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Kit and method for detecting K-RAS (K-rat sarcoma) gene polymorphism by pyrophosphoric acid sequencing method

A pyrosequencing method and gene polymorphism technology, which is applied in the field of molecular biology to achieve the effect of small sample size and easy operation

Inactive Publication Date: 2012-11-28
周宏灏
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At this time, although EGFR inhibitors or monoclonal antibodies block the downward transmission of EGFR activation signals, KRAS mutations do not depend on the continuous activation of upstream EGFR signals, so that downstream signals are still activated, and tumor cells are resistant to EGFR inhibitors or monoclonal antibodies. antagonism, i.e. insensitivity to

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  • Kit and method for detecting K-RAS (K-rat sarcoma) gene polymorphism by pyrophosphoric acid sequencing method
  • Kit and method for detecting K-RAS (K-rat sarcoma) gene polymorphism by pyrophosphoric acid sequencing method

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Embodiment 1

[0030] K-RAS exon 2 codon 12, codon 13 upstream primer: 5'-TGA CTG AAT ATA AAC TTG TGG TAG TTG -3' (SEQ ID NO.2);

[0031] K-RAS exon 2 codon 12, codon 13 downstream primer: 5'- TCG TCC ACA AAA TGA TTC TGA A -3' (SEQ ID NO.3);

[0032] Wherein, the 5' of the downstream primer is biotinylated;

[0033] K-RAS exon 2 codon 12 and codon 13 sequencing primers: 5'- CTT GTG GTA GTT GGA GCT -3' (SEQ ID NO.4);

[0034] 1. DNA extraction

[0035] 1.1 The preparation and inspection of reagent materials before the experiment are as follows:

[0036] (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 marks on the bottle; (2) Isopropanol (if not available, absolute ethanol can be used instead) and 75% ethanol ; (3) 1.5mL Eppendorf tubes and various pipette tips within the validity period of autoclaving.

[0037] 1.2 Take out the EDTA anticoagulant tube containing whole blood from the refrigerator at 4...

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Abstract

The invention discloses a kit for detecting the gene polymorphism of monoclonal antibody personalized medicine of an anti-epidermal growth factor receptor by a pyrophosphoric acid sequencing method and a method for detecting the gene polymorphism of the monoclonal antibody personalized medicine of the anti-epidermal growth factor receptor by the pyrophosphoric acid sequencing method. The invention particularly relates to the polymorphism of No.2 exon, No.12 codon and No. 13 codon of the K-ras (K-rat sarcoma) gene. The kit comprises the primer shown in the SEQ ID NO.3-4. The kit disclosed by the invention can be used for detecting the K-ras genic mutation exactly and quickly at high throughput, so that the safe, reasonable and effective individual administration can be realized for the pharmacy of the monoclonal antibody (such as cetuximab, nimotuzumab and panitumumab) of the anti-epidermal growth factor receptor.

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 K-RAS gene polymorphism by pyrosequencing. Background technique [0002] Anti-epidermal growth factor receptor monoclonal antibodies (such as cetuximab, nimotuzumab, panitumumab, etc.) can specifically bind to EGF receptors expressed on the surface of normal cells and various cancer cells, And competitively block the binding of EGF and other ligands, such as α transforming growth factor (TGF-α). They are IgG1 and IgG2 monoclonal antibodies against the EGF receptor respectively. After the two are specifically combined, they block the intracellular signal transduction pathway through the inhibition of the tyrosine kinase (TK) binding to the EGF receptor, thereby Inhibit the proliferation of cancer cells, induce the apoptosis of cancer cells, reduce the production of matrix metalloproteinases and vascular endothelial growth factors. Gef...

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 周宏灏
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