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SNP (Single Nucleotide Polymorphism) detection liquid phase chip for KIF6 and apo E genes and chromosome 9p21 section

A liquid-phase chip and detection liquid technology, applied in the medical and biological fields, can solve the problems of no detection products, high false positive rate, easy contamination of samples, etc., and achieve the effects of improved sensitivity, avoidance of cross-reaction, and strong scalability.

Active Publication Date: 2011-04-13
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of cardiovascular disease risk at home and abroad is increasing day by day, and products for detecting apo E gene polymorphisms have been launched, such as the detection products of Celera Company and Shanghai Zhujian Bioengineering Co., Ltd. The current products are generally based on the fluorescence of PCR technology Quantitative PCR, RFLP, and DNA sequencing methods have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. At the same time, due to the limitation of detection throughput, they 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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  • SNP (Single Nucleotide Polymorphism) detection liquid phase chip for KIF6 and apo E genes and chromosome 9p21 section
  • SNP (Single Nucleotide Polymorphism) detection liquid phase chip for KIF6 and apo E genes and chromosome 9p21 section
  • SNP (Single Nucleotide Polymorphism) detection liquid phase chip for KIF6 and apo E genes and chromosome 9p21 section

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Apo E, KIF6 gene and chromosome 9p21 segment SNP detection liquid chip mainly includes:

[0041] 1. ASPE Primers

[0042] Two common SNP sites C609T (ε2, rs7412) and T471C (ε4, rs429358) of apo E, SNP site T2155C (rs20455) of the KIF6 gene, and SNP sites G22115503C (rs1333049) and A22086055G (rs10757274) of the chromosome 9p21 segment ), A22114477G (rs10757278), A22105026G (rs2383206), A22105959G (rs2383207) and A22088619G (rs2891168), respectively designed specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0043] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0044]

[0045]

[0046]

[0047] 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 ...

Embodiment 2

[0062] Example 2 Detection of samples by using the apo E, KIF6 gene and chromosome 9p21 segment SNP detection liquid chip described in Example 1

[0063] The formula of described various solutions is as follows:

[0064] 50mM MES buffer (pH5.0) formula (250ml):

[0065] Reagent

source

Final concentration

Dosage per 250ml

MES (2[N-Morpholino] ethanesulfonic acid)

Sigma M-2933

0.05M

2.44g

[0066] 5M NaOH

Fisher SS256-500

5 drops

[0067] 2×Tm hybridization buffer

[0068] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

Sigma T3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

[0069] Triton X-100

Sigma T8787

0.16％

0.4ml

[0070] Store at 4°C after filtration.

[0071] ExoSAP-IT kit was purchased from US USB Company.

[0072] Biotin-labeled dCTP was purchased from Shangh...

Embodiment 3

[0141] Example 3 Detection of apo E, KIF6 gene and chromosome 9p21 segment SNP site by liquid chip with different ASPE primers

[0142] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0143] Taking the C609T site of the apo E gene, the T2155C site of the KIF6 gene, and the G22115503C mutation detection liquid chip of the chromosome 9p21 segment as examples, the specificity of the 3' end of the ASPE primers was designed for the wild-type and mutant types of the C609T, T2155C, and G22115503C SNP sites, respectively. primer sequence, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.1-SEQ ID NO.18. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO. 37-SEQ ID NO. 54. The specific design is shown in the following table (Table 9). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplif...

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) detection liquid phase chip for KIF6 gene, mainly comprising an ASPE (Application Solid Phase Extraction) primer pair, microballons and an amplification primer, wherein each ASPE primer consists of a 5'-end tag sequence and 3'-end specific primers aiming at T2155C SNP locus; the 3'-end specific primers comprise SEQID NO.23 and SEQ IDNO.24; the tag sequence is selected from SEQID NO.1 to SEQID NO.18; and the microballons are respectively coated by a specific anti-tag sequence and have codes in different colors. The invention also discloses the SNP detection liquid phase chip for apoE and KIF6 genes and the chromosome 9p21 section. The occlusion rate of the detection method and the sequencing method which are provided in the invention is as high as 100 percent. The prepared SNP detection liquid phase chip for the apoE and KIF6 genes and the chromosome 9p21 section has favorable single-noise ratio.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a liquid phase chip for detecting SNP sites of KIF6, apoE gene and chromosome 9p21 segment. Background technique [0002] KIF6 (Kinesin-like protein 6) encodes kinesin-like protein-6, which is a member of the molecular dynamics superfamily. The three studies of CARE, WOSCOPS and PROVE IT-TIMI 22 all showed the correlation between KIF6 gene Trp719Arg (rs20455T>C) and coronary events. The Trp719Arg polymorphism of the KIF6 gene reaches 60% in the Caucasian population, and the risk of coronary heart disease (CHD) increases by 55% in people with the 719Arg polymorphism. Compared with non-carriers (HR=0.94), KIF6719Arg allele carriers benefit more from intensive statin therapy than conventional statin therapy (HR=0.59), and intensive treatment can reduce the absolute risk of coronary events in carriers by 10.0 %, non-carriers were only 0.8% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟余刚曾涛
Owner SUREXAM BIO TECH
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