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Primer used for detecting nucleotide sequence variation, composition thereof, and method

A technology of nucleic acid sequence and target sequence binding region, applied in the field of molecular biology, to achieve the effect of solving cross-reaction problems

Pending Publication Date: 2020-04-07
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention solves the cross-reaction problem existing in the detection of nucleic acid sequence variation in existing digital PCR primer-probe combinations, and has significant advantages in both specificity and sensitivity compared with the prior art

Method used

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  • Primer used for detecting nucleotide sequence variation, composition thereof, and method
  • Primer used for detecting nucleotide sequence variation, composition thereof, and method
  • Primer used for detecting nucleotide sequence variation, composition thereof, and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] In this embodiment, a common nucleic acid variation is taken as an example to test the primers and detection system of the present invention. Specifically, the V600E mutation of the human BRAF gene was used as an example to simulate clinical samples to evaluate the performance of the detection system of the present invention.

[0163] BRAF mutations are found in about 8% of human tumors, and BRAF mutations can drive tumor proliferation, growth and differentiation, especially in colorectal cancer (5%-8%), thyroid cancer (5%-20%) And melanoma (40% ~ 68%), lung cancer, liver cancer and pancreatic cancer are also found to have different proportions of BRAF mutations. About 89% of the mutations occurred in the activation region of the 15th exon, most of which were the mutation of base T to A in the 1799th amino acid, resulting in the replacement of valine in BRAF by glutamic acid (V600E).

[0164] The detection of such point mutations is generally performed on the circulati...

Embodiment 2

[0205] In this example, the test effects of kits from similar manufacturers were compared using DNA samples from the Colo 205 cell line (BRAF V600E mutation).

[0206] 1. Sample preparation: the same as in Example 1, except that a fragmented Colo 205 cell line DNA sample containing a BRAF gene V600E mutation with a mutation abundance of 65.8% was used, without serial dilution and preparation of the sample.

[0207] 2. Reaction system

[0208] The preparation of the primer system of the present invention is the same as in Example 1.

[0209] The comparison kit used "PrimePCR" from Bio-Rad TM ddPCR Mutation Assay Kit: BRAF WT for p.V600E, and BRAF p.V600E" commercially available kit (Catalog No. 1863100) was used as the comparison kit of the present invention, and the usage procedure was carried out according to its instruction manual, and the reaction preparation was shown in Table 3.

[0210] table 3

[0211]

[0212]

[0213] 3. Reaction Cell Preparation

[0214] Ad...

Embodiment 3

[0226] In this example, the detection effect of the F1 primer with a longer mismatch region was tested. The specific steps are as follows:

[0227] 1. Sample preparation: same as Example 1

[0228] 2. Preparation of reaction system

[0229] 2.1 Primers and probes

[0230] Primers and probes were synthesized by Sangon Bioengineering Co., Ltd.

[0231] Wherein, the specific sequences of the designed primers are as follows:

[0232] Table 4

[0233]

[0234] Wherein, the primer probes used are: mutant F1 (SEQ ID NO:8), wild type F1 (SEQ ID NO:9), mutant F2 (SEQ ID NO:3), wild type F2 (SEQ ID NO :4), mutant probe P (SEQ ID NO:5), wild type probe (SEQ ID NO:6) and reverse primer R (SEQ ID NO:7). The total length of the mutant F1 primer is 78bp, and the total length of the wild-type F1 primer is 77bp. The 39 bases at the 3' end of the two are the target nucleic acid binding region, the 3' end is the BRAF gene V600E mutation site, and the eighth base at the 3' end 14 mism...

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Abstract

The invention relates to the molecular biology field. Specifically, the invention provides a digital PCR (polymerase chain reaction) primer pair used for measuring nucleotide sequence variation, a composition of the digital PCR primer pair and a method for detecting the nucleotide sequence variation. The forward primer of the improved digital PCR primer pair comprises an upstream detection domainand a target sequence binding domain, wherein the target sequence binding domain contains an amplified decision locus and an upstream mispairing domain; and the upstream detection domain independentlycomprises a domain which has the same sequence with a probe, and a domain which has the same sequence with a second forward primer. Compared with the prior art, sensitivity and high specificity are obviously improved, in addition, primer cost and test cost are greatly lowered, and the invention has an extremely high application value.

Description

technical field [0001] The invention relates to the field of molecular biology, and relates to digital PCR primers and compositions thereof for nucleic acid sequence variation detection. More specifically, the present invention relates to a digital PCR primer for improving the discrimination between different nucleic acids in the detection of nucleic acid sequence variation, its composition, a kit containing the primer and its use. Background technique [0002] The polymerase chain reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without the use of living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as gene cloning, phenotyping of experimental animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, diagnosis of infectious diseases, paternity testing, etc. . Due to its unparalleled ability to replicate and be precise, P...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/156C12Q2531/113C12Q1/6858C12Q2525/161C12Q2525/185C12Q2561/101C12Q2563/159C12Q1/6827
Inventor 赵雨航王书芳葛志琪
Owner SICHUAN MACCURA BIOTECH CO LTD
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