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Method and kit for detecting human PIK3CA gene mutation

A kit and technology for detecting people, applied in the fields of biotechnology and medicine, to achieve the effect of convenient and fast detection

Inactive Publication Date: 2012-07-04
苏州科贝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Although the existing kits use the HRM method, in practice, subsequent PCR enrichment and DNA sequencing are often required to genotype the detected mutations. This is because there are many mutation sites in PIK3CA. Genotyping mutations

Method used

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  • Method and kit for detecting human PIK3CA gene mutation
  • Method and kit for detecting human PIK3CA gene mutation
  • Method and kit for detecting human PIK3CA gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Selection of samples and extraction of genomic DNA

[0062] The collected genomic DNA comes from whole blood, cells, fresh tissue, and paraffin-fixed tissue. The extraction method refers to the operation manual provided by the kit, and some optimizations have been made at the same time.

[0063] Whole blood genomic DNA of healthy people was used as the wild-type control of the experiment, and was extracted using the BloodGen Mini Kit of Kangwei Century;

[0064] The genomic DNA derived from the OMC-1 cell line was used as a control for the homozygous mutant type (1633G>A) of exon 9 of the PIK3CA gene in the experiment,

[0065] The genomic DNA derived from the MCF-7 cell line was used as the control of the PIK3CA gene codon No. 9 heterozygous mutant (1633G>A) in the experiment, and was extracted using the DNA FlexGen DNAKit of Kangwei Century;

[0066] The genomic DNA derived from the SK-OV-3 cell line was used as a control for the homozygous mutant type (31...

Embodiment 2

[0076] Example 2: Identification of mutation sites

[0077] HRM technology was used to detect the genotypes of the E542K and E545K codons of exon 9 of the PIK3CA gene and the H1047R codon of exon 20 of the PIK3CA gene.

[0078] 1. The specific primers and probes for exon 9 are as follows:

[0079] SEQ ID No: 5, Forward primer:

[0080] 5'-CAGCTCAAAGCAATTTCTACACGAGAT-3'

[0081] SEQ ID No: 6, Reverse primer: 5'-TTAGCACTTACCTGTGACTCCATAGA-3'

[0082] SEQ ID No: 3, specific probe: 5'-TCTTTCTCCTGCTCAGTGAT-3'

[0083] The specific primers and probes for exon 20 are as follows:

[0084] SEQ ID No: 11, Forward primer: 5'-GCAAGAGGCTTTGGAGTATTTCAT-3'

[0085] SEQ ID No: 12, Reverse primer: 5'-TGCTGTTTAATTGTGTGGAAGATCC-3'

[0086] SEQ ID No: 4, specific probe: 5'-CCACCATGATGTGCATCATTC-3'

[0087] 2. Amplify the fragments near the E542K and E545K codons of exon 9 and the H1047R codon of exon 20 respectively by PCR; prepare the mixed solution: add 1 μl of the genomic DNA solution p...

Embodiment 3

[0091] Example 3: Kit Sensitivity Verification

[0092] Taking exon 9 of the PIK3CA gene as an example, the homozygous mutant OMC-1 genome and the wild-type genome extracted from whole blood were mixed in a certain proportion, so that the homozygous mutant genome accounted for 0.05% of the total DNA amount. The concentration of the above mixed genomic DNA and wild-type genomic DNA was adjusted to 10 ng per microliter.

[0093] 1. The specific primers and probes for exon 9 are as follows:

[0094] SEQ ID No: 5, Forward primer:

[0095] 5'-CAGCTCAAAGCAATTTCTACACGAGAT-3'

[0096] SEQ ID No: 6, Reverse primer: 5'-TTAGCACTTACCTGTGACTCCATAGA-3'

[0097] SEQ ID No: 3, specific probe: 5'-TCTTTCTCCTGCTCAGTGAT-3'

[0098] 2. Amplify some fragments near the E542K and E545K codons of exon 9 by PCR; prepare the mixed solution: add 1 μl of the genomic DNA solution prepared before, 2 μl of PCR buffer (10×), 1.6 μl of dNTP, 0.1 μl of Taq DNA polymerization Enzyme, 0.4 μl of forward prime...

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Abstract

The invention relates to a method and kit for detecting gene mutation, and specifically relates to a method and kit for detecting PIK3CA gene mutation. The invention is characterized in that the kit comprises two specific probes for performing genotyping on a 9 exon and a 20 exon of the PIK3CA gene, wherein the specific probe for the 9 exon contains a nucleotide sequence of E545K and E542K codons, and the specific probe for the 20 exon contains a nucleotide sequence of an H1047R codon. By using the conventional PCR (polymerase chain reaction) amplification in combination with the Cold-PCR amplification product enrichment technology and the high-resolution melting curve analysis technology, the kit provided by the invention can complete the judgment on the genotyping of a sample.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a method and kit for detecting PIK3CA gene mutation. Background technique [0002] The PIK3CA gene is the intracellular homologue of the retroviral v-p3k oncogene. The encoded class I phosphatidyl 3-kinases (phosphatidylinositol 3-kinases, PI3Ks) are heterologous composed of a p110 catalytic subunit and a p85 regulatory subunit. Metadimer, activated by growth factor receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), insulin receptor, etc. After activation, PI3Ks catalyze the phosphorylation of phosphatidylinositol 4-phosphate (PI-4-P) and phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) into phosphatidylinositol 3,4-bisphosphate (PI -3,4-P2) and phosphatidylinositol 3,4,5 triphosphate (PI-3,4,5-P3, Ptd InsP3). Thereby activating a series of proteins, including regulation of cell proliferation, survival and cell cycle regulation, involving many...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张泓姬云侯青
Owner 苏州科贝生物技术有限公司
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