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Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer

A technology of A22105026GSNP, G22115503CSNP, applied in the field of molecular biology, can solve the problems of no detection products, easy contamination of samples, high false positive rate, and achieve the effect of improving sensitivity, strong scalability, and avoiding cross-reaction

Active Publication Date: 2013-08-28
成都益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of cardiovascular disease risk at home and abroad is increasing day by day. The current products are generally based on PCR technology, such as fluorescent quantitative PCR method, RFLP method and DNA sequencing method, which have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate. Due to the limitation of detection flux, it cannot meet the needs of practical applications
As for the SNP site of the chromosome 9p21 segment, there are almost no related detection products at home and abroad

Method used

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  • Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer
  • Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer
  • Chromosome 9p21 section and KIF6 gene SNP (single nucleotide polymorphism) detection liquid phase chip and specific primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 KIF6 gene and chromosome 9p21 segment SNP detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032] 针对KIF6基因的SNP位T2155C(rs20455),以及染色体9p21区段的SNP位点G22115503C(rs1333049)、A22086055G(rs10757274)、A22114477G(rs10757278)、A 22105026G(rs2383206)、A22105959G(rs2383207)和A22088619G(rs2891168) , respectively design specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0034]

[0035]

[0036] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of ...

Embodiment 2

[0050] Example 2 Using the KIF6 gene and chromosome 9p21 segment SNP detection liquid chip described in Example 1 to detect samples The formulas of the various solutions are as follows:

[0051] 50mM MES buffer (pH5.0) formula (250ml):

[0052] Reagent

source

Final concentration

Dosage per 250ml

MES(2[N-Morpholino]

ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

5M NaOH

Fisher SS256-500

---

5 drops

[0053] 2×Tm hybridization buffer

[0054] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0055] Store at 4°C after filtration.

[0056] ExoSAP-IT kit was purchased from US USB Company.

[0057] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technolog...

Embodiment 3

[0122] Example 3 Detection of KIF6 gene and chromosome 9p21 segment SNP site by liquid chip with different ASPE primers

[0123] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0124] Taking the KIF6 gene T2155C site and the 9p21 gene G22115503C site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of T2155C and G22115503C respectively, and the Tag sequence at the 5' end of the ASPE primer It is selected from SEQ ID NO.1-SEQ ID NO.14, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.29-SEQ ID NO.42. The specific design is shown in the following table (Table 9). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

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Abstract

The invention discloses a chromosome 9p21 section SNP (single nucleotide polymorphism) detection liquid phase chip and a specific primer. The chip mainly comprises an ASPE (allele specific primer extension) primer pair, microspheres which are respectively coated with specific anti-tag sequence and have different color codes and an amplification primer, each ASPE primer comprises tag sequence of 5' end and the specific primer at the 3' end against an SNP site, and each specific primer is SEQ ID (sequence identity) No. 17 and SEQ ID No. 18 against chromosome 9p21 section G22115503C SNP, SEQ ID No. 19 and SEQ ID No. 20 of A22086055G SNP, SEQ ID No. 21 and SEQ ID No. 22 of A22114477G SNP, SEQ ID No. 23 and SEQ ID No. 24 of A22105026G SNP, SEQ ID No. 25 and SEQ ID No. 26 of A22105959G SNP, and / or SEQ ID No. 27 and SEQ ID No. 28 of A22088619G SNP; and the tag sequence is selected from SEQ ID No. 1-SEQ ID No.14. The invention further discloses the chromosome 9p21 section and KIF6 gene SNP detection liquid phase chip. The coincidence rate between the detection method provided by the invention and the sequencing method is as high as 100%. The prepared KIF6 gene and chromosome 9p21 section SNP detection liquid phase chip has a very good signal-noise ratio.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a chromosome 9p21 segment, a KIF6 gene SNP site detection liquid chip and specific primers. Background technique [0002] WTCCC conducted a genome-wide association study on 1926 patients with coronary heart disease / myocardial infarction or severe family history and 2938 control subjects in 2007, showing that the polymorphic site rs1333049 (G >C) Located in the 9p21 region (P=1.80×10-14). The findings were validated in East Asian populations in 2008. Confirmed in a Chinese population in 2009. The PROVEIT-TIMI 22 study also confirmed that rs1333049C allele carriers benefit more from intensive statin therapy than conventional statin therapy. At the same time, there are A22086055G (rs10757274), A22114477G (rs10757278), A22105026G (rs2383206), A22105959G (rs2383207) and A22088619G (rs1383207) and A22088619G (rs1383207)2 polymorp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森秦会娟余刚曾涛
Owner 成都益善医学检验所有限公司