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Primers and probes for detecting human K-ras gene mutation as well as use method thereof

A probe, a human technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems that cannot meet the needs of non-invasive mutation detection, and achieve fast detection speed and selectivity Strong, highly sensitive effects

Active Publication Date: 2009-12-23
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods cannot meet the increasing needs of non-invasive mutation detection, so the ability to accurately and non-invasively detect such rare mutations poses a challenge to existing detection technologies

Method used

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  • Primers and probes for detecting human K-ras gene mutation as well as use method thereof
  • Primers and probes for detecting human K-ras gene mutation as well as use method thereof
  • Primers and probes for detecting human K-ras gene mutation as well as use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Use the reagents in the kit to detect K-ras gene G in clinically collected paraffin tissue samples G T>G A T mutation as an example. According to the comparative analysis of the wild-type gene sequence and the mutant gene sequence of exon 12 of K-ras gene published by Cosmic data, a pair of primers and probes were designed as K-ras-M1-F, K-ras-M1-R, K-ras-P. According to the wild-type gene sequence analysis of the HGH gene published by Cosmic data, a pair of primers and probes were designed: HGH-F, HGH-R, and HGH-P. According to the wild-type gene sequence analysis of exon 4 of the human gene published by Cosmic data, a pair of primers and probes were designed as EX-4-F, EX-4-R,

[0096] K-ras-M1-F: TGGTAGTTGGAGCTGA

[0097] K-ras-M1-R: CTGCACCAGTAATATG

[0098] K-ras-M2-F: TGGTAGTTGGAGCTGC

[0099] K-ras-M2-R: CTGCACCAGTAATATG

[0100] K-ras-M3-F: TGGTAGTTGGAGCTGT

[0101] K-ras-M3-R: CTGCACCAGTAATATG

[0102] K-ras-M4-F: TGGTAGTTGGAGCTA

[0103] K-ras-M4-R: ...

Embodiment 2

[0145] Take the reagents in the kit to detect the K-ras gene GGT>AGT mutation in clinically collected paraffin tissue samples as an example. According to the comparative analysis of the wild-type gene sequence and mutant gene sequence of exon 12 of K-ras gene published by Cosmic data, a pair of primers and probes were designed as K-ras-M4-F, K-ras-M4-R, K-ras-M4-R, K-ras-P. According to the wild-type gene sequence analysis of the HGH gene published by Cosmic data, a pair of primers and probes were designed: HGH-F, HGH-R, and HGH-P. According to the wild-type gene sequence analysis of exon 4 of the human gene published by Cosmic data, a pair of primers and probes were designed as EX-4-F, EX-4-R, and K-ras-M1-F: TGGTAGTTGGAGCTGA

[0146] K-ras-M1-R: CTGCACCAGTAATATG

[0147] K-ras-M2-F: TGGTAGTTGGAGCTGC

[0148] K-ras-M2-R: CTGCACCAGTAATATG

[0149] K-ras-M3-F: TGGTAGTTGGAGCTGT

[0150] K-ras-M3-R: CTGCACCAGTAATATG

[0151] K-ras-M4-F: TGGTAGTTGGAGCTA

[0152] K-ras-M4-R:...

Embodiment 3

[0194] Use the reagents in the kit to detect K-ras gene G in clinically collected paraffin tissue samples G C>G A C mutation as an example. According to the comparative analysis of the wild-type gene sequence and mutant gene sequence of exon 13 of K-ras gene published by Cosmic data, a pair of primers and probes were designed as K-ras-M7-F, K-ras-M7-R, K-ras-M7-R, K-ras-P. According to the wild-type gene sequence analysis of the HGH gene published by Cosmic data, a pair of primers and probes were designed: HGH-F, HGH-R, and HGH-P. According to the wild-type gene sequence analysis of exon 4 of the human gene published by Cosmic data, a pair of primers and probes were designed as EX-4-F, EX-4-R, and K-ras-M1-F: TGGTAGTTGGAGCTGA

[0195] K-ras-M1-R: CTGCACCAGTAATATG

[0196] K-ras-M2-F: TGGTAGTTGGAGCTGC

[0197] K-ras-M2-R: CTGCACCAGTAATATG

[0198] K-ras-M3-F: TGGTAGTTGGAGCTGT

[0199] K-ras-M3-R: CTGCACCAGTAATATG

[0200] K-ras-M4-F: TGGTAGTTGGAGCTA

[0201] K-ras-M4-R...

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Abstract

The invention relates to primers and probes for detecting human K-ras gene mutation as well as use method thereof, relating to detection of gene mutation. In the invention, the fluorescent quantitative PCR technology and the fluorescence dual ring probes technology are combined to develop a multiple real-time fluorescent PCR method capable of fast detecting gene mutation and design the relevant primers. The method is used for detecting mutation of the twelfth and the thirteenth codons of the K-ras gene, mutant type primers designed specific to different mutation types are respectively adopted to detect samples to be detected, only the mutation type samples can be smoothly amplified to obtain double-strand DNA products which can be combined with DNA probes to send out fluorescence signals to be detected. The invention can detect mutate type genes with content being 1 / 1000 in the samples to be detected, has the advantages of high speed, convenience and simplicity, safety, high sensitivity, high throughput and low cost and can be applied to K-ras gene mutation screening of numerous clinical samples.

Description

technical field [0001] The invention relates to gene mutation detection primers and a method for using the primers, in particular to a primer capable of detecting seven hotspot mutations in human K-ras genes and a method for using the same. Background technique [0002] The important factors of tumor occurrence and development are uncontrolled cell proliferation and differentiation, cell apoptosis disorder and abnormal angiogenesis pathway, which is a process of accumulation of multiple gene changes. Studies in recent years have shown that K-ras gene activity is closely related to cell proliferation and apoptosis. Mutation of the K-ras gene prolongs the survival of cells that should normally die. Overexpression of the K-ras gene can also increase drug resistance and ultraviolet light-induced apoptosis. The possible mechanism is that the K-ras gene enhances the cell's ability to break down peroxidative The ability of hydrogen to inhibit apoptosis. K-ras gene can induce cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 阮力何东华郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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