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Multi-SNP locus genetic typing method based on nMALDI-TOF technology

A genotyping method and locus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to produce test results on the same day, and overcome the problem of non-specific binding of primers, multi-gene loci The effect of shortening detection and experiment time

Pending Publication Date: 2022-05-24
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0003] The conventional protocol for genotyping using this technology takes more than 7.3 hours to perform PCR amplification, SAP reaction purification and single base extension reaction, and the test results cannot be obtained on the same day

Method used

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  • Multi-SNP locus genetic typing method based on nMALDI-TOF technology
  • Multi-SNP locus genetic typing method based on nMALDI-TOF technology
  • Multi-SNP locus genetic typing method based on nMALDI-TOF technology

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Embodiment 1

[0032] Example 1: Multi-SNP locus genotyping method based on nMALDI-TOF technology

[0033] 1. Hybridization reaction

[0034] 1.1 Take out the ice box, sample DNA to be tested, probe MIX, and hybridization buffer, thaw at room temperature, vortex and mix well after thawing, centrifuge briefly, and place on ice box for use. Prepare and label the corresponding number of PCR tubes.

[0035] 1.2 Prepare a hybridization reaction system (10ul) in a 200ul PCR tube according to the following table:

[0036] Table 1 Hybridization reaction system

[0037]

[0038] Note: When the sample is peripheral blood, "X" indicates the sample volume of 10-50ng DNA, and 50ng is recommended.

[0039] 1.3 Mix by vortexing or pipetting, centrifuge briefly, place it on the PCR machine, and run the following program:

[0040] Table 2 Hybridization reaction program

[0041]

[0042] Note: The running time of the instrument is about 95min.

[0043] 2, extension, connection

[0044] 2.1 Take o...

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Abstract

The invention belongs to the technical field of SNP (Single Nucleotide Polymorphism) locus genotyping, and particularly relates to a multi-SNP locus genotyping method based on an nMALDI-TOF (Independent Matrix Assisted Laser Desorption / Ionization Time of Flight) Comprising the following steps: designing and synthesizing a probe and a PCR (Polymerase Chain Reaction) amplification primer aiming at a plurality of SNPs of DNA of a sample to be detected; hybridizing the probe and the sample DNA; adding extension and connection reaction liquid into a hybridization reaction product, uniformly mixing, and carrying out extension and connection; adding excision enzyme mixed liquor into extension and connection reaction products, uniformly mixing, and carrying out enzyme digestion; adding S5amp into an enzyme digestion reaction product; uniformly mixing the mixed solution of the N7 primer, purified water and the PCR premixed solution, and carrying out PCR reaction; and after desalting a PCR reaction product, loading a sample to MALDI-TOF-MS for detection to obtain a detection result. On the basis that the result is accurate, the operation time can be shortened by half.

Description

technical field [0001] The invention belongs to the technical field of SNP site genotyping, in particular to a multi-SNP site genotyping method based on nMALDI-TOF technology. Background technique [0002] Nucleic acid mass spectrometry detection technology is based on the principle of MALDI-TOF, combined with primer extension analysis method and base-specific cleavage analysis method, and is specially optimized for the characteristics of double-stranded DNA, so that the sample does not produce or produces less during the ionization process. Fragment ions, which can detect the molecular weight of nucleic acids in biological samples, are an important method to study single nucleotide polymorphism (SNP) in the genome. This technology has been widely used in precision medical testing projects in recent years. Nucleic acid mass spectrometry detection is completed in four steps: ① DNA extraction and DNA quality inspection of the sample; ② gene locus selection and primer design, s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/125C12Q2533/101C12Q2521/319C12Q2565/627C12Q2547/101
Inventor 李赟刘文兰
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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