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Method for extracting and purifying target nucleic acid and use thereof

An extraction method and technology of target nucleic acid, applied in the field of bioengineering, can solve the problems of inappropriateness and high sensitivity requirements

Active Publication Date: 2008-12-31
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the above technologies and inventions are not suitable in the fields of blood screening and food safety that require high sensitivity and high-efficiency extraction of RNA and DNA targets at the same time. The market urgently needs a method that can simultaneously and efficiently extract RNA from the same sample and DNA-targeted methods

Method used

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  • Method for extracting and purifying target nucleic acid and use thereof
  • Method for extracting and purifying target nucleic acid and use thereof
  • Method for extracting and purifying target nucleic acid and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1, utilize 2 '-deoxy oligonucleic acid (DNA) capture probe and 2 '-oxygen-methylated oligonucleic acid (2'-OME-RNA) capture probe to capture the comparison of HCV RNA target efficiency

[0066] Taking HCV RNA (sequence 2 in the sequence listing) as an example, using Biotin (biotin) as the marker B and Strepavidin (streptavidin) as the marker B specific binder A to detect the 2'-deoxy oligonucleotide capture probe and 2'-oxygen-methylated oligonucleotide capture probes to the capture efficiency of RNA target nucleic acid, the specific method comprises the following steps:

[0067] 1. Reagent preparation

[0068] 1. Lysis solution (A) with 2'-deoxyoligonucleotide (DNA) capture probe: 250ug / mL streptavidin-coated superparamagnetic magnetic beads with a diameter of 1 μm, 50mM Tris, pH 7.0, 0.1% (V / V) NP-40, 1% (V / V) TritonX-100, 0.1% (V / V) SDS, 0.2uM biotinylated capture probe, wherein the capture probe is: HCV-DNA TCO: 5 '-AGACAGTAGTTCCTCACAGGGG-Biotin 3' (seq...

Embodiment 2

[0096] Embodiment 2, utilizing 2'-oxygen-methylated oligonucleotide (2'-OME-RNA) capture probe and 2'-deoxy oligonucleotide (DNA) capture probe to capture the comparison of HBV DNA target efficiency

[0097] Taking HBV DNA (sequence 1 in the sequence listing) as an example, using Biotin (biotin) as marker B and Strepavidin (streptavidin) as marker B specific binder A to detect 2'-oxo-methylated oligo The capture efficiency of the nucleic acid capture probe and the 2'-deoxy oligonucleotide capture probe to the DNA target nucleic acid, the specific method comprises the following steps:

[0098] 1. Reagent preparation

[0099] 1. the lysate (A) that has 2'-deoxyoligonucleic acid (DNA) capture probe: Same as embodiment 1, wherein, capture probe is: HBV-DNA TCO: 5'-CACGGGACCATGCAAGACCTGCACG-Biotin-3' (Sequence 6 in the sequence listing, 2'-deoxyoligonucleotide (DNA) capture probe).

[0100] 2. Lysate (B) with 2'-oxygen-methylated oligonucleotide (2'-OME-RNA) capture probe: same a...

Embodiment 3

[0112] Embodiment 3, the 2'-deoxyoligonucleotide (DNA) capture probe that is used for HBV DNA capture in embodiment 2 and the 2'-oxygen-methylated oligonucleotide (2') that is used for HCV RNA capture in embodiment 1 '-OME-RNA) capture probes were used simultaneously, and the samples in Example 1 and Example 2 were detected in combination with SAT.

[0113] The detection method comprises the following steps:

[0114] 1. Reagent preparation

[0115] 1. the lysate with 2'-deoxy oligonucleotide (DNA) capture probe and 2'-oxygen-methylation oligonucleotide (2'-OME-RNA) capture probe: same as embodiment 1, wherein, The capture probes are: HBV-DNA-TCO: 5'-CACGGGACCATGCAAGACCTGCACG-Biotin3', HCV-RNA-TCO: 5'-AGACAGUAGUUCCUCAGGGG-Biotin-3'.

[0116] 2. Washing solution: 150mM NaCl solution.

[0117] 3. Preparation of HCV RNA samples and HBV DNA samples: S1 is HCV positive plasma containing about 100,000 copies / mL, S2 is ten-fold dilution of S1 with negative plasma, S3 is ten-fold di...

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Abstract

The invention discloses a method for extracting and purifying target nucleic acid and an application thereof. The extraction method comprises a marker B and a specific conjugate A, wherein, capturing nucleic acid marked by the marker B and target nucleic acid in sample are combined to a solid phase carrier combined with the marker B and the specific conjugate A to form a compound of capturing nucleic acid- target nucleic acid of solid phase carrier-A-B; and the capturing nucleic acid at least comprises a 2`-oxygen-methylation oligoclonal nucleic acid which is specifically combined with the target nucleic acid RNA and is marked by the marker B. The invention also provides a kit for extracting the target nucleic acid, which at least comprises a 2`-oxygen-methylation oligoclonal nucleic acid which is specifically combined with the target nucleic acid RNA and is marked by the marker B, and the 2`- oxygen- methylation oligoclonal nucleic acid is used as the capturing nucleic acid. The method has the advantages of high specificity and purity, less pollution, constant temperature in the reaction process, high sensitivity, fast detecting speed, low requirements on instruments and low cost. Thus, the method is particularly applicable to blood screening.

Description

technical field [0001] The invention relates to a nucleic acid extraction and purification method and its application in the field of bioengineering, in particular to a method for extracting and purifying a target nucleic acid by capturing the target nucleic acid in a sample and its application in nucleic acid detection. Background technique [0002] In recent years, due to the huge demand of the nucleic acid detection market, in-depth research has been carried out at home and abroad. Most of the existing detection technologies only perform direct detection or amplified detection of specific nucleic acid fragments in samples, and the nucleic acid fragments need to be purified. At present, there are many nucleic acid extraction methods, such as SDS method, CTAB method, spin column method, and phenol-chloroform extraction method. Since these methods are not aimed at specifically capturing the target nucleic acid, the extracted product is often the total amount in the sample. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10C12N15/11
Inventor 张常娥
Owner SHANGHAI RENDU BIOTECH
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