BRAP and PSMA6 gene SNP detection specific primer and liquid phase chip

A detection solution and specific technology, applied in the field of molecular biology, can solve problems such as inability to meet and cannot be used, and achieve the effect of consistent detection effect, simple steps, and avoidance of uncertain factors.

Active Publication Date: 2014-01-01
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Thirdly, both PCR-based detection methods (PCR-RFLP) and direct sequencing methods have limitations in detection throughput, and only one mutation can be detected at a time, which cannot meet the needs of practical applications.

Method used

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  • BRAP and PSMA6 gene SNP detection specific primer and liquid phase chip
  • BRAP and PSMA6 gene SNP detection specific primer and liquid phase chip
  • BRAP and PSMA6 gene SNP detection specific primer and liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 BRAP and PSMA6 gene SNP detection liquid chip mainly includes:

[0026] 1. ASPE Primers

[0027] Specific primer sequences were designed for the two common genotypes A96G and A80G of the BRAP gene and the wild type and mutant type of the common genotype C157G of the PSMA6 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0028] Table 1 ASPE primer sequences of BRAP and PSMA6 genes (Tag sequence + specific primer sequence)

[0029]

[0030] 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.

[0031] 2. M...

Embodiment 2

[0043] Example 2 Detection of samples using BRAP and PSMA6 gene detection liquid chip

[0044] The formula of described various solutions is as follows:

[0045] 50mM MES buffer (pH5.0) formula (250ml):

[0046]

[0047] 2×Tm hybridization buffer:

[0048] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

[0049] Triton X-100

Sigma T8787

0.16％

0.4ml

[0050] Store at 4°C after filtration.

[0051] ExoSAP-IT kit was purchased from US USB Company.

[0052] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0053] 1. Sample DNA extraction

[0054] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0055] 2. PCR amplification of samples to be tested

[0056] Three pairs...

Embodiment 3

[0113] Example 3 Detection of BRAP and PSMA6 gene SNP sites by liquid chip with different ASPE primers

[0114] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0115] Taking BRAP gene A96G and C157G 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 A96G and C157G respectively, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.4, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.16. 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.

[0116] Table 7 Design of liquid phase chip preparation

[0...

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PUM

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Abstract

The invention discloses BRAP and PSMA6 gene SNP detection specific primers and a liquid phase chip. The liquid phase chip comprises an ASPE primer which is composed of a tag sequence at a 5' terminal and specific primers mutated for a target gene at a 3' terminal, wherein the specific primers respectively comprise a SEQ ID NO.7 and a SEQ ID NO.8 for a BRAP gene A96G SNP locus, a SEQ ID NO.9 and a SEQ ID NO.10 for a BRAP gene A80G SNP locus, and / or a SEQ ID NO.11 and a SEQ ID NO.12 for a PSMA6 gene C157G SNP locus; microspheres coated by an anti-tag sequence; and amplification primers. The coincidence rate between detection results of the BRAP and PSMA6 gene SNP detection liquid phase chip provided by the invention and results of a sequencing method is up to 100%.

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 detecting BRAP and PSMA6 gene SNP and a liquid phase chip. Background technique [0002] Breast cancer susceptibility gene 1 associated protein gene (breast cancer susceptibility gene 1 associated protein, BRAP), located at 12q24 of the human genome, is named because its encoded protein can bind to the nuclear localization signal region of BRCA1. BRAP is a cytoplasmic protein whose main physiological function is to regulate the nuclear targeting of other proteins; in addition, BRAP is also associated with inflammation-related molecules lymphotoxin-α and galectin-2. [0003] Studies have shown that lymphotoxin-α is an important pro-inflammatory factor, involved in the release of chemokines, adhesion and aggregation between cells, etc., and plays an important role in the occurrence and development of cardiovascular diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟陈少贤刘志明
Owner SUREXAM BIO TECH
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