Loop type enrichment detection primer of low-abundance mutation DNA and application thereof

A detection primer, low abundance technology, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc., can solve the problems of low sensitivity, low content of mutated nucleic acid, high detection cost, etc. Highly specific and accurate effects

Inactive Publication Date: 2018-11-16
CAPITALBIO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the content of mutant nucleic acids in circulating DNA in peripheral blood is low. Taking the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer as an example, the sensitivity of Sanger sequencing and ARMS-PCR method is low, and sequencing can only detect 5 More than 100% of EGFR single-base mutations, ARMS-PCR method can detect one-tenth to one-thousandth of EGFR single-base mutations, it is difficult to detect mutations by Sanger sequencing and ARMS-PCR
This requires a detection technology with high detection sensitivity. At present, Digital PCR is commonly used. This technology can detect mutations in one thousandth to one hundred thousandth, but this technology relies on specific instruments for detection, and the detection cost is high.

Method used

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  • Loop type enrichment detection primer of low-abundance mutation DNA and application thereof
  • Loop type enrichment detection primer of low-abundance mutation DNA and application thereof
  • Loop type enrichment detection primer of low-abundance mutation DNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Enrichment detection primers for detection of T790M mutation and L858R mutation in the present invention

[0071] Table 1

[0072]

[0073]

[0074] In Table 1, T790M-Loop-F and L858R-Loop-F are the enrichment detection primers for T790M mutation and L858R mutation, where the underline is the base of the mutation site; T790M-R and L858R-R are the corresponding Downstream primers, the sequences of which are shown in SEQ ID NO:5-6.

[0075] In T790M-Loop-F, the degree of polymerization of Oligo (dT) is 32, C12 means C12Spacer modification, TCATCATGCAGCTCA is the auxiliary primer nucleotide sequence (shown in SEQ ID NO: 1), and CACCGTGCAGC is the amplification primer nucleotide sequence (SEQ ID NO: 2), and the 3' end of the amplification primer and the 5' end of the auxiliary primer are separated from the template binding site by 0 bases. For the schematic diagram, see figure 2 .

[0076]In L858R-Loop-F, the degree of polymerization of Oligo (dT) is 28...

Embodiment 2

[0077] Embodiment 2: The present invention detects the enrichment detection kit (common PCR) of T790M mutation and L858R mutation

[0078] Table 2

[0079]

[0080]

[0081] In Table 2, CX-T790M-F and CX-T790M-R are T790M sequencing upstream and downstream amplification primers (shown in SEQ ID NO: 17-18), CX-L858R-F and CX-L858R-R are L858R sequencing The downstream amplification primers (shown in SEQ ID NO: 19-20), CX-F and CX-R are upstream and downstream general primers (shown in SEQ ID NO: 21-22) for upper-machine sequencing.

[0082] For T790M and L858R Mutation Enrichment Detection Kit (General PCR) consists of the following components: T790M Mutation Enrichment Primer Mix, L858R Mutation Enrichment Primer Mix, T790M Sequencing Amplification Primer Mix, L858R Sequencing Amplification Primer Mixture, sequencing upstream universal primer CX-F, sequencing downstream universal primer CX-R, mutation enrichment amplification reagent, sequencing amplification reagent, p...

Embodiment 3

[0093] Embodiment 3: The common PCR method that the present invention detects T790M mutation and L858R mutation

[0094] 1. Detection method

[0095] The kit in Example 2 was used to perform two rounds of amplification for detection. The first round of amplification was used to enrich low-abundance mutations, and the second round of amplification was used to add general sequencing primers to prepare for on-machine sequencing.

[0096] For the preparation of the first round of amplification system, take an eight-row PCR reaction tube, add 12.5 μl of the 2× detection system, 2.5 μl of the corresponding primer components, and 5-10 μl of the corresponding template, add water to 25 μl, mix well, Place the prepared PCR reaction tube in a PCR amplification instrument for amplification.

[0097] The first round of amplification procedure:

[0098] 95°C for 5min, cycle once

[0099] 95°C for 5s, 56°C for 10s, 68°C for 30s, cycle 15 times

[0100] 95°C 5s, 60°C 5s, 68°C 30s, cycle 3...

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Abstract

The invention relates to the field of biotechnology and discloses a Loop type enrichment detection primer of low-abundance mutation DNA and application thereof. According to the Loop type enrichment detection primer disclosed by the invention, two short nucleotide sequences are connected by a Loop type structure, mutant type and wild type differential amplification is realized through the base accumulation effect, the concentration can be lowered to 10<-4> mutation enrichment to 1% to 10%, and the detection range of Sanger sequencing is reached so that the mutation enrichment can be combined with the Sanger sequencing to realize low-abundant mutation detection. The Loop type enrichment detection primer disclosed by the invention is suitable for detecting peripheral blood free DNA with lowmutation content, and realizes simple and convenient sampling while causing little pain of the patients; the detection technology has the advantages of high specificity, high sensitivity, easiness inoperation, short detection period, low detection cost and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Loop-type enrichment detection primer for low-abundance mutant DNA and its application. Background technique [0002] Detection of DNA mutations or trace alleles in biological samples is of great significance in the fields of tumor diagnosis, screening and prognosis detection. Currently, PCR-based technologies for detecting DNA mutations mainly include ARMS-PCR technology (amplification refractory mutation system PCR), enzyme-based PCR enrichment technology, ligation-based PCR enrichment technology, Blocker-based PCR enrichment technology, and low denaturation temperature. Composite PCR enrichment technology (co-amplification at lower denaturation temperature PCR, COLD-PCR) and Digital PCR technology. [0003] Since some advanced cancer patients, such as advanced lung cancer patients, are difficult to obtain tissue samples, their detection has certain limitations. Therefore, it is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2531/113C12Q2563/107C12Q2545/101C12Q2535/101
Inventor 盖伟张岩邢婉丽宋翠丹马桂红程京
Owner CAPITALBIO CORP
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