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Method for preparing a nucleic acid with high precision used in detecting a nucleic acid with a nucleic acid polymerase

A nucleic acid polymerase, polymerase technology, applied in biochemical equipment and methods, DNA preparation, and microbial assay/inspection, etc., can solve problems such as difficulty in detecting target nucleic acids, false positives, etc.

Active Publication Date: 2015-02-11
BIONEER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even though various types of nucleic acid extraction automation equipment for automatically performing nucleic acid extraction have been developed, since the template nucleic acid itself is non-specifically primed, in the detection of trace nucleic acid, the use of nucleic acid polymerase and primers for amplification Nucleic acid is predominantly amplified in reactions such as PCR reactions or real-time quantitative PCR reactions, so false positives may occur, or target nucleic acids may be difficult to detect

Method used

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  • Method for preparing a nucleic acid with high precision used in detecting a nucleic acid with a nucleic acid polymerase
  • Method for preparing a nucleic acid with high precision used in detecting a nucleic acid with a nucleic acid polymerase
  • Method for preparing a nucleic acid with high precision used in detecting a nucleic acid with a nucleic acid polymerase

Examples

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Effect test

preparation example Construction

[0087] In the method for preparing nucleic acid according to the present invention, a known integrated device for analyzing nucleic acid that manages a known automated device for extracting nucleic acid and a gene amplification device as a whole can be used, preferably, Exiprep 16DX (manufactured by Bioneer Corporation) can be used, but the present invention is not limited thereto.

[0088] More specifically, a process for isolating and purifying nucleic acids is performed, and then, a nucleic acid polymerase and a nucleic acid polymerization terminator are added to the extracted nucleic acids to perform a nucleic acid polymerization termination reaction, thereby inactivating the priming function of all nucleic acids. Since the terminator remaining after the reaction acts as an inhibitor of the subsequent reverse transcriptase reaction or PCR reaction, two methods can be used to eliminate the remaining terminator.

[0089] One of the two methods is to perform RNA purification ...

Embodiment 1

[0173] [Example 1] Non-specific RT-PCR inhibitory effect of single-stranded RNA terminated by 3'-deoxyadenosine 5'-triphosphate (3'-dATP)

[0174] (1) RNA sheet by poly(A) polymerase and 3'-deoxyadenosine 5'-triphosphate (3'-dATP) Segment termination reaction

[0175] To suppress the nonspecific priming reaction of single-stranded RNA, a termination reaction was performed using poly(A) polymerase (Takara, Japan) and 3'-deoxyadenosine 5'-triphosphate (3'-dATP).

[0176] Add 5 μl of 10X reaction buffer, 0.1 mM 3'-deoxyadenosine 5'-triphosphate (3'-dATP), 2 units of poly(A) polymerase and 1 nmol of poly(A) polymerase synthesized in Comparative Example 1 with twelve random Base sequence (12-mer) single-stranded RNA was then added with distilled water to have a final volume of 50 µl, followed by reaction at 37°C for 15 minutes.

[0177] After the reaction, purification was performed by AccuPower PCR Purification Kit (Bioneer, Korea). In the purified product, it was confirmed ...

Embodiment 2

[0204] [Example 2] Inhibition effect of non-specific reaction in real-time PCR by RNA termination present in human serum

[0205] (1) RNA termination by poly(A) polymerase and 3'-deoxyadenosine 5'-triphosphate (3'-dATP) reaction

[0206] After RNA was extracted from human serum as described in Example 1, the extracted RNA (3 μg) was terminated by poly(A) polymerase and 3'-deoxyadenosine 5'-triphosphate (3'-dATP) 3' end (treatment group).

[0207] 3'-deoxyadenosine 5'-triphosphate (3'-dATP), left unreacted during RNA termination, must be eliminated because it acts as an inhibitor of RT-PCR and PCR reactions.

[0208] RNA terminated by 3'-deoxyadenosine 5'-triphosphate (3'-dATP) was purified by AccuPrep PCR Purification Kit (Bioneer, Korea). The unterminated RNA at the 3' end (non-treated group) was also purified by the same method as above, and as described in Example 1 above, the same amount of 2 μg of each RNA in the treated group and non-treated group was quantified, a...

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Abstract

The present invention relates to a method for preparing a nucleic acid with high sensitivity, wherein a nucleic acid polymerase is used to add a terminator to the nucleic acid to be used for analysis prior to a nucleic acid polymerization reaction such as a PCR reaction, a real time quantitative PCR reaction, or the like, for detecting a trace of nucleic acid, such that a non-specific priming occurring competitively with an amplification reaction of a target nucleic acid may be basically eliminated, thereby precisely detecting only the trace of target nucleic acid and precisely measuring a concentration of the target nucleic acid.

Description

technical field [0001] The present invention relates to a method for producing a nucleic acid having high sensitivity used when detecting nucleic acid by a nucleic acid polymerase, and more specifically, the present invention relates to a method for producing a nucleic acid by preventing non-specific nucleic acid polymerization While capable of detecting trace amounts of nucleic acid, the non-specific nucleic acid aggregation is induced due to self-priming by single-stranded nucleic acid contained in nucleic acid extracted from a biological sample, preparing a nucleic acid for detecting nucleic acid in nucleic acid detection The sample method uses the nucleic acid polymerase and primers required for PCR, RT-PCR, quantitative PCR, quantitative RT-PCR, transcriptional amplification, or nucleic acid amplification at room temperature. Background technique [0002] Various methods for detecting nucleic acid using nucleic acid polymerase have been used for various inspections, suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/00
CPCC12Q1/6848C12Q1/68C12Q1/00C12Q1/686C12Q2525/186C12Q2531/113C12Q1/6806C12N15/1096C12Q1/34C12Q1/48
Inventor 朴翰浯李晙凞金悬书崔昭罗金锺勲
Owner BIONEER
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