Liquid phase chip and specific primer to test apo E gene and single nucleotide polymorphism (SNP) of 9p21 segment of chromosome

A liquid phase chip and detection solution technology, which is applied in the field of molecular biology, can solve the problems of no detection products, easy contamination of samples, high false positive rate, etc., and achieve the effects of improving detection accuracy, strong scalability, and avoiding cross-reaction

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, the detection of cardiovascular disease risk is increasing day by day at home and abroad, and products for detecting apo E gene polymorphism 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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  • Liquid phase chip and specific primer to test apo E gene and single nucleotide polymorphism (SNP) of 9p21 segment of chromosome
  • Liquid phase chip and specific primer to test apo E gene and single nucleotide polymorphism (SNP) of 9p21 segment of chromosome
  • Liquid phase chip and specific primer to test apo E gene and single nucleotide polymorphism (SNP) of 9p21 segment of chromosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 The apo E gene and chromosome 9p21 segment SNP detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032] For apo E two common SNP sites C609T (ε2, rs7412) and T471C (ε4, rs429358) and SNP sites G22115503C (rs1333049), A22086055G (rs10757274), A22114477G (rs10657278), A2221 , A22105959G (rs2383207) and A22088619G (rs2891168), respectively design specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0034]

[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 s...

Embodiment 2

[0052] Example 2 Detection of samples using apo E gene and chromosome 9p21 segment SNP detection liquid chip

[0053] The formula of described various solutions is as follows:

[0054] 50mM MES buffer (pH5.0) formula (250ml):

[0055] 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

[0056] 2×Tm hybridization buffer

[0057] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

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 Technology Service Co., Ltd.

[0061] 1. Sam...

Embodiment 3

[0127] Example 3 Detection of SNP sites of apo E and chromosome 9p21 segment by liquid chip with different ASPE primers

[0128] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0129] Taking the C609T site of the apo E gene and the G22115503C site mutation detection liquid chip of the chromosome 9p21 segment as examples, the specific primer sequences of the 3' end of the ASPE primer were designed for the wild type and mutant type of C609T and G22115503C, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.16. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.33-SEQ ID NO.48. The specific design is shown in the following table (Table 9). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example ...

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Abstract

The invention discloses a specific primer and a liquid phase chip to test apo E gene and single nucleotide polymorphism (SNP) of the 9p21 segment of chromosome. The liquid phase chip mainly comprises a pair of allele specific primer extension (ASPE) primers. The specific primer has the SEQ ID No.17 and SEQ ID No.18 corresponding to the C609T SNP site, extension primers and / or SEQ ID No. 19 and SEQ ID No.20 corresponding to the T471C SNP site. The tag sequence is from SEQ ID No.1 to SEQ ID No.16. Micro particles which contain specific anti-tag sequence and have different color codes are respectively contained, and the anti-tap sequence is from SEQ ID No.33 to SEQ ID No.48. The invention also discloses a liquid phase chip to test the apo E gene and the SNP of the 9p21 segment of chromosome. The test result of the liquid phase chip fully matches that of a sequencing method. The prepared liquid phase chip to test the apo-E gene and the SNP of the 9p21 segment of chromosome 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 liquid phase chip for detecting apoE gene and chromosome 9p21 section SNP sites and specific primers. Background technique [0002] Apolipoprotein E (Apolipoprotein E, apo E) is one of the important apolipoproteins in plasma. Apo E is mainly involved in the transport, storage and excretion of lipids in the process of liver synthesis, secretion and metabolism, and has the functions of repairing tissues, inhibiting platelet aggregation and immune regulation. The human apo E gene is located on the long arm of chromosome 19, region 13, band 2 (19q132). The full length of the gene is 3.7 kb, containing 4 exons and 3 introns, and its cDNA length is 1.163 kb. The precursor of apo E protein consists of 317 amino acids and contains 18 amino acid signal peptides, and the mature Apo E protein consists of 299 amino acids. The Apo E gene has three al...

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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