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Kit for quantitative detection of K-ras mutation

A kit and chain reaction technology, applied in the field of kits for quantitative detection of K-ras mutations, can solve problems such as inability to quantify

Active Publication Date: 2011-07-06
BEIJING ACCB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other methods, such as allele-specific oligonucleotide probe hybridization, are very sensitive to hybridization conditions and require strict control of experimental conditions; restriction fragment length polymorphism experiments require considerable manpower and cannot be quantified

Method used

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  • Kit for quantitative detection of K-ras mutation
  • Kit for quantitative detection of K-ras mutation
  • Kit for quantitative detection of K-ras mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Human fresh tumor tissue, paraffin-embedded tissue, peripheral blood, pleural effusion, human Genomic DNA Extraction from Cell Lines

[0028] The human cancer cell lines we tested included non-small cell lung cancer (NSCLC) (A549, H460, H838 and H1703), breast cancer (MCF-7, BT474 and HuL 100), malignant multiple mesothelioma cell lines (H513, H2052 , H290, MS-1 and H28), colon cancer cell lines (SW480), head and neck cancer cell lines (U87), cervical cancer (Hela), sarcoma cell lines (Mes-SA, Saos-2 and A204).

[0029]Fresh human tumor tissues, peripheral blood, and paraffin-embedded tissues tested included NSCLC, mesothelioma, colon cancer, malignant melanoma, renal cancer, esophageal cancer, thyroid cancer, malignant tumor, and ovarian cancer.

[0030] Specimen DNA Extraction

[0031] Can use the DNA extraction kit of Qiagen Company, Promega Company, Roche Company to extract sample genomic DNA, use Gene Company Nanodrop ND1000 type nucleic acid trace ...

Embodiment 2

[0086] Example 2: Preparation of plasmid standards containing mutant and wild-type detection sequences

[0087] 1. Wild-type plasmid construction ( figure 1 ， figure 2 )

[0088] 1.1 Preparation of the carrier

[0089] The TA cloning vector pMD18-T was purchased from TAKARA Company.

[0090] 1.2 Preparation of insert fragments

[0091] Use the PCR method to prepare the insert fragment. The template for PCR is the sample genomic DNA extracted in step 1. The reaction system and amplification conditions are as follows (Table 1, Table 2, Table 3):

[0092] Table 1: PCR reaction system (50μl)

[0093]

[0094] Table 2: PCR Primers

[0095]

[0096] Table 3: PCR Amplification Conditions

[0097]

[0098] 1.3 After using the QIAgen Gel Recovery Kit to recover the target fragment, it was ligated into the pMD18-T vector (purchased from TAKARA Company) by TA cloning.

[0099] 1.4 The newly constructed plasmid was massively amplified in Escherichia coli DH5α strain, an...

Embodiment 3

[0111] Example 3: Taking lung cancer and cervical cancer samples as examples: human cell lines, human fresh tumors Genomic DNA K-ras mutation detection in tumor tissue, peripheral blood, and paraffin-embedded tissue

[0112] 1. The fluorescent quantitative PCR reaction template is the genomic DNA of lung cancer and cervical cancer samples extracted in Example 1 and the standard prepared in Example 2, and double distilled water is used as a negative control. In order to make a standard curve, standard dilutions were 1ng / μl, 0.5ng / μl, 0.25ng / μl, 0.125ng / μl, 0.0625ng / μl, 0.03125ng / μl.

[0113] 2. Reaction system and reaction conditions (table 2, table 5, table 6, table 7), wherein labeling probe fluorescent emission group is selected from: FAM, TET, HEX, ROX; Fluorescence quencher group is selected from: BHQ, TAMARA.

[0114] Table 5: Real-time quantitative PCR reaction system (20μl / tube)

[0115]

[0116] To detect mutations in codons 12 and 13 of the K-ras gene, six sy...

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Abstract

The invention relates to a kit for quantitative detection of K-ras mutation. The invention specifically relates to a detecting method and a detecting kit for K-ras gene mutation relevant with the curative effect of molecular targeted anti-cancer drugs. The invention especially relates to fluorescence quantification PCR detecting method for K-ras gene mutational hotspot region detecting mutational, kit and the application of the fluorescence quantification PCR detecting method. The method of the invention detects the mutation of K-ras gene at a particular locus, predicts the curative effect of molecule targeting anticancer medicament EGFR tyrosine kinases inhibitor, furthermore provides clinic individualized medication scheme for a tumour patient with guidance.

Description

Background of the invention [0001] The K-ras gene belongs to the ras oncogene family, and is also known as the p21 gene because of its encoded 21kD ras protein. K-ras gene is one of the important molecules in the EGFR signaling pathway, and its encoded product is located downstream of the EGFR pathway, and plays an important "switch" role in the growth, proliferation, angiogenesis and other processes of tumor cells. When a mutation occurs, the K-ras protein is in a continuous active state and loses normal regulation of cell growth, which can lead to continuous cell growth and prevent cell apoptosis. The mutation rate of K-ras gene in different tumor tissues is different, among which 82% of pancreatic cancer, 43% of colon cancer, 30% of lung cancer, 29% of thyroid cancer, 10% or less than 10% of bladder, liver, kidney and uterine cancer (BosJ. L. et al, Cancer Res, 1989, 49(17): 4682-4689). Since the continuous activation of mutant K-ras has nothing to do with the inhibition ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/63G01N21/64
CPCC12Q2600/156C12Q1/6886C12Q2600/106
Inventor 许军普陈钊
Owner BEIJING ACCB BIOTECH
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