel experiments to ensure its accuracy, and the reaction mixture of each experimental group was placed in the following first temperature control program for reaction:

[019...

Embodiment 2

[0230] Example 2: Further verification of amplification effects using different linear amplification primer mixtures

[0231] Human epidermal fibroblasts were isolated and lysed according to the method described in implementation 1b) to obtain single-cell genomic DNA, and the primer mix used in the experimental group 11 / 12 in Table 1 and the primer mix used in the experimental group 9 / 10 were used, respectively. The primer mixture was used for amplification, and 10 parallel experiments were performed for each primer mixture (represented as 1_1, 1_2...1_10 and 2_1, 2_2...2_10). Amplify according to the program described in embodiment 1a) and obtain the amplified product, and carry out gel electrophoresis detection to the amplified product, the electrophoresis detection result is as follows Image 6 shown. Wherein the concentration of the amplified product in the experimental group 2_1, 2_2...2_10 is slightly lower than the concentration of the amplified product in the experime...

Embodiment 3

[0239] Example 3: Detection of pathogenic sites and quality inspection primers

[0240] Pathogenic site detection

[0241] 35 disease-causing loci were randomly selected (see Table 8 below for the selected loci), and primers were designed. The selected pathogenic loci and their corresponding primers are shown in Table 8 and Table 9, respectively.

[0242] Table 8: 35 randomly selected disease-causing loci

[0243] Pathogenic site name chromosome location 1 SMN1-1 chr5 2 SMN1-2 chr5 3 SMN1-3 chr5 4 SMN1-4 chr5 5 SMN1-1R chr5 6 SMN1-2R chr5 7 SMN1-3R chr5 8 SMN1-4R chr5 9 PDS-IV15 chr7 10 PDS-EXON5 chr7 11 PDS-EXON7+8 chr7 12 PDS-EXON10 chr7 13 PDS-EXON17 chr7 14 PDS-EXON19 chr7 15 HBB3 chr11 16 HBB chr11 17 MMACHC chr1 18 HBA2 chr16 19 GJB2 chr13 20 GJB2-C796 chr13 21 ATP7B-8 chr13 22 PKHD1-3...

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Abstract

The present application relates to a method for amplifying genomic DNA, comprising: (a) providing a first reaction mixture, including a sample comprising genomic DNA, a first primer, a nucleotide monomer mixture, and a nucleic acid polymerase, wherein the first The primer comprises a universal sequence and a first variable sequence including a first random sequence from the 5' end to the 3' end; (b) placing the first reaction mixture in a first temperature cycle program to obtain a pre-amplification product; (c) providing A second reaction mixture comprising preamplified products, a second primer, a mixture of nucleotide monomers and a nucleic acid polymerase, wherein the second primer comprises or consists of a specific sequence and the general sequence from the 5' end to the 3' end Composition; (d) placing the second reaction mixture in a second temperature cycle program for amplification to obtain an amplification product. The present application also relates to a kit for amplifying genomic DNA.

Description

technical field [0001] The invention relates to a method for amplifying DNA, in particular to a method for amplifying single cell whole genome DNA and sequencing it. Background technique [0002] Single-cell whole-genome sequencing technology is a new technology for amplifying and sequencing the whole genome at the single-cell level. The principle is to amplify a small amount of whole-genome DNA from a single isolated cell, obtain a complete genome with high coverage, and perform high-throughput sequencing. [0003] At present, there are four main types of whole-genome amplification techniques: Primer Extension Preamplification-Polymerase Chain Reaction (PEP-PCR for short). For specific methods, see Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N.1992.Wholegenome amplification from a single cell:implications for genetic analysis.Proc Natl Acad Sci U S A.89(13):5847-51.), degenerated oligonucleotide primer polymerase chain Degenerate Oligonucleotide–Primed Polymera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6869C12Q1/6844C12Q1/6853C12Q1/6848C12Q2525/155C12Q2525/161C12Q2525/179C12Q2525/301C12Q2535/122C12Q1/68C12Q1/6874
Inventor 高芳芳陆思嘉任军
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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