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Method for constructing yeast cell-based gene detection standard and its kit

A construction method and gene detection technology, applied in the field of molecular biology, can solve the problems of inconvenient sample acquisition, expensive cell lines, precious samples, etc., and achieve simple and easy DNA extraction process, high homologous recombination efficiency, amplification and Easy to detect effects

Active Publication Date: 2020-11-17
上海泽因生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

For example, clinical patient samples have defects such as inconvenient sample acquisition, precious samples, poor uniformity, and inability to prepare a large number of standard products; cell lines for genetic diseases are often immortalized through patient cells, and the genetic characteristics of immortalized cell lines are difficult to maintain for a long time; The establishment of tumor mutation cell lines is difficult and unstable, and the construction process of genetic mutation-containing cell lines is cumbersome and requires a complicated screening process. At the same time, cell lines are expensive and require high culture conditions; plasmid sample construction is simple and fast, but the number of plasmid copies in the system is high , Diluting and simulating genomic DNA and mixing of lower mutation percentage standards will cause large errors and easy contamination

Method used

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  • Method for constructing yeast cell-based gene detection standard and its kit
  • Method for constructing yeast cell-based gene detection standard and its kit

Examples

Experimental program
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Embodiment 1

[0067] Taking the "important gene mutation c.35G>C (hereinafter referred to as G12A mutation) of human non-small cell lung cancer" as an example, a yeast cell standard (positive standard) for detecting G12A mutation is constructed.

[0068] Step (a): Constructing a gene editing plasmid targeting Saccharomyces cerevisiae gene URA3 based on the CRISPR / Cas9 gene editing system;

[0069] In step (a), the inventor selects a yeast-Escherichia coli shuttle vector, specifically, p425-Sap-TEF1p-Cas9-CYC1t-2xSap vector (commercially available);

[0070] In step (a), the inventors designed the sequence of the gRNA targeting Saccharomyces cerevisiae gene URA3, specifically, the sequence of the gRNA is: cattacgaatgcacacggtgtgg (as shown in SEQ ID No.1); two reverse reactions were designed according to the gRNA sequence To the complementary primers (in addition, specifically in this embodiment, in order to be able to match with the SapI digestion gap of the p425-Sap-TEF1p-Cas9-CYC1t-2xSap v...

Embodiment 2

[0121] Embodiment 2: Build the kit of gene detection standard

[0122] The test kit of embodiment 2 mainly comprises:

[0123] 1), the gene editing plasmid targeting the Saccharomyces cerevisiae gene URA3 obtained in step (a) of the above-mentioned Example 1 (the gene editing plasmid targeting the Saccharomyces cerevisiae gene URA3 constructed based on the CRISPR / Cas9 gene editing system), wherein the gene editing The plasmid targets the Saccharomyces cerevisiae gene URA3 and makes the DNA double-strand break site an editing site;

[0124] 2) The homologous recombination vector obtained in (b-3) in step (b) of the above-mentioned Example 1 (constructed based on the CRISPR / Cas9 gene editing system, containing the upstream homologous repair arm of the targeted editing site and the downstream homologous repair arm; Homologous recombination vector of the source repair arm);

[0125] 3) Saccharomyces cerevisiae cell line; in this embodiment, Saccharomyces cerevisiae cell W303-1A ...

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Abstract

The invention relates to a construction method and a kit for a gene detection standard substance based on yeast cells. The yeast cells are creatively used as a genetic background for the first time, to-be-detected target genes such as a human body cell mutation gene are integrated into a yeast cell genome through a homologous recombination mode, and therefore, obtained recombination yeast cells can be used as positive contrast standard substances in genetic detection of target genes.

Description

technical field [0001] The invention relates to a method for constructing a gene detection standard product based on yeast cells and a kit thereof, belonging to the technical field of molecular biology. Background technique [0002] In clinical genetic diagnosis, the accuracy of genetic testing is very important. Taking prenatal testing as an example, false positive results can wrongly guide patients to terminate normal embryos, and false negative results can lead to optimistic expectations and wrong diagnoses for sick babies. Clinically, genetic testing is often the basis for guiding the formation of a diagnosis and treatment plan. More and more people use genetic testing to obtain the susceptibility of individuals in the population, so as to take effective disease prevention measures, including interventional diagnosis and treatment methods and lifestyle changes. [0003] In order to ensure the reliability and accuracy of genetic testing results, each experiment must hav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N9/22C12Q1/6806
CPCC12N9/22C12N15/905C12Q1/6806C12Q2521/327
Inventor 卢大儒陈红岩丁嘉琦卢德颐
Owner 上海泽因生物科技有限公司
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