Method and kit for detecting individualized medication related gene in tumor chemotherapy

An individual and gene technology, applied in the field of molecular biology, can solve the problems of low throughput, low resolution, high background signal, etc., and achieve the effect of reducing usage, simple and stable reagent consumables

Active Publication Date: 2017-04-05
DALIAN GENTALKER BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0044] However, the current molecular detection methods are mainly based on restriction fragment length polymorphism analysis, PCR...

Method used

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  • Method and kit for detecting individualized medication related gene in tumor chemotherapy
  • Method and kit for detecting individualized medication related gene in tumor chemotherapy
  • Method and kit for detecting individualized medication related gene in tumor chemotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] The design of embodiment 1 primer

[0096] (1) PCR amplification primers:

[0097] According to the individualized drug-related genes of the selected tumor chemotherapy individualized drugs to be detected, the amplification primers specific for each individualized drug-related gene are designed, and the amplification primers can amplify a segment including the mutation site. DNA sequence. The amplification primer has at least 15 bases at the 3' end that completely match the gene sequence it is targeting, and has a 10 base (acgttggatg) tag sequence at the 5' end to distinguish the amplification primer from the extension primer.

[0098] (2) Mass spectrometry extension primer:

[0099] Design an extension primer, the length of the extension primer is 15-28 bases, and its 3' end is located at the last base in the extension direction of the drug-related mutation site, and the extension primer only extends when the extension reaction occurs One base, the extended base is ...

Embodiment 2

[0115] The preparation of embodiment 2 detection template

[0116] Mutation samples are tumor tissues (numbered 2-A and 2-B) of 2 tumor patients in the hospital and 4 normal human tissue samples (numbered 3-A, 4-A, 5-A, 6-A) DNA was extracted (extracted using Tiangen DNA kit, and the operation method was extracted according to the instructions of the kit).

Embodiment 3

[0117] Example 3 Detection method of genes related to individualized medication of tumor chemotherapy

[0118] (1) Using the DNA extracted in Example 2 as a template, use the PCR amplification primers in Example 1 to amplify by PCR to obtain the target sequence amplification product. See Table 4 for the PCR amplification reaction system. Among them, all reagents were purchased from Agena Bioscience.

[0119] Table 4: PCR amplification reaction system

[0120]

[0121]

[0122] The PCR reaction conditions were 94°C for 2 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and a total of 45 cycles of amplification; finally, extension at 72°C for 5 minutes. In this example, the DNA extracted in Example 2 was used as the DNA template for PCR amplification. At the same time, sterile double distilled water was used as a negative control. The control sample and the test sample were reacted and tested according to t...

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Abstract

The invention provides a method and a kit for detecting individualized medication related genes in tumor chemotherapy. Compared with the prior art, the method and the kit have the following advantages: 1) the reagent consumables are simple and stable, and expensive reagents, such as fluorescent dyes, special enzymes and the like, are avoided; 2) the reaction can be carried in a micro scale system, thus reducing usage of the sample and the consumables; 3) theoretically, the genes can be recognized as long as one copied amplified DNA fragment is amplified since mass spectrum technology can directly detect the molecular weight (mass-to-charge ratio) of DNA and can directly determine the types of basic groups (i.e., signal conversion in any form is unnecessary), so that the method has high sensitivity; and 4) the mass spectrum technology also is automatic and allows high-throughput detection, so that when the mass spectrum is combined with multi-primer extension technology, several gene loci can be detected in the same reaction system, thereby greatly reducing work load and increasing detection throughput.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to multiple PCR amplification technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). And more specifically, the present invention relates to a method and a kit for detecting genes related to individualized medicine of tumor chemotherapy. Background technique [0002] Chemotherapy drugs are drugs for the treatment of diseases caused by malignant tumors. These drugs can act on different links of tumor cell growth and reproduction, destroy the cell cycle of tumor cells, and inhibit or kill tumor cells. For example, first-line chemotherapy drugs such as 5-fluorouracil and cisplatin kill tumor cells by interfering with tumor cell DNA synthesis and destroying tumor cell DNA. Chemotherapy is one of the main means of treating tumors at present. [0003] Clinically used anti-tumor chemotherapeutic drugs all have different degr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/16C12Q2533/101C12Q2565/627C12Q2537/143
Inventor 董新波刘琦赵金银方楠于闯许立志李杰
Owner DALIAN GENTALKER BIO-TECH CO LTD
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