High-throughput method for simultaneously detecting gene mutation and copy number change

Abstract

The invention relates to the technical field of biological medicines, in particular to a high-throughput method for simultaneously detecting gene mutation and copy number change, which comprises the following steps: respectively designing a pair of positive chain probe and negative chain probe for a plurality of sites or regions to be detected; combining the positive chain probe and the negative chain probe to obtain a combined probe, and performing a first round of PCR amplification on DNA to be detected based on the combined probe; for a product obtained by the first round of PCR amplification, carrying out a second round of PCR amplification on the product obtained by the first round of PCR amplification by utilizing a pair of PCR primers matched with the sequencing primers of the second-generation sequencing platform; performing high-throughput double-end sequencing on a product obtained by the second round of PCR amplification; and performing target site genotype judgment and copynumber analysis on the high-throughput double-end sequencing data. The problem that gene region point mutation and copy number variation cannot be simultaneously detected in one technology in the prior art is solved.

Description

technical field

[0001] The invention relates to the technical field of biomedicine, in particular to a high-throughput method for simultaneously detecting gene mutations and copy number changes. Background technique

[0002] Gene is the material basis of heredity, and it is a specific nucleotide sequence with genetic information on DNA or RNA molecules. Except for some viruses whose genetic material is RNA, the genetic material of almost all non-viral organisms is DNA. Different species have their specific gene sequences, so by detecting the gene sequences in a sample, the biological species in the sample can be judged. During the life process of cells, genes are transcribed into mRNA through DNA, and then cells use mRNA as a template to translate biologically active protein molecules, thereby expressing the genetic information stored in the DNA sequence. On the one hand, genes can faithfully replicate themselves to maintain the basic characteristics of organisms; on the o...

Examples

Embodiment 1

[0021] Example as figure 1 As shown, a high-throughput method for simultaneously detecting gene mutations and copy number changes includes: designing a pair of positive-strand probes and negative-strand probes for multiple sites or regions to be detected, and each pair of positive-strand The positive strand probe in the probe and the negative strand probe is located on the positive strand of the genome sequence, and the negative strand probe in each pair of positive strand probe and negative strand probe is located on the negative strand of the genome sequence; The length of the strand probe or negative strand probe is 18-36bp, preferably 20-27bp.

[0022] Use the positive-strand probe and the negative-strand probe to combine to obtain a combined probe, and perform the first round of PCR amplification on the DNA to be tested based on the combined probe; for the product obtained by the first round of PCR amplification, use a Amplify the universal amplification primers that mat...

Embodiment 2

[0025] Example 2 Design probes for the coding regions of 4 genes (ATP7B, NF1, TSC1, TSC2), the probes and general primer information are shown in Table 1:

[0026] Table 1

[0027]

[0028]

[0029] The three positive samples of the known mutant type were amplified by PCR using a mixture of positive-strand probes and negative-strand probes at each site to obtain amplified products; the amplified products of the two panels were mixed, and then universally used with illumina with different tag sequences PCR primers compatible with the sequencing platform were used for amplification, and the products of each sample were uniformly mixed and then sequenced on an illumina sequencing instrument in PE150 mode, and the sequencing data were subsequently analyzed.

[0030] Experimental procedure: (1) Quantify the concentration of wild-type samples and mutant samples; (2) Configure two panels of positive-strand probe and negative-strand probe mixture, each primer concentration is 2uM;...

technical field

[0001] The invention relates to the technical field of biomedicine, in particular to a high-throughput method for simultaneously detecting gene mutations and copy number changes. Background technique

[0002] Gene is the material basis of heredity, and it is a specific nucleotide sequence with genetic information on DNA or RNA molecules. Except for some viruses whose genetic material is RNA, the genetic material of almost all non-viral organisms is DNA. Different species have their specific gene sequences, so by detecting the gene sequences in a sample, the biological species in the sample can be judged. During the life process of cells, genes are transcribed into mRNA through DNA, and then cells use mRNA as a template to translate biologically active protein molecules, thereby expressing the genetic information stored in the DNA sequence. On the one hand, genes can faithfully replicate themselves to maintain the basic characteristics of organisms; on the o...

Examples

Embodiment 1

[0021] Example as figure 1 As shown, a high-throughput method for simultaneously detecting gene mutations and copy number changes includes: designing a pair of positive-strand probes and negative-strand probes for multiple sites or regions to be detected, and each pair of positive-strand The positive strand probe in the probe and the negative strand probe is located on the positive strand of the genome sequence, and the negative strand probe in each pair of positive strand probe and negative strand probe is located on the negative strand of the genome sequence; The length of the strand probe or negative strand probe is 18-36bp, preferably 20-27bp.

[0022] Use the positive-strand probe and the negative-strand probe to combine to obtain a combined probe, and perform the first round of PCR amplification on the DNA to be tested based on the combined probe; for the product obtained by the first round of PCR amplification, use a Amplify the universal amplification primers that mat...

Embodiment 2

[0025] Example 2 Design probes for the coding regions of 4 genes (ATP7B, NF1, TSC1, TSC2), the probes and general primer information are shown in Table 1:

[0026] Table 1

[0027]

[0028]

[0029] The three positive samples of the known mutant type were amplified by PCR using a mixture of positive-strand probes and negative-strand probes at each site to obtain amplified products; the amplified products of the two panels were mixed, and then universally used with illumina with different tag sequences PCR primers compatible with the sequencing platform were used for amplification, and the products of each sample were uniformly mixed and then sequenced on an illumina sequencing instrument in PE150 mode, and the sequencing data were subsequently analyzed.

[0030] Experimental procedure: (1) Quantify the concentration of wild-type samples and mutant samples; (2) Configure two panels of positive-strand probe and negative-strand probe mixture, each primer concentration is 2uM;...

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