Low abundance gene mutation enriching method based on removing wild sequences

A wild-type, low-abundance technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., to solve problems such as inability to detect DNA mutations

Active Publication Date: 2018-04-10
CHONGQING UNIV OF POSTS & TELECOMM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, high-throughput sequencing cannot detect DNA mutations with an abundance of less than ~2%. The method of removing a large n...

Method used

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  • Low abundance gene mutation enriching method based on removing wild sequences
  • Low abundance gene mutation enriching method based on removing wild sequences

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0017] 1. Sample DNA extraction

[0018] The DNA of the sample can be extracted using the CTAB method, of course, the DNA of the sample can also be extracted using a DNA extraction kit, and the extraction steps refer to the corresponding instructions.

[0019] 2. Sample processing

[0020] The extracted sample DNA is processed according to the method of the present invention, specifically:

[0021] ①Hybridization: Mix the sample DNA with the hybridized strand to form a hybridization mixture, and denature it at high temperature (usually above 94 degrees Celsius for at least 2 minutes) to melt the sample DNA from double-stranded DNA to single-stranded DNA (such as figure 1 (a) in step), and then lower the temperature (usually to about 70 degrees Celsius for 1-2 minutes) to make the hybridized strand and the wild-type sequence in the melted single-stranded DNA form a fully complementary paired homologous double-stranded DNA and Non-perfectly complementary heteroduplex DNA that ...

Embodiment

[0028] Example: Enrichment and detection of low-abundance mutation samples (5% mutation content)

[0029] In order to verify the feasibility of the method of the present invention in actual samples, a sample with a mutation content of 5% was used for identification. In this embodiment, the samples were subjected to (1) digestion with double-strand specific nuclease by the method of the present invention, followed by PCR amplification with the primers of the present invention and (2) without any treatment, and then with common primers Perform PCR amplification. The enrichment effect of the amplified products was detected and compared with a pyrosequencing platform. The specific method is:

[0030] 1. Sample preparation

[0031] A wild-type plasmid and a mutant plasmid were constructed by a biotechnology company, and the mutation sites corresponding to the two plasmids were G>A. A plasmid mixture of the mutant plasmid and the wild-type plasmid was then prepared, wherein the ...

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Abstract

The invention discloses a low abundance gene mutation enriching method based on removing wild sequences. According to the method, before PCR amplification, a sample DNA used as a DNA template is processed, so that nucleotide single chains which contain mutation loci and are sealed at the 3' tail end are used as hybrid chains to be hybridized with wild and mutation type sequences, and duplex-specific nuclease can specifically remove a homoduplex DNA completely formed by the hybrid chains and the mutation type sequences in complete complementary pairing with the wild sequences but cannot removea heteroduplex DNA formed by the hybrid chains and the mutation type sequences in incomplete complementary pairing. Removed products are subjected to the PCR amplification, the 3' tail end of a primerused by the PCR amplification is located on the mutation loci and is sealed, under the effect of a polymerase with a 3'-5' proofreading activity, the wild sequences cannot be amplified, but the mutation type sequences can be amplified in an index mode, therefore the low abundance gene mutation sequences are effectively enriched, and the enriching sensitivity and accuracy are improved; meanwhile,according to the method, multiple mutation type sequences can also be enriched simultaneously, and therefore, the amplification efficiency is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a low-abundance gene mutation enrichment method based on removing wild-type sequences. Background technique [0002] The completion of the Human Genome Project has promoted the rapid development of gene mutation research. In recent years, the proposal of the "Precision Medicine" program has further made the study of gene mutations and their detection methods a hot spot and basis for biological research and clinical precision medicine. With the continuous development of detection technology, researchers have found that most gene mutations exhibit low abundance (content ranges from 0 to 100%), and these low abundance gene mutations have important research significance in the fields of medicine and biology. Emerging new methods and technologies provide reliable prospects for the enrichment of low-abundance gene mutations, most of which are based on the principle of PCR technology. PCR...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6806
CPCC12Q1/6806C12Q1/6869C12Q2535/101C12Q2531/113C12Q2565/301C12Q2521/327
Inventor 浦丹赵珊舒坤贤
Owner CHONGQING UNIV OF POSTS & TELECOMM
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