Antisense interference oligonucleotide used for inhibiting primer non-specific amplification

An antisense oligonucleotide, non-specific technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of delay, limited degradation, and inability to eliminate

Inactive Publication Date: 2013-07-17
北京万达因生物医学技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Generally, dUTP is used to replace the dTTP product for aerosol recontamination of the amplified product, combined with uracil-DNA-glycosylase (UDG / UNG, US Patent 6,090,553) to selectively degrade the contaminated product. UDG / UNG is subsequently thermally denatured and inactivated by PCR, but In actual work, the degradation effect on a large number of primer-dimer-containing dUTP amplification products is limited, and it cannot eliminate or delay the Ct value of the SYBR Green I real-time fluorescent PCR background primer

Method used

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  • Antisense interference oligonucleotide used for inhibiting primer non-specific amplification
  • Antisense interference oligonucleotide used for inhibiting primer non-specific amplification
  • Antisense interference oligonucleotide used for inhibiting primer non-specific amplification

Examples

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example 1

[0083] Example 1: Real-time fluorescent PCR of human hepatitis B virus SYBR Green I:

[0084] Hepatitis B virus (referred to as hepatitis B) is a worldwide infectious disease caused by hepatitis B virus (Hepatitis B virus, HBV). The infection rate of hepatitis B in our country is very high, which greatly endangers people's health. At present, the detection methods of hepatitis B mainly include enzyme immunoassay, radioimmunoassay, chemiluminescence, immunofluorescence, nucleic acid amplification (PCR) fluorescence quantitative method and so on. Enzyme immunoassay is widely used, but real-time fluorescent PCR analysis can accurately detect the virus gene of hepatitis B patients, and plays an irreplaceable important role in judging the virus replication level of infected patients, monitoring the infectivity of the disease and the efficacy of antiviral drugs.

[0085] Hepatitis B virus (HBV) is a partially double-stranded DNA virus. It mainly has three type-specific conserved re...

example 2

[0118] Example 2: HBV modified TaqMan probe method real-time fluorescent PCR:

[0119] Probe-based real-time fluorescent PCR is mainly represented by TaqMan probes, and also includes MGB probes with increased binding force and locked nucleic acid LNA base probes. The increased signal of the amplified product is detected by a fluorescent probe with a quencher group. Generally, the 5' end of TaqMan probes is labeled with fluorescent groups such as FAM, VIC, NED, and the 3' end is labeled with quenching groups such as TAMRA, DABCYL&BHQ, etc., and the quenched TaqMan probes are hydrolyzed by the 5' exonuclease of Taq enzyme. Free fluorophores generate fluorescence. TaqMan probe design respects the following general principles: 1) The T of the probe m Value ratio of primer T m The value should be higher than 10°C; 2) The 5' end of the probe cannot be a G base, and the degraded G still has the effect of quenching the reporter fluorescence; 3) The G in the probe cannot be more tha...

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Abstract

The invention relates to an antisense interference oligonucleotide used for inhibiting primer non-specific amplification. Through combining a primer 3' end antisense oligonucleotide, the antisense interference oligonucleotide interferes PCR primer 3' end non-specific amplification. The antisense oligonucleotide is a section of oligonucleotide with 4-14 base composed of antisense nucleotide. The basic characteristics are that the antisense base oligonucleotide maintains a Watson base pairing hybrid property, but loses the functions as PCR amplification template and primers. Antisense oligonucleotide is immobilized by using nanospheres, such that primers are indirectly immobilized to a PCR system, and hot start is sustained-released. Mineral oil is used for sealing PCR, such that aerosol cross-contamination is avoided, and PCR reaction is prevented from non-specific amplification. The antisense oligonucleotide is suitable for various PCR technologies with primer annealing and extension, including array amplification technology.

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid amplification in the field of molecular biology and molecular testing, and specifically relates to the technical field of interfering with non-specific amplification such as primer polymerization by antisense "dead" oligonucleotides competing for non-specific templates. Background technique: [0002] Nucleic acid amplification technology originated in 1971. Korana first proposed the idea of ​​in vitro nucleic acid amplification: "After DNA denaturation, hybridization with suitable primers, extension of primers with DNA polymerase, and continuous repetition of this process, tRNA genes can be cloned." The seeds of its ideas will take root and germinate when they enter the soil of the development of modern molecular biology. Finally, in 1983, Kary Mullis of the Human Genetics Laboratory of PE-Cetus Company in the United States had the inspiration of nucleic acid amplification when studying nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/68
Inventor 江洪岳素文阮志强江必胜江雨康
Owner 北京万达因生物医学技术有限责任公司
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