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Liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection and detection method thereof

A technology for detecting liquids and genes, applied in the field of molecular biology, can solve the problems of poor repeatability, low sensitivity, and easy contamination of samples, so as to achieve more accurate test results, overcome low sensitivity, and improve detection accuracy. rate effect

Active Publication Date: 2010-07-21
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are products on the market to detect CYP19A1 gene polymorphisms, such as Applied Biosystems' SNPGenotyping Assays series products are allelic difference analysis methods based on TaqMan technology, but they can only detect one mutation at a time, which is time-consuming and laborious; the traditional solid-phase chip method is expensive and has low sensitivity. poor repeatability
However, other PCR-based techniques for detecting SNPs, such as direct sequencing, semi-quantitative PCR, and PCR-single-strand conformational polymorphism (SSCP) detection, have low sensitivity, easy sample contamination, and high false positive rates. Due to the limitations of detection throughput, ordinary PCR methods and fluorescent quantitative PCR cannot meet the needs of detection and promotion.

Method used

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  • Liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection and detection method thereof
  • Liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection and detection method thereof
  • Liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 CYP19A1 gene SNP detection liquid chip, mainly includes:

[0029] 1. ASPE primer

[0030] 9 common SNP loci for CYP19A1: rs4646 (G>T), rs10046 (C>T), rs700519 (C>T), rs1870050 (C>A), hCV1664178 (A>C), rs12900137 (G>C) ), rs730154 (G>A), rs936306 (T>C), rs1902586 (A>G), design specific primer sequences. ASPE primers consist of "Tag + specific primer sequence". The ASPE primer sequence is shown in the following table:

[0031] Table 1 ASPE primer sequence (Tag + specific primer sequence)

[0032]

[0033]

[0034]

[0035] Each ASPE primer includes two parts, the 5'end is the specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3'end is the mutant or wild-type specific primer sequence (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. After synthesis, each primer was prepared with 10mmol / L Tris Buffer to prepare 100pmol / mL stock solution.

[0036...

Embodiment 3

[0116] Example 3 Detection of CYP19A1 gene SNP by liquid chip with different ASPE primers

[0117] 1. Design of liquid-phase chip preparation (choice of Tag sequence and Anti-Tag sequence)

[0118] Taking the detection liquid chip of CYP19A1 gene rs4646 (G>T) site mutation as an example, the specific primer sequence of the 3'end of ASPE primer was designed for the wild-type and mutant type of rs4646 (G>T), and the 5'end of ASPE primer The tag sequence is selected from 6 of SEQ ID NO.1-SEQ ID NO.18. Correspondingly, the anti-tag sequence that is coated on the microsphere and is complementary to the corresponding tag sequence is selected as SEQ ID NO.37-SEQ ID NO.54. The specific design is shown in the following table (table 6). The synthesis of ASPE primers, anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0119] Table 6 Design of liquid phase chip preparation

[0120]

[0121]

[0122] 2. Sample det...

Embodiment 4

[0127] Example 4 Detection of SNP of CYP19A1 gene by liquid chip with different spacer arms

[0128] 1. Design of liquid phase chip preparation (choice of spacer arm)

[0129] Take the detection liquid chip of CYP19A1 gene rs10046 (C>T) site mutation as an example, to explore the influence of different spacer arm liquid chip on the detection of CYP19A1 gene SNP, targeting the wild type and mutant type of rs10046 (C>T) Design the specific primer sequence at the 3'end of the ASPE primer, and the Tag sequence at the 5'end of the ASPE primer is selected from 4 of SEQ ID NO. 1-SEQ ID NO. 18. Correspondingly, the and corresponding ones coated on the microspheres The ant i-tag sequence of complementary pairing of tag sequence selects SEQ ID NO. 37-SEQ ID NO. 54. The spacer arm of this embodiment is (CH 2 ) 12. The specific design is shown in the following table (table 8). The synthesis of ASPE primers, anti-tag sequence coated microspheres, amplification primers, detection methods, etc....

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Abstract

The invention discloses a liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection, which comprises wild type and mutant type specific ASPE primers designed targeting CYP19A1 gene SNPs of rs4646, rs10046C>T, rs700519C>T, rs1870050C>A, hCV1664178A>C, rs12900137G>C, rs730154G>A, rs936306T>C and rs1902586A>G, microballoon spheres respectively coated with specific anti-tag sequences and primers used for amplifying CYP19A1 gene SNPs with target sequences to be detected. Each ASPE primer comprises a tag sequence at 5' end and a specific primer sequence at 3' end, wherein the specific primers are selected from SEQ ID NO. 19-36 and the tag sequences are selected from SEQ ID NO.1-18; and the anti-tag sequences can be correspondingly in complementary pairing with the tag sequences. The invention also discloses a CYP19A1 gene SNP detection method. The coincidence rate of the detection method provided by the invention and a sequencing method is as high as 100%; and the prepared liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection has excellent signal-to-noise rate, and basically no cross reaction exists between a design probe and the anti-tag sequences.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to a CYP19A1 gene SNP detection liquid phase chip and a detection method. Background technique [0002] In recent years, aromatase inhibitors (AIs) have received more and more attention and applications in the treatment of breast and ovarian cancer due to their strong specificity, high curative effect, and low side effects. Aromatase belongs to the cytochrome P450 family. It can catalyze the conversion of androgens to estrogen. It is the rate-limiting enzyme in the synthesis of estrogen. It is widely present in normal tissues and organs such as ovary, placenta, testis, brain, fat, bone, and breast. The occurrence of cancer is closely related. Therefore, AIs can reduce the level of estrogen by inhibiting aromatase, thereby eliminating the stimulating effect of estrogen on tumor growth. Both in vivo and in vitro studies have shown tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森李国强杨惠夷
Owner SUREXAM BIO TECH
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