LSP (linear scorpion primer) and Kit for detecting human BRCA1 (breast cancer susceptibility gene 1) mutations

A kit and human technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of high requirements for materials and technology, environmental and operator hazards, and limited sensitivity, etc. The effect of reducing primer dimers, reducing the amount of primers, and high fluorescence signal-to-noise ratio

Active Publication Date: 2017-11-03
厦门安普利生物工程有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

It needs to amplify, purify, and sequence the sample, which takes a long time, is costly, and has relatively high requirements for materials and technology. The most important thing is that the sensitivity is not high, and it can only detect mutations with a content greater than 30%. Gene detection is harmful to the environment and operators, so it has certain limitations in clinical application

Method used

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  • LSP (linear scorpion primer) and Kit for detecting human BRCA1 (breast cancer susceptibility gene 1) mutations
  • LSP (linear scorpion primer) and Kit for detecting human BRCA1 (breast cancer susceptibility gene 1) mutations
  • LSP (linear scorpion primer) and Kit for detecting human BRCA1 (breast cancer susceptibility gene 1) mutations

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Experimental program
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Embodiment 1

[0049] (1) Sample

[0050] Plasmid preparation: The following gene fragments were synthesized by General Biosystems (Anhui) Co., Ltd. (respectively, the sequences shown in Table 2), and TA cloned into PGEM-T-EASY vector to obtain recombinant bacteria.

[0051] Table 2 Sequence list of plasmids and their corresponding gene fragments

[0052]

[0053]

[0054] Expand the culture of the recombinant bacteria, and use TaKaRa MiniBEST Plasmid Purification Kit to extract the plasmids with the target gene fragments: S4P plasmid, A7C plasmid, G10L plasmid, M18T plasmid, C24A plasmid, H1732del plasmid, G1731dup plasmid, G1735L plasmid, G1738G plasmid, A1739G Plasmid, H1746A plasmid, L1750A plasmid.

[0055] 1. Negative reference (for different normal people's genomic DNA)

[0056] The blood DNA of different normal Asians can be extracted.

[0057] 2. Positive reference product (for a sample with a mutation content of 0.1%)

[0058] The concentrations of plasmids and normal hum...

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Abstract

The invention discloses an LSP (linear scorpion primer) and kit for detecting human BRCA1 (breast cancer susceptibility gene 1) mutations, wherein 5' end of the LSP is provided with a linear probe having different dual-fluorescent labels, 3' end is provided with a sequence fragment complementary to a template, and the two sequence fragments are linked via spacer 18; when the template exists, the LSP and the template can be extended by annealing to synthesize a template complementary chain; after the template complementary chain is synthesized, the 5' end probe portion of the LSP may bind with the chain, forming a scorpion-like shape; a downstream primer is extended along the 3' end of the template complementary chain; the probe portion is sheared under the action of a polymerase so as to emit light. The LSP of the invention has good specificity and high sensitivity, approximate genes are free of mutual interference, and detection results show higher fluorescent signal-to-noise ratio.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an LSP primer and a kit for detecting human BRCA1 gene mutation. Background technique [0002] Breast cancer susceptibility gene 1 (breast cancer susceptibility gene 1, BRCA1) is a tumor suppressor gene, the gene is located in the 17q12-21 region, the genome length is about 80Kb, the coding region is 5711bp, a total of 24 exons, encoding a gene with 1863 protein of amino acid residues. BRCA1 protein has five characteristic domains, including N-terminal zinc finger domain, BRCA1 C-terminal motif (BRCT), Rad51 binding region, nuclear localization region and transcriptional active region. BRCA1 protein is involved in DNA damage repair, transcriptional activation and repression, regulation of cell cycle, negative regulation of centrosome duplication, negative regulation of tumor growth, and plays an important role in maintaining genome stability. If the BRCA1 gene is mutated, the damag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 雷彩霞魏劭魏超
Owner 厦门安普利生物工程有限公司
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