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Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection

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

Inactive Publication Date: 2011-04-20
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current detection products for SLCO1B1 polymorphism are generally PCR-restriction fragment length polymorphism and direct sequencing methods based on PCR technology, which have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate. limitations and cannot meet the needs of practical applications

Method used

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  • Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection
  • Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection
  • Specific primmer and liquid phase chip for SLCO1B1 gene SNP detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 The SLCO1B1 gene SNP detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Specific primer sequences were designed for wild-type and mutant types of five common genotypes of SLCO1B1 gene, T521C (rs4149056), T89595C (rs4363657), A388G (rs2306283), C463A (rs11045819) and A1929C (rs34671512). ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] Table 1 ASPE primer sequence of SLCO1B1 (Tag sequence + specific primer sequence)

[0025]

[0026]

[0027] 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 100pmol / mL stock solution with 10mmol / LTris...

Embodiment 2

[0040] Example 2 Detection of samples using SLCO1B1 gene detection liquid chip

[0041] The formula of described various solutions is as follows:

[0042] 50mM MES buffer (pH5.0) formula (250ml):

[0043] 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

[0044] 2×Tm hybridization buffer

[0045] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0046] Store at 4°C after filtration.

[0047] ExoSAP-IT kit was purchased from US USB Company.

[0048] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0049] 1. Sample DNA extractio...

Embodiment 3

[0111] Example 3 Detection of the SNP site of SLCO1B1 gene by liquid chip with different ASPE primers

[0112] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0113] Taking the T521C site mutation detection liquid chip of the SLCO1B1 gene as an example, the specific primer sequences at the 3' end of the ASPE primer were designed for the wild type and mutant type of T521C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.10, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30. 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.

[0114] Table 7 Design of liquid phase chip preparation

[0115]

[0...

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Abstract

The invention discloses a specific primmer and a liquid phase chip for solute carrier organic anion transporter 1B1 (SLCO1B1) gene signal nucleotide polymorphism (SNP) detection. The liquid phase chip mainly comprises allele specific primer extension (ASPE) primer pairs, microspheres and amplifiers, wherein each ASPE primer consists of a tag sequence at a 5' end and specific primers at a 3' end aiming at an SNP locus; the specific primers comprise SEQ ID NO.11 and SEQ ID NO.12 aiming at T521C SNP locus, SEQ ID NO.13 and SEQ ID NO.14 aiming at T89595C SNP locus, SEQ ID NO.15 and SEQ ID NO.16 aiming at A388G SNP locus, SEQ ID NO.17 and SEQ ID NO.18 aiming at C463A SNP locus, and / or SEQ ID NO.19 and SEQ ID NO.20 aiming at A1929C SNP locus; and the tag sequence is selected from SEQ ID NO.1 to SEQ ID NO.10. The coincidence rate of the liquid phase chip provided by the invention and the detection result of a sequencing method reaches 100 percent. And the prepared liquid phase chip for the SLCO1B1 gene SNP detection has high signal-to-noise ratio.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for SLCO1B1 gene detection and a liquid phase chip. Background technique [0002] Solute carrier organic anion transporter family member 1B1 (Solute carrier organic anion transporter family, member 1B1, SLCO1B1), the gene encodes the protein organic anion transporting polypeptide OATPIB1 (also known as OATP-C, OATP2, LST1), which regulates the hepatic uptake of statins effect. OATP1B1 is a transport protein specifically distributed on the basement membrane of liver cells, and is closely related to the hepatic uptake of many endogenous or exogenous substances in the body, such as bile acids, sulfurized and glucuronidated conjugates, thyroid Hormones, peptides, methotrexate, and statins (including pravastatin, pitavastatin, rosustatin, and simvastatin, etc.) are all substrates for OATP1B1 transport. In recent years, multipl...

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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