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NPPA (natriuretic proatrial peptide A) gene SNP (Single Nucleotide Polymorphisms) detection specific primer and liquid phase chip

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

Inactive 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

[0003] Currently, detection methods for detecting NPPA mutations are generally PCR-based RT-PCR, fluorescent quantitative PCR, PCR-RFLP, etc., which have the disadvantages of low sensitivity, easy sample contamination, and high false positive rate. Limitations, can not meet the needs of practical applications

Method used

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  • NPPA (natriuretic proatrial peptide A) gene SNP (Single Nucleotide Polymorphisms) detection specific primer and liquid phase chip
  • NPPA (natriuretic proatrial peptide A) gene SNP (Single Nucleotide Polymorphisms) detection specific primer and liquid phase chip
  • NPPA (natriuretic proatrial peptide A) gene SNP (Single Nucleotide Polymorphisms) detection specific primer and liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 NPPA gene SNP detection liquid phase chip mainly includes:

[0023] 1. ASPE primers

[0024] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes of NPPA gene, rs5063 (A>G), rs5065 (T>C) and rs5067 (A>G). ASPE primers consist of "Tag sequence + specific primer sequence". The ASPE primer sequences are shown in the table below:

[0025] Table 1 ASPE primer sequence (Tag sequence+specific primer sequence) of NPPA gene

[0026]

[0027] Each ASPE primer consists of 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 primer fragment specific for mutant or wild type (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. After synthesis, each primer was prepared into 100pmol / mL stock solution with 10mmol / L Tris Buffer.

[0028] 2. Microspheres coated with anti...

Embodiment 2

[0040] Example 2 Detection of samples using the NPPA gene detection liquid chip described in Example 1

[0041] The formulations of the various solutions are 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 x Tm hybridization buffer

[0045] reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH 8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

[0046] Triton X-100

Sigma T8787

0.16％

0.4ml

[0047] After filtration, store at 4°C.

[0048] The ExoSAP-IT kit was purchased from the US USB company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technolo...

Embodiment 3

[0112] Example 3 Detection of NPPA gene SNP sites by liquid-phase chips with different ASPE primers

[0113] 1. Design of liquid chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0114] Taking the liquid chip for detection of NPPA gene rs5063 (A>G) site mutation 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 rs5063 (A > G), while the specific primer sequences at the 5' end of the ASPE primer were designed. The Tag sequence is selected from SEQ ID NO.1 to SEQ ID NO.6. Correspondingly, the anti-tag sequence that is complementary to the corresponding tag sequence coated on the microspheres is selected from SEQ ID NO.13 to SEQ ID NO.18. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0115]...

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Abstract

The invention discloses an NPPA (natriuretic proatrial peptide A) gene SNP (Single Nucleotide Polymorphisms) detection specific primer and a liquid phase chip. The liquid phase chip mainly comprises an ASPE primer, microballoon spheres and an amplimer; the ASPE primer comprises a tag sequence of a 5'end and a specific primer of a 3' end, wherein the specific primer aims at an SNP site; the specific primer aims at SEQ ID NO.7 and SEQ ID NO.8 of a rs5063SNP site, SEQ ID NO.9 and SEQ ID NO.10 of a rs5065SNP site and / or SEQ ID NO.11 and SEQ ID NO.12 of a rs5067SNP site; the tag sequence is selected from SEQ ID NO.1 to SEQ ID NO.6; and the microballoon spheres are respectively coated by specific anti-tag sequences and have different color codes. The result contact ratio of the liquid phase chip and a sequencing method is as high as 100 percent, and the prepared NPPA gene SNP detection liquid phase chip has very good signal-to-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 specific primer and a liquid phase chip for NPPA gene SNP detection. Background technique [0002] Atrial natriuretic peptide (ANP) belongs to the family of natriuretic peptides. ANP activates guanylate cyclase by binding to receptors and promotes the increase of intracellular cyclic guanylate levels, thereby exerting biological functions. Atrial natriuretic peptide has various effects such as natriuresis, diuresis, relaxation of vascular smooth muscle, inhibition of cell proliferation, etc. It plays an important role in maintaining blood pressure, water / sodium balance and in the pathophysiological process of cardiovascular disease. The gene encoding atrial natriuretic peptide (Natriuretic peptide precursor A, NPPA) is located on chromosome 1p36. Studies have shown that NPPA gene polymorphism plays an important role in the deve...

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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