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Multi-sample multi-site SNP detection method

A detection method and multi-site technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve problems such as the difficulty in optimizing the temperature difference extension conditions of the melting chain, the difficulty in multi-site SNP detection, and the difficulty in micro-sample detection, etc., to achieve Low cost, high sensitivity and specificity, high sensitivity and specificity

Inactive Publication Date: 2011-03-16
XUZHOU MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, due to the large difference in the melting temperature of different probes, the extension conditions are difficult to be optimized, which in turn makes it difficult to detect multi-site SNPs.
Based on multiphase PCR and chemiluminescence technology, a method for the detection of multi-samples and multi-site SNPs was developed with streptavidin-modified 384 microwell plates as the carrier. This method has high detection specificity and good reproducibility Advantages, but the number and size of the wells of the microplate limit the number of detection samples and SNP sites, and the detection of small samples is difficult due to the high background of the streptavidin-modified substrate

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0021] Realized the correlation study between 10 polymorphic sites of COMT gene (rs15761987, rs15761985, rs15761983, rs15761981, rs15761980, rs15761978, rs15761977, rs15761976, rs15761975, rs15761967) and postoperative fentanyl analgesic dose.

[0022] For the above 10 SNP sites, design corresponding Padlock probes (18bp at both ends: respectively complementary to the sequences on both sides of the SNP site, and the SNP site corresponds to the base at the 5' end; the middle length is 50bp (see attached table) 1(1)); specific amplification primers with acrylamide modification at the 5' end (complementary to the 18 nucleotide sequences at the 5' end of the Padlock probe) and general fluorescent detection probes (see attached table 1(2) for Detect SNP mutant sites; see attached table 1 (3) for detecting SNP wild-type sites); use different Padlock probes to capture 10 SNP sites of the same sample in the same reaction tube, and connect the Padlock probes with ligase Needle, the Pad...

Embodiment 2

[0024] Detection of 4 SNP sites (rs6929137(G / A), rs1999805(T / C), rs4870044(G / A), rs1038304(A / G)) in the chromosome 6q25.1 region of 200 people, and further research Association of gene polymorphisms with bone mineral density and vertebral fractures in postmenopausal women

[0025]For the four SNP sites of rs6929137(G / A), rs1999805(T / C), rs4870044(G / A), rs1038304(A / G), corresponding Padlock probes, specific amplification primers and universal fluorescence detection were designed Probes (sequence features are all the same as in Example 1); in the same reaction tube, use different Padlock probes to capture four SNP sites of the same sample, and connect the Padlock probes with ligase, and there are Padlock probes corresponding to the SNP sites Circularization occurs, otherwise no circularization; then use specific amplification primers to hybridize with Padlock probes and perform constant temperature rolling circle amplification with high-fidelity Bst, circularized Padlock probes ...

Embodiment 3

[0027] Realize the correlation between the pain degree of patients with rheumatoid arthritis and the SNP of HLA-II gene

[0028] For 3 SNP sites (rs1059576, rs1059582, rs1059586) of HLA-DRB1 gene, 2 SNP sites (rs2071282, rs2071283) of HLA-NOTCH4 gene and 1 SNP site (rs1049110) of HLA-DQB2 gene, design corresponding Padlock probes (18bp at both ends and 50bp in the middle), specific amplification primers and universal fluorescent detection probes with biotin modification at the 5' end (sequence features are the same as in Example 1); in the same reaction tube Use different Padlock probes to capture 6 SNP sites of the same sample, and connect the Padlock probes with ligase, and the Padlock probes with corresponding SNP sites will be circularized, otherwise they will not be circularized; then use biotin-modified Specific amplification primers were hybridized with Padlock probes, and high-fidelity Bst was used for constant temperature rolling circle amplification. The circularized...

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Abstract

The invention discloses a multi-sample multi-site SNP (single nucleotide polymorphism) detection method, and belongs to microarray chip-based SNP multi-sample multi-site detection technology. The method comprises the following steps of: designing an oligonucleotide DNA chip; capturing different SNP sites of the same sample by using different Padlock probes in a single tube, then hybridizing specific amplification primers and the Padlock probes and performing rolling circle amplification, and finally fixing different SNP amplification products of different samples on the same chip in certain order to prepare a multi-sample multi-site SNP microarray to be detected; and finally, performing detection by using a general fluorescent probe, and judging the parting of the SNP according to the color of microarray point fluorescence. The method has the advantage of realizing parallel detection of different SNP sites of different samples and detection of different SNP sites of the same sample. Accuracy and sensitivity of detecting SNP site information are high. Because of the specificity and the accuracy of identifying the SNP sites by the Padlock probes, the accuracy of identifying the SNP sites is ensured.

Description

technical field [0001] The invention belongs to the multi-sample and multi-site detection technology of gene polymorphism (SNP) based on a microarray chip, in particular to a multi-sample and multi-site SNP detection method with high sensitivity and high throughput. Background technique [0002] The research of SNP detection method is the premise and foundation of the study of disease-related SNP. The detection method of SNP is different from the determination of nucleic acid sequence. It needs to detect the changes of upconversion, transversion, insertion or deletion of bases at a specific nucleotide position in genomic DNA, which has great influence on the sensitivity, throughput and accuracy of detection. There are extremely high requirements, and the detection method is more complicated and cumbersome than DNA sequencing. Up to now, there are about 70 kinds of SNP detection methods reported. For the analysis of single or multiple target genes, the more commonly used me...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 潘志强张励才张成标邵翠杰
Owner XUZHOU MEDICAL COLLEGE
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