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Primers for detecting hot-spot mutation sites of KRAS gene, kit and PCR (polymerase chain reaction) method for primers or kit

A mutation site and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high detection cost, inability to detect clinical specimens on a large scale at the same time, and high price of detection instruments. Achieve the effect of increasing PCR differentiation

Active Publication Date: 2015-08-26
沈阳优吉诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] In view of this, the present invention provides a primer, a kit and a PCR method for detecting KRAS gene hotspot mutation sites, to at least solve the complex interpretation of results, high price of detection instruments, difficult operation, and certain false positives existing in previous kits. One or more problems such as negative and false positive, high testing cost, low clinical popularity, inability to test clinical specimens on a large scale at the same time

Method used

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  • Primers for detecting hot-spot mutation sites of KRAS gene, kit and PCR (polymerase chain reaction) method for primers or kit
  • Primers for detecting hot-spot mutation sites of KRAS gene, kit and PCR (polymerase chain reaction) method for primers or kit
  • Primers for detecting hot-spot mutation sites of KRAS gene, kit and PCR (polymerase chain reaction) method for primers or kit

Examples

Experimental program
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Effect test

Embodiment 1

[0121] Example 1: Preparation of wild-type and mutant-type positive plasmids directed at 7 hotspot mutations of the KRAS gene

[0122] The KRAS gene encodes a 21kDa KRAS protein, which plays an important regulatory role in the signal transduction pathways of tumor cell growth and angiogenesis. The most common mutations of the KRAS gene are point mutations, and the mutation sites are mainly in the 12th codon, 13th codon of exon 2 and the 61st codon of exon 3, which will lead to the prognosis of patients with colorectal cancer and lung cancer Poor, and resistant to targeted drugs—KRAS tyrosine kinase inhibitors and anti-KRAS antibodies. KRAS gene mutation detection has been included in the 2011 edition of the NCCN (National Comprehensive Cancer Network) Chinese version of the clinical practice guidelines for colorectal cancer and the 2011 edition of the clinical practice guidelines for non-small cell lung cancer to help doctors choose cancer treatments The most effective treatm...

Embodiment 2

[0153] Example 2: Design and specificity screening of allele-specific primers (ASP)

[0154] For Gly12Asp of the KRAS gene, wild-type and a series of mutant-specific primers were designed as follows:

[0155] 1. KRAS Gly12Asp-WT-F: aaacttgtggtagttggagcagt (SEQ No. 26)

[0156] 2. KRAS Gly12Asp-Mut-F: aaacttgtggtagttggagcaga (SEQ No. 27)

[0157] 3. KRAS Gly12Asp-Mut-F1: aacttgtggtagttggagcaga (SEQ No. 28)

[0158] 4. KRAS Gly12Asp-Mut-F2: acttgtggtagttggagcaga (SEQ No. 29)

[0159] 5. KRAS Gly12Asp-Mut-F3: aaacttgtggtagttggagctca (SEQ No. 30)

[0160] 6. KRAS Gly12Asp-Mut-F4: aacttgtggtagttggagctca (SEQ No. 31)

[0161] 7. KRAS Gly12Asp-Mut-F5: acttgtggtagttggagctca (SEQ No. 32)

[0162] At the same time, a specific Taqman probe was designed and synthesized: FAM-tgtggacgaatatgatccaacaatagagg-BHQ1 (SEQ No.33).

[0163] Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0164] Then use the above 7 primers to pair with the common d...

Embodiment 3

[0166] Embodiment 3: ASP sensitivity screening

[0167] Then use the No. 7 primer to pair with the KRAS Gly12Asp-R primer respectively, and use the mutant recombinant plasmid according to 10 6 、10 5 、10 4 、10 3 、10 2 , 10, and 0 for serial dilution, plus Taqman specific probes, for sensitivity verification on a fluorescent quantitative PCR instrument. No. 7 mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the mutation site of the KRAS Gly12Asp gene screened according to our method, as shown in Table 3.

[0168] By analogy, we respectively designed and screened KRAS-Gly12Ala, Gly12Val, Gly12Ser, Gly12Arg, Gly12Cys and Gly13Asp mutation-specific primers, as shown in Table 3.

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Abstract

The invention discloses primers for detecting hot-spot mutation sites of a KRAS gene, a kit and a PCR (polymerase chain reaction) method for the primers or the kit. The primers and the kit include a wild-type specific upstream primer, a specific upstream primer of a Gly12Asp mutation site, a specific upstream primer of a Gly12Ala mutation site, a specific upstream primer of a Gly12Val mutation site, a specific upstream primer of a Gly12Ser mutation site, a specific upstream primer of a Gly12Arg mutation site, a specific upstream primer of a Gly12Cys mutation site, a specific upstream primer of a Gly13Asp mutation site and a downstream primer shared by the wild-type specific upstream primer and a mutant-type specific upstream primer. The kit has the advantages of simplicity, quickness, accuracy and low price for detection and the like, and provides a powerful tool for scientific research and clinical analysis of the hot-spot mutation sites of the KRAS gene.

Description

technical field [0001] The invention relates to the field of molecular biology gene detection, and in particular provides a primer, a kit and a PCR method for detecting hot spot mutation sites of KRAS gene. Background technique [0002] There are three genes in the RAS gene family that are closely related to human tumors—HRAS, KRAS, and NRAS, which are located on chromosomes 11, 12, and 1, respectively. The KRAS gene encodes a 21kDa KRAS protein, also known as the p21 gene. In the RAS gene family, KRAS has the greatest impact on human cancer. It is like a molecular switch and plays an important regulatory role in the signal transduction pathway of tumor cell growth and angiogenesis. Normal KRAS gene can inhibit tumor cell growth. , and once a mutation occurs, the normal KRAS protein cannot be produced, causing intracellular signal transduction disorder, cell proliferation out of control and cancerous. [0003] The KRAS mutated gene is a common oncogene, and its mutations c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2535/125C12Q2561/101
Inventor 高劲松张英杰李星颐田密魏潇魏奇
Owner 沈阳优吉诺生物科技有限公司
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