Primer combination and its application for detecting braf gene mutation in trace tissues

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

Active Publication Date: 2019-12-03
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 amplification (NASBA), self-sequencing sequence replication (3SR) and strand displacement replication (SDA) are all isothermal amplification methods, but their specificity for the amplification of target sequences is not strong
[0006] despite this, BRAF The detection method of mutation is still based on the detection of tumor tissue samples as the gold standard, but in clinical practice, the detection is often limited due to insufficient tissue samples

Method used

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  • Primer combination and its application for detecting braf gene mutation in trace tissues
  • Primer combination and its application for detecting braf gene mutation in trace tissues
  • Primer combination and its application for detecting braf gene mutation in trace tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: In the DNA template of 1000pg BRAF Gene Mutation Detection

[0033] 1. Experimental materials

[0034] 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.

[0035] 2. Experimental procedures and results

[0036] 2.1 Pre-amplification

[0037] (1) Primer dilution

[0038] 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:

[0039] ;

[0040] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put them on ice for 20 minutes;

[0041] (3) Then add the following system: ...

Embodiment 2

[0058] Embodiment 2: In the DNA template of 500pg BRAF Gene Mutation Detection

[0059] 1. Experimental materials

[0060] 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.

[0061] 2. Experimental steps

[0062] 2.1 Pre-amplification

[0063] (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:

[0064] ;

[0065] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put it on ice for 20 minutes.

[0066] (3) Then add the following system:

[0067] ;

[0068] Af...

Embodiment 3

[0083] Example 3: In the DNA template of 100pg BRAF Gene Mutation Detection

[0084] 1. Experimental materials

[0085] 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.

[0086] 2. Experimental steps

[0087] 2.1 Pre-amplification

[0088] (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.

[0089] Configure the following system:

[0090] ;

[0091] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put it on ice for 20 minutes.

[0092] (3) Then add the following system:

[0093] ;

[00...

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PUM

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Abstract

The invention discloses a primer combination for detecting mutation of a BRAF gene in trace tissue. The primer combination comprises a pre-amplification primer group, a primer group for detecting No.11 exon mutation and a primer group for detecting No.15 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 BRAF 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 BRAF gene, the DNA concentration of the detectable sample is low up to 100 pg, and all mutation of a No.11 exon and a No.15 exon of the BRAF gene can be simultaneously detected. The detection method is high in sensitivity and specificity, low in cost and suitable for BRAF 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 BRAF Primer combination for gene mutation detection and its application in tumor drug selection and disease diagnosis. Background technique [0002] BRAF The gene is an active DNA sequence that can transfect NIH3T3 cells and was first discovered and cloned in human Ewing sarcoma by Ikawa et al. in 1988. The BRAF gene belongs to the RAF family together with the ARAF and CRAF genes. It is named as the murine sarcoid virulence carcinogenic homologue B1. It is located on human chromosome 7q34 and is about 190 kb long. It encodes a protein of 783 amino acids with a relative molecular mass of 84436. CR1, CR2 and CR3 three conserved regions. BRAF is an important transduction factor in the Ras-Raf-MEK-ERK signal transduction pathway. Its functional coding region consists of 2510 base pairs, and it mainly acts through the serine-threonine protein kinase in the silk prote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113
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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