Probe for detecting K-ras gene mutation, primer, kit and method

A kit and probe technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of low sensitivity, simultaneous qualitative and quantitative, cumbersome operation, etc.

Inactive Publication Date: 2015-06-10
SHANGHAI CHANGHAI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention aims to solve the technical problems that the current methods for detecting K-ras gene mutations cannot be qualitative and quantitative at the same time, the operation is cumbersome, and the sensitivity is low, and a probe, primer, a

Method used

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  • Probe for detecting K-ras gene mutation, primer, kit and method
  • Probe for detecting K-ras gene mutation, primer, kit and method
  • Probe for detecting K-ras gene mutation, primer, kit and method

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Embodiment 1

[0039] 1. Main source of reagents:

[0040] 1.1 Designing PNAs

[0041] PNA is a PNA designed for the 12th and 13th codons of the wild-type K-ras gene, synthesized by Panagene Company in South Korea, and its composition is: NH2-CCTACGCCACCAGCTCC-COOH (SEQ ID NO: 1).

[0042] 1.2 Design of Taqman MGB probe

[0043] The K-ras-M13-FAM probe and the K-ras-M13-VIC probe were synthesized by AppliedBiosystems, USA, and their compositions are:

[0044] K-ras-M13-FAM probe:

[0045] FAM-TTGCCTACGCDACCAGCTCCAACT-MGB (SEQ ID NO: 2) (Note D stands for A or G or T);

[0046] K-ras-M13-VIC probe:

[0047] VIC-TTGCCTACGDCACCAGCTCCAACT-MGB (SEQ ID NO: 3) (Note D stands for A or G or T).

[0048] 1.3PCR primers

[0049] PCR primers were synthesized by AppliedBiosystems, USA.

[0050] Upstream primer F: 5'-CCTTATGTGTGACATGTTCTAATATAGTC-3' (SEQ ID NO: 4);

[0051] Downstream primer R: 5'-TTCTGAATTAGCTGTATCGTCAAGG-3' (SEQ ID NO: 5).

[0052] 1.4 TaqMan Gene Expression Master Mix (TaqMan ...

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Abstract

The invention discloses a probe for detecting K-ras gene mutation. The probe is Taqman MGB fluorescence probe in specific binding with a mutant type sequence of the 13th code of K-ras gene. The invention further discloses a primer and a kit for detecting K-ras gene mutation, the kit comprises the Taqman MGB fluorescent probe in specific binding with the mutant type sequence of the 13th code of the K-ras gene, a primer pair for amplifying the nucleotide sequence of the 13th code containing the K-ras gene; and PNA designed aiming at the 13th code GCC of the wild type K-ras gene. The invention further discloses a dual-fluorescent mark probe real-time quantitative detection method in K-ras gene mutation. The kit and the detection disclosed by the invention are capable of qualitatively and quantitatively detecting the mutation of the 13th code of K-ras gene at the same time, the sensitivity is high, the operation is simple and convenient, and wide application prospect is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology and is applied to biological science research and clinical molecular diagnosis, in particular to a probe, primer, kit and method for detecting the mutation of the 13th codon of K-ras gene. Background technique [0002] K-ras gene (GenBank: NC_000012) mutations are expressed in a variety of cancers, such as pancreatic cancer, colon cancer, blood cancer, thyroid cancer, bile duct cancer, lung cancer, endometrial cancer, ovarian cancer, skin cancer, spermatogonia Among them, K-ras gene mutation plays an important role in the occurrence and development of pancreatic cancer and colon cancer. It has been reported in the literature that point mutations at codon 12, 13 or 61 of the K-ras gene can be detected in pancreatic cancer tissue, pancreatic juice or feces, blood, and colon cancer tissue. Studies have shown that the detection of K-ras gene mutation in somatic DNA can be used as a method for early diagn...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6851
Inventor 李兆申高军金晶李泉江唐旖旎朱艳萍丁佳寅诸娴王玉琼
Owner SHANGHAI CHANGHAI HOSPITAL
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