Liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 (glutathione S transferase pi) genes

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

Active Publication Date: 2011-04-20
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection products of GSTM1, GSTT1 and GSTP1 polymorphisms are generally based on PCR technology, such as multiplex PCR, PCR-RFLP, real-time fluorescent quantitative PCR and DNA sequencing, which have the disadvantages of low sensitivity, easy sample contamination and high false positive rate. , at the same time, due to the limitation of the detection flux, it cannot meet the needs of practical applications

Method used

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  • Liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 (glutathione S transferase pi) genes
  • Liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 (glutathione S transferase pi) genes
  • Liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 (glutathione S transferase pi) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] GSTM1, GSTT1 and GSTP1 gene polymorphism detection liquid chip mainly includes:

[0025] 1. ASPE Primers

[0026] Specific primer sequences were designed for GSTM1 and GSTT1 gene deletions, A313G mutation (Ile105Val, rs1695) and C341T mutation (Ala114Val, rs1138272) of GSTP1 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0027] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0028]

[0029] 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.

[0030] 2. Microspheres coated with anti-tag...

Embodiment 2

[0041] Example 2 Detection of samples using GSTM1, GSTT1 and GSTP1 gene detection liquid chip

[0042] The formula of described various solutions is as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045] 2×Tm hybridization buffer

[0046] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0047] Store at 4°C after filtration.

[0048] ExoSAP-IT kit was purchased from US USB Company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0050] 1. Sample DNA extraction:

[0051] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0052] 2. PCR amplification of samples to be tested

[0053] Using Primer5.0 to design four pairs of primers, m...

Embodiment 3

[0114] The liquid phase chip of embodiment 3 different ASPE primers detects the mutation of GSTM1, GSTT1 and GSTP1 gene

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

[0116] Taking the A313G site mutation detection liquid chip of the GSTP1 gene 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 A313G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 ~SEQ ID NO.6, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.15~SEQ ID NO.20. 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.

[0117] Table 7 Design of liquid phase chip preparation

[...

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Abstract

The invention discloses a liquid phase chip for detecting mutation of GSTP1 (glutathione S transferase pl) genes, mainly comprising an ASPE primer consisting of a tag sequence at 5' end and a specific primer at 3' end and aiming at a mutant site, micro-balloons and an amplification primer; wherein the specific primer is SEQ ID NO.11 and SEQ ID NO.12 aiming at the A313G mutant site, and SEQ ID NO.13 and SEQ ID NO.14 aiming at the C341T mutant site; the tag sequence is selected from the group of SEQ ID NO.1 to SEQ ID NO.6; and the micro-balloons different color codes are respectively covered with the special anti-tag sequences. The liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 genes in the invention has a great signal-noise ratio; no cross reaction exists between the designed probes and the anti-tag sequences basically; the tag-tag sequence, the selection of the anti-tag tag sequence and the combinationof the tag-tag sequence with the specific ASPE primer can avoid the cross reaction, and realize parallel detection of a plurality of mutant sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a GSTM1, GSTT1 and GSTP1 gene detection liquid phase chip. Background technique [0002] Glutathione S transferases (glutathione S transferases, GSTs) are a multifunctional biphasic metabolic enzyme family that mainly catalyze the binding reaction between GSH (reduced glutathione) and electrophilic compounds, making the latter lose their binding The activity of DNA in the body, it is the main detoxification system for cells to resist damage and cancer. In addition, GSTs can also be combined with some ligands such as bilirubin and steroids to participate in the physiological reactions of the human body. Tumor cells express GSTs to protect themselves from chemotherapy drugs, and gene mutations can change the activity of GSTs, thereby changing the pharmacokinetic characteristics of drugs. In recent years, the relationship between GSTs and tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森刘志明
Owner SUREXAM BIO TECH
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