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Kit and method for detecting CDA (cytidine deaminase) genetic polymorphism by use of pyrosequencing technique

A pyrosequencing method and gene polymorphism technology, applied in the field of molecular biology, can solve the problems of decreased clearance rate of gemcitabine, decreased concentration of metabolite dFdU, decreased serum CDA activity, etc., to facilitate the construction of standardized operating procedures and sample volume Small, easy-to-operate effect

Inactive Publication Date: 2012-08-22
湖南宏灏基因生物科技有限公司
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Problems solved by technology

Further studies have found that CDA*3 can lead to decreased clearance of gemcitabine in patients with pancreatic cancer, increased serum gemcitabine concentration, decreased serum CDA activity, decreased metabolite dFdU concentration, and reduced clearance of gemcitabine in vivo

Method used

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  • Kit and method for detecting CDA (cytidine deaminase) genetic polymorphism by use of pyrosequencing technique
  • Kit and method for detecting CDA (cytidine deaminase) genetic polymorphism by use of pyrosequencing technique
  • Kit and method for detecting CDA (cytidine deaminase) genetic polymorphism by use of pyrosequencing technique

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Embodiment 1

[0025] CDA-pyroF (upstream primer): 5'-AGG GTG CAA CAT AGA AAA T-3' (SEQ ID NO.2);

[0026] CDA-pyroR (downstream primer): 5'-TTG CCC TGA AAT CCT TGT ACC-3' (SEQ ID NO.3);

[0027] Sequencing primer: 5'-TGT GCT GAA CGG ACC-3' (SEQ ID NO.4);

[0028] 1. DNA extraction

[0029] 1.1 The preparation and inspection of reagent materials before the experiment are as follows:

[0030] (1) Check the shelf life of the kit and ensure that ethanol has been added to Wash Buffer 1 and 2, and tick the corresponding mark on the bottle; (2) Isopropanol (if not available, it can be replaced by absolute ethanol) and 75% ethanol ; (3) 1.5mL Eppendorf tubes and various pipette tips within the validity period of autoclaving.

[0031] 1.2 Take out the EDTA anticoagulant tube containing whole blood from the refrigerator at 4°C, and mix it upside down several times;

[0032] 1.3 Make a mark on the 1.5mL Eppendorf tube corresponding to the unique identification of the specimen;

[0033] 1.4 Pipett...

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Abstract

The invention discloses a kit and method for detecting CDA (cytidine deaminase) genetic polymorphism by use of the pyrosequencing technique. The kit checks the CDA genetic polymorphism which specifically refers to the rs60369023 (G>A) single nucleotide polymorphism. The kit comprises the primer shown by SEQ ID NO.2-4. The kit and method disclosed by the invention can realize accurate, quick and high-flux detection on the CDA genetic polymorphism so as to realize safe, reasonable and effective personalized administration of substrate gemcitabine.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit and a method for detecting CDA gene polymorphism by pyrosequencing. Background technique [0002] Gemcitabine is an antimetabolite drug clinically used to treat tumors such as pancreatic cancer, lung cancer, breast cancer and bladder cancer. Gemcitabine is a cytidine nucleoside derivative that enters cells through the human balanced nucleoside carrier 1 (hENT1). In the cytoplasm, gemcitabine is phosphorylated by deoxycytidine kinase (dCK) to difluorodeoxycytidine monophosphate (dFdCMP), which is further activated to difluorodeoxycytidine diphosphate (dFdCDP) and difluorodeoxycytidine triphosphate (dFdCDP). Phosphate (dFdCTP) activity product. dFdCDP inhibits the activity of nucleotide reductase (RR), reducing the raw material deoxynucleoside diphosphates necessary for DNA synthesis and repair in cells, thereby further inhibiting DNA synthesis; dFdCTP is a DNA ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 周宏灏
Owner 湖南宏灏基因生物科技有限公司
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