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Method for separating low copy genes

A separation method and low-copy technology, applied in the field of gene separation, to achieve the effects of high detection success rate, simple operation, and improved detection efficiency and accuracy

Inactive Publication Date: 2013-09-25
JIANGSU LIFETIME BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective method and kit to solve this problem

Method used

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  • Method for separating low copy genes
  • Method for separating low copy genes
  • Method for separating low copy genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: RNase T1 specific digestion of single-stranded RNA experiment

[0060] 1 Main reagents, materials and instruments

[0061] 1.1 ssRNA: si-NC sense strand and antisense strand, synthesized by Biomaike Biotechnology Co., Ltd.

[0062] si-NC sequence (5'-3') (the terminal "dTdT" is a DNA base):

[0063] Sense strand siNC F: UUCUCCGAACGUGUCACGUdTdT (SEQ ID NO: 1);

[0064] Antisense strand siNC R: ACGUGACACGUUCGGAGAAdTdT (SEQ ID NO: 2).

[0065] 1.2 Reagent: RNase T1 (Fermentas company), 40% polyacrylamide, 10× Tris-borate-EDTA (TBE) buffer (containing 890mM Tris-H 3 BO 3 and 20mM EDTA, pH=8.3), 10% (W / V) ammonium persulfate (APS), tetramethyldiethylamine (TEMED), DEPC-H 2 O (all reagents are autoclaved), etc.

[0066] 1.3 Instruments: polyacrylamide gel electrophoresis system (Bio-Rad Company); gel imaging system (Biosens Company), etc.

[0067] 2 RNase T1 digestion reaction

[0068] Dilute the sense strand and antisense strand of si-NC to a certain conce...

Embodiment 2

[0076] Example 2: RNase T1 has no sequence-dependent experiment on the effect of dsRNA

[0077] 1 Main reagents, materials and instruments

[0078] 1.1 The RNA sequence in the following table is taken as an example, synthesized by Biomaike Biotechnology Co., Ltd.

[0079]

[0080] 1.2 Reagent: RNase T1 (Fermentas Company), 40% polyacrylamide, 10× TBE buffer (containing 890mM Tris-H 3 BO 3 and 20mM EDTA, pH=8.3), 10% (W / V) ammonium persulfate, tetramethyldiethylamine, DEPC-H 2 O (all reagents are autoclaved), etc.

[0081] 1.3 Instruments: polyacrylamide gel electrophoresis system (Bio-Rad Company); gel imaging system (Biosens Company), etc.

[0082] 2 RNase T1 digestion reaction

[0083] Dilute the sense and antisense strands of the RNA to a certain concentration, and take a part to anneal into double strands. Take 0.1OD ssRNA and dsRNA and react with 100U RNase T1 at 37°C for 10min.

[0084] 3 Polyacrylamide gel electrophoresis detection

[0085] 3. Preparation of ...

Embodiment 3

[0091]Embodiment 3: Experiment of the effect of RNase T1 on long single-stranded RNA

[0092] 1 Main reagents, materials and instruments

[0093] 1.1 Reagents: RNase T1 (Fermentas Company), T7 in vitro transcription kit (Biomark Biotechnology Co., Ltd.), 40% polyacrylamide, 10×TBE buffer (containing 890mM Tris-H 3 BO 3 and 20mM EDTA, pH=8.3), 10% (W / V) ammonium persulfate, tetramethyldiethylamine, DEPC-H 2 O (all reagents are autoclaved), etc.

[0094] 1.2 Instruments: polyacrylamide gel electrophoresis system (Bio-Rad Company); gel imaging system (Biosens Company), etc.

[0095] 2 Preparation of long single-stranded RNA (Long single-strand RNA, LRNA)

[0096] Using the CDS sequence of the gene bank number NM 198159 in the NCBI database of the United States as the experimental template, LRNA was synthesized by T7 in vitro transcription method, with a length of 1563nt.

[0097] The primers used for in vitro transcription template preparation are:

[0098] Upstream primer ...

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Abstract

The invention provides a method for separating low copy genes. The method is characterized in that particular steps comprises: a first step, taking a single-strand RNA genetic group containing at least two kinds of copy numbers or at least one low copy single-strand RNA in a sample as target genes to be separated, and designing a segment of a driver RNA molecule complemented with the target genes to be separated; a second step, annealing the driver RNA molecule in the first step and the single-strand RNA (the target genes) to be separated to form a double-strand structure; a third step, digesting the annealed product obtained in the second step by a ribonuclease; and a fourth step, purifying a double-strand target gene product that is undigested by the ribonuclease and is obtained in the third step, to obtain molecules of the target genes to be separated. The advantages of the method are that: the method can overcome a shortcoming that low copy gene detection is affected by high copy genes in the prior art, and greatly improves specificity and sensitivity of subsequent gene detection, thereby improving detection efficiency and accuracy. The method has the advantages of simple operation, strong specificity, and high success rate of the subsequent detection.

Description

technical field [0001] The invention relates to a gene isolation method, in particular to a low-copy gene isolation method. Background technique [0002] At present, gene extraction and separation methods are often to purify the total genome or messenger RNA (mRNA) in biological samples such as blood and tissues to achieve the purpose of separation, and many domestic and foreign biological companies have also successfully developed commercial kits. It is used by many researchers. In these extracted and isolated total genomes or messenger RNAs, due to the different enrichment levels of genes, low-copy genes are often only one ten-thousandth, or even one-millionth, of high-copy genes. In the process of gene detection such as needle hybridization, it will be missed or buried, so it cannot truly reflect the existence or non-existence of low-copy genes, resulting in misreading of samples, which seriously affects the results of gene analysis in the post-genome era. These missed g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 朱远源李铁军
Owner JIANGSU LIFETIME BIOLOGICAL TECH
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