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Primers for detecting mutation of human B-raf gene V600E, primer probe composition and kit

A primer-probe and kit technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve the problems of poor wild-type background tolerance, low amplification efficiency, and high detection cost , to achieve the effect of short detection time, high amplification efficiency and high sensitivity

Pending Publication Date: 2020-09-11
河南赛诺特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of low sensitivity, low amplification efficiency, high detection cost, and poor tolerance of wild-type background in the common clinical B-raf gene V600E mutation detection method

Method used

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  • Primers for detecting mutation of human B-raf gene V600E, primer probe composition and kit
  • Primers for detecting mutation of human B-raf gene V600E, primer probe composition and kit
  • Primers for detecting mutation of human B-raf gene V600E, primer probe composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] This embodiment provides a primer for detecting the V600E mutation region of the B-raf gene, including a forward primer and a reverse primer. The forward primer includes three parts arranged in the following order along the 5' end to the 3' end:

[0067] 1) The first sequence, the peptide nucleic acid PNA sequence used to identify mutation hotspots and hybridize with the wild-type template, which completely matches the wild-type template sequence and has one base mismatch with the mutant template sequence, the nucleotide sequence is as follows Shown in SEQ ID NO.2;

[0068] SEQ ID NO.2: 5'-ATCGAGATTTCACT-3' (Tm: 65°C)

[0069] 2) Spacer, which connects the 3' end of the first sequence and the 5' end of the second sequence, is C6;

[0070] 3) The second sequence is combined with the sequence upstream of the mutation site, 3-6 bp of its 3' end sequence overlaps with the 5' end of the first sequence, and the nucleotide sequence is shown in SEQ ID NO.4;

[0071] SEQ ID NO...

Embodiment 2

[0076] This embodiment provides a primer for detecting the V600E mutation region of the B-raf gene, including a forward primer and a reverse primer. The forward primer includes three parts arranged in the following order along the 5' end to the 3' end:

[0077] 1) The first sequence, the peptide nucleic acid PNA sequence used to identify mutation hotspots and hybridize with the wild-type template, which completely matches the wild-type template sequence and has one base mismatch with the mutant template sequence, the nucleotide sequence is as follows Shown in SEQ ID NO.1;

[0078] SEQ ID NO.1: 5'-TCGAGATTTCACTGTAGCTA-3' (Tm: 76°C)

[0079] 2) Spacer, connecting the 3' end of the first sequence and the 5' end of the second sequence, which is C3;

[0080] 3) The second sequence, combined with the sequence upstream of the mutation site, 3-6 bp of its 3' end sequence overlaps with the 5' end of the first sequence, and the nucleotide sequence is shown in SEQ ID NO.5;

[0081] SEQ...

Embodiment 3

[0086] This embodiment provides a primer for detecting the V600E mutation region of the B-raf gene, including a forward primer and a reverse primer. The forward primer includes three parts arranged in the following order along the 5' end to the 3' end:

[0087] 1) The first sequence, the peptide nucleic acid PNA sequence used to identify mutation hotspots and hybridize with the wild-type template, which completely matches the wild-type template sequence and has one base mismatch with the mutant template sequence, the nucleotide sequence is as follows Shown in SEQ ID NO.3;

[0088] SEQ ID NO.3: 5'-CATCGAGATTTCACT-3'

[0089] 2) Spacer, which connects the 3' end of the first sequence and the 5' end of the second sequence, is C18;

[0090] 3) The second sequence, combined with the sequence upstream of the mutation site, 3-6 bp of its 3' end sequence overlaps with the 5' end of the first sequence, and the nucleotide sequence is shown in SEQ ID NO.6;

[0091] SEQ ID NO.6: 5'-CTGA...

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Abstract

The invention discloses primers for detecting mutation of human B-raf gene V600E, primer probe composition and a kit. The kit comprises a B-raf gene V600E mutation detection primer pair and detectionprobe, an internal quality control primer pair and a quality control probe. A forward primer of the detection primer pair comprises three parts arranged in the following order from the 5' end to the 3' end: 1) a first sequence: the first sequence is used for identifying mutation hot spots, is a peptide nucleic acid PNA sequence hybridizing with a wild type template, is completely matched with thesequence of the wild type template and has a base mismatched with the sequence of a mutant template; 2) Spacer: the Spacer is connected with the 3' end of the first sequence and the 5' end of a secondsequence; 3) the second sequence: the second sequence is bound with a sequence upstream of a mutation site, 3-6bp of the sequence of the 3' end of the second sequence overlaps with the 5' end of thefirst sequence, Tm value of the first sequence is 6.3-11.3 DEG C higher than that of the second sequence. The kit is simple and quick, has sensitivity up to 1 permillage, and has high specificity, and the false positive rate is greatly reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology gene detection, and in particular relates to a primer, a primer-probe composition and a kit for detecting the V600E mutation of human B-raf gene. Background technique [0002] The BRAF gene, located on chromosome 7q34, is a member of the RAF family and encodes a serine / threonine protein kinase. This protein plays a role in regulating the MAPK / ERK signaling pathway, affecting cell division, differentiation and secretion. BRAF gene mutations are common in some malignant tumors, such as lung cancer, colorectal cancer, melanoma, non-small cell lung cancer, non-Hodgkin's lymphoma, etc. Among them, the mutation frequency of BRAF gene V600E is the highest, because this mutation is often associated with poor clinical prognosis, so BRAF gene V600E mutation is an important target for clinical detection. [0003] At present, there are many methods for B-raf gene mutation detection, and the reported methods...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2545/101C12Q2545/113C12Q2563/107C12Q2561/101Y02A50/30
Inventor 刘超王知丰李三华李卫娟齐华刘文弟
Owner 河南赛诺特生物技术有限公司
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