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, achieve more accurate test results, overcome low sensitivity, and improve detection accuracy. rate effect

Active Publication Date: 2013-04-24
SUREXAM BIO TECH
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  • 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
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Effect test

Embodiment 1

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

[0029] 1. ASPE Primers

[0030] Nine common SNPs targeting 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". ASPE primer sequences are shown in the table below:

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

[0032]

[0033]

[0034]

[0035] 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 sequence (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.

[0036] ...

Embodiment 3

[0114] Example 3 Liquid-phase chip detection of CYP19A1 gene SNP with different ASPE primers

[0115] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0116] 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 the ASPE primer was designed for the wild type and mutant type of rs4646 (G>T), while the 5' end of the ASPE primer The Tag sequence is selected from 6 of SEQ ID NO.1-SEQ ID NO.18. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.37-SEQ ID NO.54. The specific design is shown in the following table (Table 6). 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.

[0117] Table 6 Design of liquid phase chip preparation

[0118]

[0...

Embodiment 4

[0125] Example 4 Detection of CYP19A1 Gene SNP by Different Spacer Arm Liquid Chips

[0126] 1. Design of liquid phase chip preparation (selection of spacer arm)

[0127] Taking the detection liquid chip of CYP19A1 gene rs10046 (C>T) site mutation as an example, to find out the influence of different spacer arm liquid phase chips on the detection of CYP19A1 gene SNP, aiming at 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 corresponding The anti i-tag sequence of tag sequence complementary pairing selects SEQ ID NO.37-SEQ ID NO.54. The spacer arm of the present embodiment is (CH 2 )12, the specific design is shown in the following table (Table 8). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described...

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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, in particular to a CYP19A1 gene SNP detection liquid chip and a detection method. Background technique [0002] In recent years, aromatase inhibitors (Aromatase inhibitors, AIs) have received more and more attention and application in the treatment of breast and ovarian cancer due to their strong specificity, high efficacy, and small side effects. Aromatase belongs to the cytochrome P450 family, which can catalyze the conversion of androgen into estrogen. It is the rate-limiting enzyme in the synthesis of estrogen. closely related to the occurrence of cancer. 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 that the third-generation AIs drugs, such as letrozole and anastrozole, can effectively inhibit the transform...

Claims

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Application Information

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