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Method for amplifying DNA

A technology for pre-amplification and amplification products, applied in the field of DNA amplification, can solve the problems of long time, cumbersome operation, complicated preparation process, etc.

Active Publication Date: 2016-09-07
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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  • Method for amplifying DNA
  • Method for amplifying DNA
  • Method for amplifying DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] Example 1: Preliminary verification of amplification effects using different linear amplification primer mixtures

[0186] a) Validation using standard genomic DNA samples

[0187] 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).

[0188] linear amplification

[0189] 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

[0229] Example 2: Further verification of amplification effects using different linear amplification primer mixtures

[0230] 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 Figure 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 experim...

Embodiment 3

[0238] Example 3: Detection of pathogenic sites and quality inspection primers

[0239] Pathogenic site detection

[0240] 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.

[0241] Table 8: 35 randomly selected disease-causing loci

[0242]

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

...

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Abstract

The invention relates to a method for amplifying genome DNA, wherein the method comprises the following steps: (a) providing a first reaction mixture which includes a sample containing the genome DNA, a first primer, a nucleotide monomer mixture and nucleic acid polymerase, wherein from 5' terminal to 3' terminal, the first primer comprises a universal sequence and a first variable sequence including a first random sequence; (b) applying the first reaction mixture to a first temperature cyclic program, so that a pre-amplified product is obtained; (c) providing a second reaction mixture which includes the pre-amplified product, a second primer, a nucleotide monomer mixture and nucleic acid polymerase, wherein from 5' terminal to 3' terminal, the second primer comprises or is composed of a special sequence and the universal sequence; and (d) applying the second reaction mixture to a second temperature cyclic program, so that a amplified product is obtained. The invention also relates to a kit for amplifying the genome 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 Applications(China)
IPC IPC(8): C12Q1/68
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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