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Specific sequence, liquid phase chip and method for SNP detection of TPMT gene

A detection solution and specificity technology, applied in the field of molecular biology, can solve problems such as poor repeatability of detection results, failure to meet clinical needs, easy contamination of samples, etc., to achieve accurate and reliable detection results, improve detection accuracy, and strong expansion sexual effect

Active Publication Date: 2010-03-17
广州益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current products are mainly based on traditional solid-phase chips, which are expensive, and the sensitivity is not high, and the repeatability of the test results is poor.
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 the detection throughput, ordinary PCR methods and fluorescent quantitative PCR cannot meet the clinical needs
However, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis technology and the allelic difference analysis method based on TaqMan technology can only detect one mutation at a time, which is time-consuming and laborious.

Method used

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  • Specific sequence, liquid phase chip and method for SNP detection of TPMT gene
  • Specific sequence, liquid phase chip and method for SNP detection of TPMT gene
  • Specific sequence, liquid phase chip and method for SNP detection of TPMT gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1TPMT gene SNP detection liquid phase chip mainly includes:

[0031] 1. ASPE primers

[0032] Specific primer sequences were designed for three common SNP sites of TPMT: G238C, G460A and A719G, respectively. ASPE primers consist of "Tag + specific primer sequences". The ASPE primer sequences are shown in the table below:

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

[0034]

[0035]

[0036]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 mutant or wild type specific primer sequence (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.

[0037] 2. Microspheres coated with anti-tag sequences

[0038] According to the designed ASPE-specific primer...

Embodiment 2

[0050] Embodiment 2 uses the TPMT gene SNP detection liquid chip described in embodiment 1 to detect the various solutions described in the detection of clinical samples. The formulations are as follows:

[0051] 50mM MES buffer (pH5.0) formula (250ml):

[0052] 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

[0053] 2 x Tm hybridization buffer

[0054] reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH 8.0

SigmaT3038

0.2M

50ml

5M NaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0055] After filtration, store at 4°C.

[0056] The ExoSAP-IT kit was purchased from the US USB company.

[0057] Biotin-labeled dCTP was purchased from Shanghai San...

Embodiment 3

[0116] Example 3: Detection of TPMT gene SNP sites by liquid-phase chips using different ASPE primers

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

[0118] Taking the liquid chip for the detection of the G238C site mutation of the TPMT gene as an example, the specific primers at the 3' end of the ASPE primer were designed for the wild type and mutant type of G238C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1- 3 in SEQ ID NO. 6, correspondingly, the anti-tag sequences that are coated on the microspheres and are complementary to the corresponding tag sequences are selected as SEQ ID NO. 13-SEQ ID NO. 18. The specific design is shown in the following table (Table 6). 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.

[0119] Table 6

[0120]

[0121]

[0122...

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Abstract

The invention discloses a liquid phase chip, a specific prime and a method for the SNP detection of TPMT gene, the liquid phase chip comprises wild-type and mutant-type specific ASPE primer pairs designed respectively in accordance with mutant sites of each type, microspheres respectively enveloped with specific anti-tag sequences, and primers for amplifying target sequences having SNP sites of TMPT gene G238C, G460A and / or A719G. The prepared liquid phase chip for the SNP detection of TPMT gene has quite excellent signal-to-noise ratio and, basically, no cross reaction is present between thedesigned probe and the anti-tag sequence. The ASPE primers designed according to the invention has quite excellent specificity and can accurate distinguish the mutant sites of each type. The detectionmethod has simple steps and convenient operation, can complete the detection of 3 types of mutant sites in one step, and can avoid plenty of uncertain factors existing in the process of repeated operation, therefore, the accuracy of the detection can be enhanced enormously.

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 sequence, liquid phase chip and method for detecting SNP of TPMT gene. Background technique [0002] Thiopurines have been used for nearly 40 years in clinical anti-cancer treatment of leukemia, solid tumors and immunosuppressive treatment after organ transplantation, including 6-mercaptopurine (6-MP), sulfur Azathioprine (AZA) and 6-thioguanine (6-thioguanine, 6-TG). For example, 6-MP is one of the main drugs for maintenance therapy of acute lymphoblastic leukemia (ALL), while AZA is one of the most commonly used drugs for the treatment of rheumatoid arthritis and Crohn's disease in recent years. However, these drugs can produce severe and even life-threatening hematological toxicity, leading to forced interruption of treatment, resulting in treatment failure, so their rational use has attracted much attention in cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森何嘉英杨惠夷
Owner 广州益善医学检验所有限公司
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