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Primer group for detecting PIK3CA gene mutation and application method thereof

A primer set and gene technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of low throughput of PIK3CA gene mutation detection, reduce detection cost, improve detection efficiency, The effect of cost saving

Inactive Publication Date: 2020-07-03
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, since each PCR reaction targets only one exon of the PIK3CA gene, the detection throughput of PIK3CA gene mutations is low

Method used

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  • Primer group for detecting PIK3CA gene mutation and application method thereof
  • Primer group for detecting PIK3CA gene mutation and application method thereof
  • Primer group for detecting PIK3CA gene mutation and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Design and synthesize primer sets

[0065] Step 1.1: According to the sequences of exons 1, 9 and 20 of the PIK3CA gene, design upstream and downstream primers for specifically amplifying gene exons.

[0066] For the design of primers, Primer Quest and Primer Premier 5.0 were used to design primers and analyze dimers and stem-loop mismatches. Primers were designed at both ends containing mutation sites, and the annealing temperatures of the three pairs of primers were basically consistent.

[0067] The primer set provided in this example covers at least one exon of exon 1, exon 9 and exon 20 of the PIK3CA gene. Since small sequence changes will lead to a significant decrease in primer amplification efficiency and poor specificity, multiple PCR primer sets were designed for different sites / exons, and after pre-experimental screening, the length and position of the product fragments were integrated. According to the inclusion situation, the primer set with the best ampli...

Embodiment 2

[0073] Extract DNA from the sample to be tested as an amplification template

[0074] Step 2.1: The sample to be tested can be: serum, plasma sample or collected tumor tissue sample collected from fresh peripheral blood, or oral exfoliated cells collected from a human body with a buccal swab, or fresh peripheral blood collected from a human body.

[0075] Step 2.2: Specifically, use the Tiangen Oral Swab DNA Extraction Kit (DP322), or use the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) to extract DNA from the sample, and use the NP80-touch (Germany IMPLEN) measure the concentration and purity of DNA, and preserve the DNA.

Embodiment 3

[0077] Preparation of Multiplex PCR Amplification Reaction System

[0078] Step 3.1: Using the DNA obtained in step 2.2 as an amplification template, and using the primer set synthesized in step 1.2, prepare a multiplex PCR amplification reaction system.

[0079] In this example, the DNA polymerase and buffer in the KOD FX enzyme system (article number: KFX-101) of TOYOBO Co., Ltd. were used as the basic raw materials. concentration, buffer concentration, and enzyme dosage to prepare a multiplex PCR amplification system. The specific composition of this reaction system is shown in Table 3 below.

[0080] It can be understood that the proportional amplification / reduction of the reaction system is within the protection scope of the embodiment of the present invention; the purpose of amplification can also be achieved by replacing other DNA polymerase systems and adjusting the appropriate ratio.

[0081] table 3

[0082]

[0083]

[0084] Primer Mix is ​​a mixture of prim...

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Abstract

The invention provides a primer group for detecting PIK3CA gene mutation and an application method thereof. The primer group comprises at least one primer pair of the following three primer pairs: a primer pair for detecting the No.1 exon of a PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2; a primer pair for detecting the No.9 exon of the PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primeris shown as SEQ ID NO.4; and a primer pair for detecting the No.20 exon of the PIK3CA gene, wherein the nucleotide sequence of an upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6. The gene detection flux is improved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer set for detecting PIK3CA gene mutation and an application method thereof. Background technique [0002] The PIK3CA gene is a highly mutated gene in colon cancer, gastric cancer and breast cancer, accounting for about 32%, 25% and 8% of the mutations respectively. High-frequency mutations in PIK3CA have kinase activity, resulting in dysregulation of PI3K signaling pathways at different levels. The wild type had no tumor formation, while the mutant type formed tumors at different sites with micrometastasis and invasion. Among the discovered PIK3CA gene mutations, a small part occurred in exon 1, and most of the gene mutations were concentrated in the specific sites of exons 9 and 20. [0003] At present, polymerase chain reaction (Polymerase Chain Reaction, PCR) detection tests can be designed respectively for the No. 1, No. 9 and No. 20 exons of the PIK3CA gene t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 李晓娇赵方圆智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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