Kit for quantitatively detecting breast cancer resistance protein (BCRP) mutation

A kit, chain reaction technology, applied in DNA/RNA fragments, introduction of foreign genetic material using vectors, fluorescence/phosphorescence, etc., can solve problems such as inability to quantify

Inactive Publication Date: 2011-07-27
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 quantitatively detecting breast cancer resistance protein (BCRP) mutation
  • Kit for quantitatively detecting breast cancer resistance protein (BCRP) mutation
  • Kit for quantitatively detecting breast cancer resistance protein (BCRP) mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Human fresh tumor tissue, paraffin-embedded tissue, peripheral blood, pleural effusion, human Genomic DNA Extraction from Cell Lines

[0029] 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, S1-M1-80), head and neck cancer cell lines (U87), cervical cancer (Hela), sarcoma cell lines (Mes-SA, Saos-2 and A204).

[0030]Human fresh tumor tissues, peripheral blood, and paraffin-embedded tissues that we tested included NSCLC, mesothelioma, colon cancer, malignant melanoma, renal cancer, esophageal cancer, thyroid cancer, malignant tumor, and ovarian cancer.

[0031] Specimen DNA Extraction

[0032] Can use the DNA extraction kit of Qiagen Company, Promega Company, Roche Company to extract sample genomic DNA, use Gene Company Nanodrop ND1000 type ...

Embodiment 2

[0087] Example 2: Preparation of plasmid standards containing mutant and wild-type detection sequences

[0088] 1. Wild-type plasmid construction ( figure 1 ， figure 2 )

[0089] 1.1 Preparation of the carrier

[0090] The TA cloning vector pMD18-T was purchased from TAKARA Company.

[0091] 1.2 Preparation of insert fragments

[0092] 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):

[0093] Table 1: PCR reaction system (50μl)

[0094]

[0095]

[0096] Table 2: PCR Primers

[0097]

[0098] Table 3: PCR Amplification Conditions

[0099]

[0100] 1.3 After recovering the target fragment using the QIAgen gel recovery kit, it was ligated into the pMD18-T vector (purchased from TAKARA Company) by TA cloning.

[0101]1.4 The newly constructed plasmid was massively amplified in Escherichia coli DH5α...

Embodiment 3

[0113] Example 3: Taking lung cancer and cervical cancer samples as examples: human cell lines, human fresh tumors Detection of BCRP mutation in tumor tissue, peripheral blood, and paraffin-embedded tissue genomic DNA

[0114] 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.

[0115] 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.

[0116] Table 5: Real-time quantitative PCR reaction system (20μl / tube)

[0117]

[0118] To detect the mutation of codon 482 of the BCRP gene, three sys...

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Abstract

The invention relates to a method and a kit for detecting breast cancer resistance protein (BCRP) gene mutation related to treatment effect of molecular targeted anti-cancer medicines, in particular to a fluorescence quantitative polymerase chain reaction (PCR) detection method and a kit for detecting mutation in a BCRP genic mutation hotspot region, and application thereof. By the invention, the mutation of a specific BCRP gene locus is detected, the treatment effects of anti-tumor medicines such as mitoxantrone MX, adriamycin, daunorubicin, etoposide, topotecan, Irinotecan, CPT-11, cis-platinum, taxol, vinblastine and the like are predicted, and guidance is provided for clinical individualized medication schemes for patients suffering from tumors.

Description

Background of the invention [0001] Multidrug resistance (MDR) is an important drug resistance mechanism of tumors and one of the main reasons for the failure of tumor chemotherapy. In human drug-resistant tumor cells, MDR includes several transporters: P-g protein (P-glyeoprotein, P-gp), multidrug resistance-accosiated protein (MRP1-7) and breast cancer resistance protein (breast cancer resistance protein, BCRP), these proteins belong to the ATP binding box (adenosine triphosphate binding cassette, ABC) membrane transport protein superfamily. They act as drug pumps, which can reduce the concentration of cytotoxic drugs in cells and make tumor cells resistant to a variety of tumor drugs. As a newly discovered family member, BCRP plays an important role in multidrug resistance. [0002] The human ABCG2 gene contains 16 exons with a total length of 66kb, located on chromosome 4q22, and is the only ABC transporter found on chromosome 4 so far (AllikmetsR, et al, Cancer Res, 1998...

Claims

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Application Information

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