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Sequencing primer for qualitative detection of TPMT genetic typing and kit thereof

A technique for sequencing primers and qualitative detection, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve strong specificity, high sensitivity and specificity, and intuitive results

Active Publication Date: 2013-01-30
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There are currently no products on the market that utilize pyrophosphate technology for TPMT genotyping

Method used

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  • Sequencing primer for qualitative detection of TPMT genetic typing and kit thereof
  • Sequencing primer for qualitative detection of TPMT genetic typing and kit thereof
  • Sequencing primer for qualitative detection of TPMT genetic typing and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of kit

[0041] 1. Design and synthesis of primers and probes

[0042] Using PyroMark Assay Design2.0 software, for exon 7 and exon 10 of TPMT and the intron region in the middle, the research shows that the current genotyping of TPMT in Han people is mainly concentrated in *3A / 3B / 3C ( William ZF et al.J Gastrointestin Liver Dis.2011;20(3).247-253.Shuta U et al.Pharmacogenetics and Genomics.2008;18.887-893.William EE et al.Journal of Clinical Oncology.2001;19( 8).2293-2301.Oreste E et al.Pharmacogenetics and Genomics.2005;15.801-815.), select specific mutation sites——TPMT G460A, A719G, in which amplification primers and sequencing primers are first purified by PAGE, and then Purified by HPLC, one of the amplification primers was labeled with biotin at the 5' end.

[0043] The amplified sequence is shown in Table 1:

[0044] Table 1. Specific amplification primers and sequencing primer sequences

[0045]

[0046] 2. Control substance...

Embodiment 2

[0055] Embodiment 2: the use of kit

[0056] 1. sample testing

[0057] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, UNG enzyme, and Taq DNA polymerase, aliquot the system, add sample DNA, and a blank control as templates to form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0058] The main components of the TPMT460 system are as follows:

[0059]

[0060] The main components of the TPMT719 system are as follows:

[0061]

[0062]

[0063] The system reaction procedure is as follows:

[0064] Table 4: PCR reaction program

[0065]

[0066] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0067] 2. Pyrosequencing

[0068] The sequencing operation was carried out according to the standar...

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Abstract

The invention provides a sequencing primer for qualitative detection of TPMT genetic typing and a kit thereof, and belongs to the field of in vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, a PCR reaction liquid, PCR primers, pyrosequencing primers and a reference substance. The kit provided by the invention has high sensitivity and good specificity; the PCR products can be simply treated for sequencing on a phosphate sequenator; and the kit has advantages of simple operation, short reaction time, and higher sensitivity than a gold standard-capillary electrophoresis sequencing, and is more suitable for SNP analysis.

Description

technical field [0001] The invention relates to the field of in vitro nucleic acid detection, in particular to a pair of sequencing primers for distinguishing *3A / 3B / 3C gene subtypes in TPMT genes in clinical samples and a kit thereof. Background technique [0002] Thiopurine S-methyltransferase TPMT (thiopurine S-methyltransferase, TPMT), is a cytoplasmic enzyme widely distributed in liver, kidney, heart, brain, lung, skeletal muscle and blood cell tissue, mainly catalyzing aromatic and heterocyclic compounds S-methylation reaction of mercapto compounds. Mercaptopurines, such as 6-mercaptopurine, 6-thioguanine and azathioprine, are clinically used as antineoplastic drugs for the treatment of acute leukemia, or as immunosuppressants widely used in organ transplantation, inflammatory bowel disease Waiting for treatment. TPMT plays a key role in the metabolism of this class of drugs. The efficacy and toxicity of mercaptopurines are related to the enzyme activity of TPMT in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈艳
Owner CHANGSHA 3G BIOTECH
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