Sequencing primer pair for qualitatively detecting human BRAF V600E gene mutation and kit thereof

A technique for sequencing primers and qualitative detection, applied in DNA/RNA fragments, recombinant DNA technology, etc., to achieve strong specificity, high sensitivity and specificity, and good accuracy

Active Publication Date: 2014-07-09
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no product on the market that uses pyrophosphate technology to detect the genetic polymorphism of BRAF V600E

Method used

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  • Sequencing primer pair for qualitatively detecting human BRAF V600E gene mutation and kit thereof
  • Sequencing primer pair for qualitatively detecting human BRAF V600E gene mutation and kit thereof
  • Sequencing primer pair for qualitatively detecting human BRAF V600E gene mutation and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of kit

[0035] 1. Design and synthesis of primers

[0036] Using QIAGEN PyroMark Assay Design software, design upstream and downstream amplification primers and sequencing primers for the 1799 site (T>A) of exon 15 of the BRAF gene. The most important thing in the kit that affects the accuracy of the kit is the primers, including amplification primers and sequencing primers. In the early stage of design, we designed multiple sets of primers for comparison (see Figure 5); the primers were all synthesized by professional companies. Purified by HPLC, wherein the 5' end of the forward amplification primer shown in SEQ ID NO.1 is labeled with biotin.

[0037] Table 1 Mutation site and type

[0038] Mutation

[0039] The amplified sequence is shown in Table 2:

[0040] Table 2. Sequences of specific amplification primers and sequencing primers

[0041]

[0042] 2. Selection of reference substance

[0043] A synthetic oligonucle...

Embodiment 2

[0048] Embodiment 2: the use of kit

[0049] 1. Sample amplification

[0050] Take the blank control, quality control substance, positive control substance and sample DNA respectively as PCR reaction templates, add UNG enzyme, Taq polymerase, specific PCR reverse amplification primers, and PCR reaction solution containing PCR forward amplification primers In the process, a PCR reaction system is formed for PCR amplification.

[0051] The main components of the system are as follows (Table 4):

[0052] Table 4

[0053]

[0054] 2. PCR reaction procedure

[0055] After setting up the PCR reaction program, put the reaction tube into the fluorescent PCR instrument (ABI7500) to start amplification. The reaction program is as follows (Table 5):

[0056] Table 5. PCR reaction program

[0057]

[0058] 3. Pyrosequencing

[0059] The sequencing operation was performed according to the standard operating procedure of pyrophosphate, and the main steps were: sample preparation...

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PUM

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Abstract

The invention provides a sequencing primer pair for qualitatively detecting human BRAF V600E gene mutation and a kit thereof, belonging to the field of detection for in-vitro nucleic acid. The kit comprises a uracil DNA (deoxyribonucleic acid) glycosylase, a Taq polymerase, PCR (polymerase chain reaction) solution, a PCR amplification primer, a pyrosequencing primer and a positive reference substance. The kit provided by the invention is high in sensitivity, good in specificity, capable of sequencing a PCR product on a pyrosequencer after performing a simple treatment, simple and convenient in operation, short in reaction time, higher in sensitivity compared with golden standard-capillary electrophoresis sequencing, and more suitable for mutation analysis.

Description

technical field [0001] The invention relates to the field of in vitro nucleic acid detection, in particular to a sequencing primer set and kit for distinguishing human BRAFV600E gene mutations in clinical samples. Background technique [0002] The full name of the BRAF gene is human mouse sarcoma virus (v-raf) oncogene homolog B1 (v-raf mourine sarcoma viral oncogene homolog B1, B-raf), which encodes a B-type mitogen-activated protein kinase-dependent kinase RAF kinase (RAF) is a key component of the RAS / RAF / MEK / MARK signaling pathway, which regulates cell growth, proliferation, and apoptosis, and when altered, may lead to tumor formation. BRAF missense mutations occur in nearly 8% of human tumors, mainly in colorectal cancer, melanoma and papillary thyroid carcinoma. Different frequency of BRAF gene mutation exists in various gastrointestinal tumors. Therefore, this mutation can be used as a diagnostic and prognostic molecular biological marker and a gene target for the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 周姗
Owner CHANGSHA 3G BIOTECH
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