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JAK2 gene specific mutation detection kit and JAK2 gene specific mutation detection method based on single-stranded RNA peptide ligase

A detection kit and detection method technology, applied in the field of molecular biology, can solve the problem of inability to distinguish between wild-type genes and mutant genes

Active Publication Date: 2020-04-14
黄志清
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of them are PCR-based methods, however, conventional PCR amplification cannot distinguish wild-type genes from mutant genes, and requires specific design or analysis

Method used

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  • JAK2 gene specific mutation detection kit and JAK2 gene specific mutation detection method based on single-stranded RNA peptide ligase
  • JAK2 gene specific mutation detection kit and JAK2 gene specific mutation detection method based on single-stranded RNA peptide ligase
  • JAK2 gene specific mutation detection kit and JAK2 gene specific mutation detection method based on single-stranded RNA peptide ligase

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Experimental program
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Effect test

Embodiment 1

[0035]Embodiment 1 system and method construction

[0036] 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 JAK2 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 JAK2 gene mutation (known) of the patients detected by next-generation gene sequencing is shown in Table 2. Since the expression of the JAK2 mutant gene with the g.[146452_146455delATGA] mutation is abnormally elevated (abnormal expression of cancer mutation genes), choosing to start mutatio...

Embodiment 2

[0056] Embodiment 2 sample verification

[0057] A further collection of 30 male patients with colorectal cancer diagnosed clinically and pathologically, and no metastases were found at the time of diagnosis, and the postoperative tumor tissue was collected after obtaining the full informed consent of the patient and meeting the requirements of clinical diagnosis and treatment, and tissue RNA from Qiagen, Germany and DNA extraction reagents to extract total RNA and genomic DNA from the sample. Using the extracted RNA as a template, the g.[146452_146455delATGA] mutation of the JAK2 gene was detected by using the method of the present invention and the conventional PCR-Sanger sequencing method with reference to Example 1, and the results are shown in Table 3. There are 7 patients with JAK2 gene g.[146452_146455delATGA] mutation detected by the method of the present invention, but conventional PCR-Sanger sequencing only detects 2 of them. Following up these 30 patients, among th...

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Abstract

The invention provides a JAK2 gene g.[146452_146455 delta ATGA] mutation detection kit and a JAK2 gene g.[146452_146455 delta ATGA] mutation detection method based on single-stranded RNA peptide ligase, wherein a target mutant gene mRNA fragment is directly captured and enriched before exponential amplification, and then the target gene mutation fragment is specifically amplified and detected. TheJAK2 gene g.[146452_146455delta ATGA] detection kit mainly comprises sRPlasseI ligase, a sRPlasseI ligase buffer solution, RNase H enzyme, magnetic polypeptide Ps, short DNA fragments P1 and P2, andprimers Pn and Pm, wherein the amino acid sequence of the sRPlasseI ligase is represented by SEQ ID NO:1. The detection method mainly comprises the following three parts of hybridization and RNase H enzyme digestion, sRPlaseI enzyme linking 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 JAK2 gene g.[146452_146455delta ATGA] mutation with the content as low as 1% in a sample, and canbe applied to prediction and diagnosis and treatment of pulmonary metastasis of male colorectal cancer patients.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a JAK2 gene g.[146452_146455delATGA] mutation detection kit and a detection method based on single-stranded RNA peptide ligase. 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/501C12Q2521/327C12Q2563/143C12Q2563/149C12Q2531/113C12Q2535/101
Inventor 黄志玲黄种山其他发明人请求不公开姓名
Owner 黄志清