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Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes

A COX-1 and detection solution technology, applied in the field of molecular biology, can solve the problems of easy contamination of samples, high false positive rate, low sensitivity, etc., and achieve the effect of strong scalability, avoiding cross-reaction, and improving sensitivity.

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

[0004] At present, products for COX-1 and GPIIIa polymorphism detection are generally based on PCR technology, such as fluorescence quantitative PCR method, RFLP method and DNA sequencing method, etc., which have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate. Quantitative limitations, can not meet the needs of practical applications

Method used

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  • Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes
  • Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes
  • Liquid chip and specific primer for detecting SNP of GPIIIa gene and liquid chip and specific primer for detecting SNP of GPIIIa and COX-1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 GPIIIa and COX-1 gene SNP detection liquid chip mainly includes:

[0032] 1. ASPE Primers

[0033]Specific primer sequences were designed for the three common SNP sites C50T (rs3842787), A842G (rs10306114) and G123A (rs3842788) of the COX-1 gene, and the SNP site T196C (rs5918) of the GPIIIa gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0034] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0035]

[0036]

[0037] 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 100 pmol / mL with 10 mmol / L Tris Buffer.

[003...

Embodiment 2

[0051] Example 2 Detection of samples using the GPIIIa and COX-1 gene detection liquid chip described in Example 1

[0052] The formula of described various solutions is as follows:

[0053] 50mM MES buffer (pH5.0) formula (250ml):

[0054] 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

[0055] 2×Tm hybridization buffer

[0056] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

[0057] 5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0058] Store at 4°C after filtration.

[0059] ExoSAP-IT kit was purchased from US USB Company.

[0060] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technolo...

Embodiment 3

[0122] Example 3 Detection of COX-1 and GPIIIa gene SNP sites 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 COX-1 gene C50T and GPIIIa gene T196C 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 C50T and T196C, and the Tag sequence of the 5' end of the ASPE primer was It is selected from SEQ ID NO.1-SEQ ID NO.8. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. The specific design is shown in the following table (Table 7). 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.

[0125] Table 7 Design of liquid phase chip prepa...

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Abstract

The invention discloses a liquid chip and specific primer for detecting SNP of a GPIIIa gene. The liquid chip mainly comprises a wild type ASPE primer and a mutant type ASPE primer which are respectively designed aiming at the SNP loci of the GPIIIa gene, microspheres which are respectively coated with specific anti-tag sequences and have different colors of codes and an amplification primer for amplifying the GPIIIa gene target sequence containing the T196C SNP loci, wherein each ASPE primer is formed by the tag sequence at the 5, terminal and the specific primer, aiming at the SNP loci of the target gene, at the 3, terminal; and the anti-tag sequences are selected from SEQ ID NO.17 to SEQ ID NO.24 and correspondingly complement and are paired with the tag sequences selected from each ASPE primer. The invention also provides a liquid chip and specific primer for detecting SNP of COX-1 and GPIIIa genes. Besides the corresponding composition of the GPIIIa gene, the liquid chip also comprises ASPE primer pair aiming at the SNP loci of the COX-1 gene, microspheres coated with specific anti-tag sequences and an amplification primer. The liquid chip has very good signal to noise ratio,can avoid cross reaction and can realize parallel detection of a plurality of SNP loci.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a COX-1 and GPIIIa gene SNP detection liquid chip and specific primers. Background technique [0002] Platelet membrane glycoprotein (Glycoprotein IIIa, GPIIIa) is the main platelet integrin and activator, which is a transmembrane glycoprotein complex. Its role is to act as a receptor to mediate the binding of fibrinogen to the surface of platelets and the subsequent aggregation of platelets. GPIIIa contains human platelet antigen 1 (human platelet antigen, HPA) or PIA, and this gene has a polymorphic site that binds to fibrinogen. A case-control study in the United States observed that the leucine-33 of the GPIIIa gene was replaced by proline Acid substitution, T196C mutation of HPA-1 (Leu33Pro, rs5918, PLA1 / A2 alloantigen system) is associated with emergency thrombosis, and its predictive significance is known in hypertension, smoking, hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森秦会娟余刚曾涛
Owner SUREXAM BIO TECH
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