Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit

A technology for gene expression and fluorescence quantification, applied in DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as reducing the sensitivity of anti-folate drugs, and achieve the effect of high sensitivity, good specificity and good application prospects

Inactive Publication Date: 2016-01-20
SUZHOU BEISIMAI MEDICAL INSTR CO LTD
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Problems solved by technology

A retrospective analysis of phase III randomized head-to-head studies of pemetrexed and docetaxel as second-line treatment of NSCLC found that the median survival for pemetrexed and docetaxel in patients with squamous cell carcinoma was 6.2 months, 7.4 months (P=0.018), docetaxel was better than pemetrexed;

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  • Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit
  • Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit
  • Fluorescent quantitative PCR (polymerase chain reaction) detection kit for TYMS (thymidylate synthase) gene expression quantity and application of kit

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Experimental program
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Embodiment 1

[0026] (1) Sample preparation: take surgically removed tissue from the patient, and extract RNA (concentration not less than 100ng, A260 / 280 not less than 1.9).

[0027] (2) Sample treatment: Take 100ng of RNA, add 5 μl of reverse transcription reagent (promega kit), add double distilled water to 20 μl, and perform reverse transcription reaction according to the following conditions: 15 minutes at 25°C; 1 hour at 50°C; 85°C 5 minutes at °C; 5 minutes at 4°C.

[0028] (3) Fluorescent quantitative PCR reaction:

[0029] Use the reverse transcription product in (2) as a template: Take 5 μl of the above reverse transcription template and add it to a 0.1 ml 8-strip tube (containing 15 μl of fluorescent quantitative PCR reagent). At the same time, add standard products with corresponding gradient concentrations into the standard tube (containing 15 μl of fluorescence quantitative PCR reaction reagent); add 5 μl of the above cDNA template into the internal reference tube (containing...

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Abstract

The invention relates to a fluorescent quantitative PCR (polymerase chain reaction) kit for detecting human TYMS (thymidylate synthase) gene mutation. The kit mainly comprises specific primers and a PCR reaction reagent, wherein sequences of the specific primers are as follows: the sequence of an upstream primer is represented as 5'-ATCTTCCTCTGATGGCGCTG-3', and the sequence of a downstream primer is represented as 5'-AAAGGTCTGGGTTCTCGCTG-3'. The kit has the main benefits as follows: the kit is used for detecting the human TYMS gene expression quantity, is good in specificity and high in sensitivity and has the better application prospect.

Description

(1) Technical field [0001] The invention relates to a thymidylate synthase (Thymidylatesynthase, TYMS) gene fluorescent quantitative PCR detection kit and application thereof. (2) Background technology [0002] The thymidylate synthase (Thymidylatesynthase, TYMS) gene is located at 18p11.32, with a full length of 16Kb and 7 exons. The expression product of TYMS is thymidylate synthase (TS). TS is a dimeric protein composed of two identical units, and the molecular weight of each subunit is 36kDa. Thymidylate synthase is widely found in the cytoplasm of the human body, and its main function is to bind 5,10-2 methylenetetrahydrofolate (CH2FH4) and catalyze the methylation of deoxyuridine acid (BUMP) into deoxythymidylate (dTMP ), is the rate-limiting enzyme for the de novo synthesis of dTMP, and dTMP is necessary for nucleic acid biosynthesis and the only source of intracellular thymidine. Inhibiting TS activity can inhibit the proliferation of tumor cells. [0003] Immunohi...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘小龙张云杨诚高运臻李雨阳
Owner SUZHOU BEISIMAI MEDICAL INSTR CO LTD
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