JAK1 gene specific mutation detection kit and detection method thereof

A technology for detection kits and detection methods, applied in the field of molecular biology, can solve problems such as the inability to distinguish between wild-type genes and mutant genes

Active Publication Date: 2020-04-07
黄志清
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of them are PCR-based methods, however, conventional PCR amplification cannot di

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  • JAK1 gene specific mutation detection kit and detection method thereof
  • JAK1 gene specific mutation detection kit and detection method thereof
  • JAK1 gene specific mutation detection kit and detection method thereof

Examples

Experimental program
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Embodiment 1

[0038] Embodiment 1 system and method construction

[0039] Collect colorectal cancer patients who have been clinically and pathologically diagnosed and underwent resection in Fujian Province, and these patients have had cancer gene or whole exome or whole genome sequencing including JAK1 gene in a third-party gene company for clinical diagnosis and treatment (next generation sequencing method). The postoperative paraffin-embedded tissue samples were collected from these colorectal cancer patients with full informed consent and clinical diagnosis and treatment, and their genetic testing reports and data were collected. A total of 26 patients with colorectal cancer met the criteria, and the JAK1 gene mutation status (known) of the patients detected by next-generation gene sequencing is shown in Table 4. In this example, the above-mentioned JAK1 gene g.[227149insA] mutation detection kit and detection method based on the specific double-stranded DNA peptide ligase dDPlaseI will...

Embodiment 2

[0064] Embodiment 2 sample verification

[0065] A further collection of 20 middle-aged female colorectal cancer patients (40-60 years old, with an average age of 53.5 years old) diagnosed clinically and pathologically, and no metastasis was found at the time of diagnosis. During the examination, an additional 5ml of peripheral venous blood was drawn, and the cell-free DNA in the blood, namely cfDNA, was extracted with the QIAamp Circulating Nucleic Acid kit from Qiagen, Germany. Using the extracted blood cfDNA as a template, the method of the present invention and the conventional PCR-Sanger sequencing method were used to detect the g.[227149insA] mutation of the JAK1 gene with reference to Example 1, and the results are shown in Table 5. There are 9 patients with JAK1 gene g.[227149insA] mutation detected by the method of the present invention, but conventional PCR-Sanger sequencing only detects 2 of them. During the follow-up of these 20 patients, 7 of the 9 patients whose...

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Abstract

The invention provides a JAK1 gene g.[227149insA] mutation detection kit based on double-stranded DNA peptide ligase and a method thereof. The invention is characterized in that a target gene mutationfragment is directly captured and enriched before exponential amplification, and then the target gene mutation fragment is specifically amplified and detected. The JAK1 gene g.[227149insA] detectionkit mainly comprises dDPlaseI ligase, a dDPlaseI ligase buffer solution, RNase H enzyme, magnetic polypeptide Ps, an RNA primer Pr, a DNA primer Pn, a DNA fragment Pd and a DNA primer Pm, wherein theamino acid sequence of dDPlaseI ligase is as shown in SEQ ID NO: 1. The detection method mainly comprises the following four parts: pre-amplification, RNase H enzyme digestion, dDPlaseI enzyme connection and magnetic bead separation, and specific amplification. The detection kit and the detection method provided by the invention have the characteristics of strong specificity, accuracy, high efficiency, simplicity, convenience and economy, can effectively detect JAK1 gene g.[ 227149insA] mutation with the content as low as 1% in a sample, and can be applied to prediction and diagnosis and treatment of liver metastasis of middle-aged female colorectal cancer patients.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a double-stranded DNA peptide ligase-based JAK1 gene g.[227149insA] mutation detection kit and a detection method. Background technique [0002] Gene is the genetic material of an organism, and the protein expressed by it bears various life functions in the organism. Mutations or mutations in genes will lead to different biological characteristics or disease states in organisms. Gene mutation is a change in the nucleotide composition or sequence of a gene in its molecular structure, mainly including point mutations, frameshift mutations, deletion mutations and insertion mutations. Gene mutations are closely related to the occurrence and development of tumors. At present, cancer has become the disease with the highest morbidity and mortality in the world. According to the data released by the National Cancer Center in 2019, there were about 3.929 million new cancer cases in my ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6869
CPCC12Q1/6886C12Q1/6869C12Q2600/156C12Q2531/113C12Q2535/101C12Q2521/327C12Q2523/308C12Q2521/501Y02A50/30
Inventor 黄志玲黄种山其他发明人请求不公开姓名
Owner 黄志清
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