Nucleic acid amplification blocking agent for detecting low-abundance mutant sequences and application

A technology of mutation sequence and blocking agent is applied in the field of detection of mutant genes, which can solve the problems of high price and low detection sensitivity, and achieve the effects of low price, high affinity and flexible base modification position.

Inactive Publication Date: 2019-04-19
SHANGHAI MAG GENE NANOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PNA-mediated nucleic acid amplification blockers are not only expensive, but also inhibit the amplification of mutant templates while inhibiting wild templates, resulting in a decrease in detection sensitivity.

Method used

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  • Nucleic acid amplification blocking agent for detecting low-abundance mutant sequences and application
  • Nucleic acid amplification blocking agent for detecting low-abundance mutant sequences and application
  • Nucleic acid amplification blocking agent for detecting low-abundance mutant sequences and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0033] Embodiment 1 (Influence of Different LNA Modification Numbers on Mutation Amplification Efficiency)

[0034] 1. Preparation of LNA-modified nucleic acid amplification blocking agent

[0035] In the case of keeping the annealing temperature of the nucleic acid amplification blocking agent constant, the effect of different LNA modification numbers on the amplification efficiency of the mutant template was verified by changing the number of LNA modifications and its length. Taking the L861Q site mutation type in EGFR as an example, different nucleic acid amplification blocking agents were designed, the sequences of which are shown in Table 1 (SEQ ID NO: 1 to SEQ ID NO: 4).

[0036] 2. PCR amplification

[0037] According to the type of L861Q mutation to be detected, we designed a pair of primers for amplification, the sequences of which are shown in Table 2 (SEQ ID NO: 8 to SEQ ID NO: 9). The detection method adopted is detected by Taqman probe, and its sequence is shown...

Embodiment approach 2

[0045] Embodiment 2 (the same length but the influence of different LNA modification positions):

[0046] 1. Preparation of LNA-modified nucleic acid amplification blocking agent

[0047] In the case of maintaining the length of the nucleic acid amplification blocking agent and the number of LNA modifications, the modification position of the LNA was changed to verify its influence on the amplification efficiency of the mutant template. According to the mutation type of L861Q site in EGFR, different nucleic acid amplification blocking agents were designed, and their sequences are shown in Table 1 (SEQ ID NO:5 to SEQ ID NO:7).

[0048] 2. PCR amplification

[0049] According to the type of L861Q mutation to be detected, we designed a pair of primers for amplification, the sequences of which are shown in Table 2 (SEQ ID NO: 8 to SEQ ID NO: 9). The detection method adopted is detected by Taqman probe, and its sequence is shown in Table 2 (SEQ ID NO: 10).

[0050] 2.1. Extract ...

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Abstract

The invention discloses a design principle of a nucleic acid amplification blocking agent for detecting low-abundance mutant sequences, and relates to the field of detection of mutant genes. The nucleic acid amplification blocking agent is oligonucleotide modified by locked nucleic acid (LNA), and a pairing region of the nucleic acid amplification blocking agent is located between amplification sequences and completely paired with wild-type gene sequences, and has at least one mispairing with the mutant sequences. The invention further discloses application of the nucleic acid amplification blocking agent in detecting the low-abundance mutant sequences. According to the nucleic acid amplification blocking agent and the application, the affinity between the nucleic acid amplification blocking agent modified by locked nucleic acid and the mutant nucleic acid sequences / wild-type nucleic acid sequences has a huge difference, and the purpose of highly-selective amplification / enrichment of the mutant sequences in a sample is achieved; the nucleic acid blocking agent has the advantages of being high in affinity, flexible in basic group modification position, good in heat stability, cheapin price and the like, and has a more significant detection effect on deletion mutation and insertion mutation.

Description

technical field [0001] The invention relates to the field of detection of mutant genes, in particular to the design principle and application of a nucleic acid amplification blocking agent for low-abundance mutant sequences. Background technique [0002] Gene mutation is a change in the gene structure caused by the addition, deletion or change of base pairs in the DNA molecule. Gene mutations are associated with many disease symptoms, such as a small amount of tumor circulating DNA in the tissues and peripheral blood of tumor patients, and the emergence of bacterial and viral drug resistance in the early stage. landmark. Nucleic acid amplification is the most common method for mutation gene detection. [0003] Nucleic acid amplification is an essential step in most nucleic acid assays. Accurate detection of nucleic acids generally refers to the ability to amplify a specific target nucleic acid in the presence of excess non-target nucleic acid, ie having a sequence similar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6827
CPCC12N15/11C12Q1/6827C12N2310/3231C12Q2525/10C12Q1/6858C12Q2525/117C12Q2527/107C12Q2531/113C12Q2537/163C12Q1/6853C12Q1/6876C12Q2525/186C12Q2525/113
Inventor 徐宏杨浩徐高连古宏晨
Owner SHANGHAI MAG GENE NANOTECH CO LTD
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