Method for preparing gene mutation reference substance

A reference product, genome technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complex process, high verification cost, long verification process, etc., and achieve fast preparation process, low verification cost, and preparation. short time effect

Pending Publication Date: 2020-03-13
JIAXING ACCB DIAGNOSTICS
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Problems solved by technology

[0005] Currently, digital PCR and high-throughput sequencing are commonly used to accurately measure the proportion of target gene mutations in gene mutation reference products, and the cost of each measurement reaction is relatively high. Therefore, when preparing mutation reference products according to conventional methods, It is necessary to prepare multiple gradients and then determine the mutation ratio one by one. This process is complicated, and the number of samples for mutation...

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  • Method for preparing gene mutation reference substance
  • Method for preparing gene mutation reference substance
  • Method for preparing gene mutation reference substance

Examples

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Embodiment

[0031] Preparation and verification of EGFR gene mutation reference product (1% mutation ratio)

[0032] 1. Adopt the method described in the present invention, extract lung cancer cell line H1975 (containing EGFR gene L858R mutation, and there is gene amplification in EGFR), lung cancer cell line H1650 (contain EGFR gene exon 19 deletion mutation, be called for short 19del, and EGFR There is gene amplification), genomic DNA (gDNA) of the 293T cell line (in which the EGFR gene is wild-type), and the genomic DNA can also be prepared into cfDNA by a sonicator (Covaris). Take R=0 and R=4 respectively to prepare two cases of reference products to be tested, and determine the target gene mutation ratio in them by NGS technology, and the results are shown in the table below.

[0033]

[0034] 2. Using the method described in the present invention, calculate the R value corresponding to the gDNA or cfDNA reference product prepared to a 1% mutation ratio (see the table below).

[...

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Abstract

The invention discloses a method for preparing a gene mutation reference substance. The method disclosed by the invention comprises the following steps: firstly, mixing mutant genome DNA and wild genome DNA according to at least two proportions, measuring the mutation proportion of the prepared reference substance, and calculating a slope and intercept of a fitting straight line; and then preparing the mutation reference substance with any mutation proportion which does not exceed the initial mutation type genome DNA mutation proportion according to the target requirement without verifying themutation proportion of the reference substance obtained by dilution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a gene mutation reference product by utilizing mutant genome DNA and wild-type genome DNA. Background technique [0002] In the field of cancer treatment, specific mutations carried by multiple genes are closely related to the efficacy of cancer targeted therapy drugs. Therefore, gene mutation detection has important clinical significance, and gene mutation detection technology has also been widely used. In the development and verification process of gene mutation detection technology, it is necessary to use reference products containing target gene mutations for the development and performance verification of detection reagents, which are characterized by simulating clinical samples as much as possible and containing different and known mutations Proportion. [0003] When mixing genomic DNA extracted from mutant and wild-type clinical samples or c...

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2531/113C12Q2535/122
Inventor 陈钊莫敏俐张艳刘阳李东霞刘玉忠
Owner JIAXING ACCB DIAGNOSTICS
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