Detection of nucleotide variation on target nucleic acid sequence

A target nucleic acid sequence and target nucleotide technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., and can solve the problem of inability to completely block the amplification of wild-type alleles

Active Publication Date: 2016-02-24
SEEGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the PCR clamp described above may not be able to completely block the amplification of the wild-type allele

Method used

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  • Detection of nucleotide variation on target nucleic acid sequence
  • Detection of nucleotide variation on target nucleic acid sequence
  • Detection of nucleotide variation on target nucleic acid sequence

Examples

Experimental program
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Effect test

example 1

[0217] [Example 1 (Interchain Interactivity-Dual Labeling)]

[0218] In Example 1 of the interactive dual labeling system, the first fragment or capture and template oligonucleotides have an interactive dual label comprising a reporter molecule and a quencher molecule, the extension dimerization in step (e) above The melting of the substance induces a change in the signal from the interacting dual label to provide the target signal in step (e). exist Figure 5 Example 1 of an interactive dual labeling system is shown diagrammatically in . Example 1 was named Interchain Interactivity-Dual Labeling.

example 1-1

[0219] [Example 1-1 (Interstrand Interactivity on Capture and Templated Oligonucleotides—Dual Labeling)]

[0220] refer to Figure 5 , to illustrate the illustrated example. The templated sites of the capture and template oligonucleotides have reporter and quencher molecules. Probing and labeling oligonucleotide-nucleotide variants that hybridize to the target nucleic acid sequence are cleaved and a first fragment is released, and the first fragment is hybridized to the capture site of the capture and templated oligonucleotides and extended to form extended dimer.

[0221] In the case where the extended dimer is formed in step (d) above, the reporter and quencher molecules of the capture and template oligonucleotides are structurally separated from each other such that the quencher molecule does not interfere with the reporter molecule from the reporter molecule. The signal is quenched, and in the case where the extension dimer is unchained in step (e), the reporter molecul...

example 1-2

[0234] [Example 1-2 (Detection and Labeling of Oligonucleotides - Interstrand Interactivity on Nucleotide Variations - Dual Labeling)]

[0235]The 5'-labeling sites that detect and label oligonucleotide-nucleotide variations can have reporter and quencher molecules. The probe and label oligonucleotide-nucleotide variants that hybridize to the target nucleic acid sequence are cleaved to release a first fragment comprising a 5'-labeled site with reporter and quencher molecules. The first fragment described above hybridizes to the capture site of the capture and templated oligonucleotides.

[0236] In the case of forming the extended dimer in the above-mentioned step (d), the reporter molecule and the quenching molecule on the above-mentioned first fragment are structurally separated from each other, so that the quenching molecule does not quench the signal from the reporter molecule, and in the In the case where the extension dimer is melted in step (e), the reporter ...

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Abstract

The present invention relates to the detection of a nucleotide variation on a target nucleic acid sequence using an amplification blocker and a VD-PTOCE (Variation Detection by PTO Cleavage and Extension) assay. The present invention is significantly effective in the detection of a minority mutation in an excess of wild-type DNA.

Description

[0001] 【Related application】 [0002] This application is based on the U.S. Provisional Patent Application No. 61 / 827966, filed on May 28, 2013, and claims the priority of the patent application. The disclosures of the above patent application are incorporated herein by reference. 【Technical field】 [0003] The present invention relates to the detection of nucleotide variations on target nucleic acid sequences using amplification blockers and Variation Detection by PTOC Leavage and Extension (VD-PTOCE, Variation Detection by PTOC Leavage and Extension) analysis based on detection and labelling oligonucleotide cleavage and extension. 【Background technique】 [0004] Deoxyribonucleic acid (DNA) hybridization is a fundamental process in molecular biology and is affected by ionic strength, base structure, length of reduced nucleic acid fragments, degree of mismatch, and the presence of denaturing agents. Deoxyribonucleic acid hybridization-based techniques will be very useful too...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6827C12Q1/6844C12Q2600/156C12Q2523/107C12Q2525/186C12Q1/6883
Inventor 千钟润李荣祚
Owner SEEGENE INC
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