MDA (Multiple Displacement Amplification)-based whole-genome amplification method

A whole-genome amplification and genomic technology, applied in DNA preparation, recombinant DNA technology, etc., can solve problems such as errors, application limitations, and efficiency deviations, and achieve the effects of facilitating clinical testing, overcoming low coverage, and reducing error rates

Inactive Publication Date: 2015-04-29
SHENZHEN HAPLOX BIOTECH
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Problems solved by technology

[0005] Among these two types, PCR amplification is more classic, but for different sequences, there is considerable deviation in the efficiency of PCR amplification, which is prone to amplification bias problems: such as CG-rich DNA sequences and corresponding genes Nonrandom loss of loci, nonrandom loss of alleles, and biases related to DNA fragment size (tend to amplify more short fragments), especially when applied to single cells where large numbers of short fragments

Method used

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  • MDA (Multiple Displacement Amplification)-based whole-genome amplification method
  • MDA (Multiple Displacement Amplification)-based whole-genome amplification method
  • MDA (Multiple Displacement Amplification)-based whole-genome amplification method

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Embodiment 1

[0029] A method for MDA-based whole-genome amplification, comprising the steps of,

[0030] 1) Separating and extracting a single tumor cell;

[0031] 2) extracting the genomic DNA of the single tumor cell by the method of Cell Search;

[0032] 3) Using the single-cell genomic DNA obtained in step 2) as a template, perform multiple displacement amplification (MDA) with the Haplox method single-cell whole-genome amplification kit, specifically, including the following steps:

[0033] Prepare mixed solution A: add random primer octamer and genomic DNA template to the amplification system, and the concentration of random primer octamer is 40 μM to obtain mixed solution A;

[0034] Prepare mixed solution B: freshly mix 10x Phi29Buffer, 1mM dNTPs, 0.1mM dUTP, H2O, and Phi29Polymerase (10U / μL, Enzymatics) to obtain mixed solution B; store at 4°C for later use;

[0035] Reaction: mix equal volumes of mixed solution A and mixed solution B into the reaction tube, then add mineral oil...

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Abstract

The invention provides an MDA (Multiple Displacement Amplification)-based whole-genome amplification method. The method is characterized by comprising the following steps: (1) separating and extracting single cells; (2) extracting DNA of the single cells; (3) carrying out multiple displacement amplification (MDA) by virtue of a Haplox-method single-cell whole-genome amplification kit by taking the DNA of the single cells obtained by the step (2) as a template; and (4) purifying a product obtained by the step (3) to obtain purified DNA.

Description

technical field [0001] The present invention relates to the technical field of DNA amplification, in particular to an MDA-based whole genome amplification method. Background technique [0002] In the past two decades, with the improvement of gene sequencing technology and the successive development of major international cooperation projects such as the Thousand Genomes Project, Cancer Genome Project, and Meta-Hit Project, genome research has gradually been pushed to a climax. However, the sequencing materials used so far have invariably been mixed DNA samples of millions or more cells. This method can obtain the whole genome sequence information, but the result obtained by studying it is only the average value of the signal in a group of cells, or only represents the information of the dominant number of cells, and the unique characteristics of a single cell are ignored. For example, it is nearly impossible for scientists to figure out which mutations are present in which ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 许明炎
Owner SHENZHEN HAPLOX BIOTECH
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