Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination

A primer combination and primer set technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem of insufficient tissue sample acquisition, complicated operation, and low specificity of target sequence amplification, etc. problem, to achieve the effect of good practical application value, high amplification efficiency and low price

Active Publication Date: 2016-09-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of the last base mismatch in ARMS technology cannot completely block the amplification of wild-type DNA, there is a risk of false positives, and it can only be detected for specific sites
In addition, such as high performance liquid chromatography, capillary electrophoresis, etc., require special equipment, and the operation is complicated, which is not conducive to large-scale clinical promotion
Nucleic acid sequence amplif

Method used

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  • Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination
  • Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination
  • Primer combination for detecting mutation of PIK3CA gene in trace tissue and application of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: In the DNA template of 1000pgPIK3CA Gene Mutation Detection

[0032] 1. Experimental materials

[0033] phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (Primer Mix), 2×Pfu Mix, primer19-primer22, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0034] 2. Experimental procedures and results

[0035] 2.1 Pre-amplification

[0036] (1) Primer dilution

[0037] Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM. Configure the following system:

[0038] ;

[0039] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put them on ice for 20 minutes;

[0040] (3) Then add the following system: ...

Embodiment 2

[0057] Embodiment 2: In the DNA template of 500pg PIK3CA Gene Mutation Detection

[0058] 1. Experimental materials

[0059] phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (Primer Mix), 2×Pfu Mix, primer19-primer22, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0060] 2. Experimental steps

[0061] 2.1 Pre-amplification

[0062] (1) Primer dilution. Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM. Configure the following system:

[0063]

[0064] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put it on ice for 20 minutes.

[0065] (3) Then add the following system:

[0066]

[0067] Afte...

Embodiment 3

[0081] Example 3: In the DNA template of 100pg PIK3CA Gene Mutation Detection

[0082] 1. Experimental materials

[0083]phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (Primer Mix), 2×Pfu Mix, primer19-primer22, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0084] 2. Experimental steps

[0085] 2.1 Pre-amplification

[0086] (1) Primer dilution. Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM.

[0087] Configure the following system:

[0088]

[0089] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put it on ice for 20 minutes.

[0090] (3) Then add the following system:

[0091]

[0092]...

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PUM

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Abstract

The invention discloses a primer combination for detecting mutation of a PIK3CA gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.9 exon mutation and a primer group for detecting No.20 exon mutation. According to a method, a trace amount of DNA is pre-amplified to obtain a high-concentration DNA template, and the mutation condition of the PIK3CA gene in a sample is detected by combining a direct sequencing method; by applying the primer combination to preparation of a detection reagent for detecting mutation of the PIK3CA gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.9 exon and a No.20 exon of the PIK3CA gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for PIK3CA gene mutation detection on a clinical tumor patient and has the very good clinical application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a PIK3CA Primer combination for gene mutation detection and its application in tumor drug selection and disease diagnosis. Background technique [0002] Phosphatidylinositol-3-kinase (PI3K) is a phosphatidylinositol kinase that can phosphorylate the third hydroxyl group of the inositol ring, and has dual activities of phosphatidylinositol kinase and serine-threonine protein kinase (Fruman , D. A. et al.2014), regulating various biological functions such as cell proliferation, survival, migration, apoptosis and cell cycle. The PIK3CA gene encodes the class IA PI3K catalytic subunit P110α, which is located at 3q26.3 and contains 21 exons (Volinia, S. et al. 1994). KANG S et al found that, PIK3CA Gene plays an oncogene role in a variety of tumors, present in about 30% of human solid tumors PIK3CA Gene mutations, 80%-90% of which occur in the 9th and 20th exons of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2531/113
Inventor 盛苗苗罗瑛唐文如李珊珊王芳赵月光张继虹贾舒婷吴晓明刘静周若宇旦菊花
Owner KUNMING UNIV OF SCI & TECH
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