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Method for production of cDNA library having reduced content of cDNA clone derived from highly expressed gene

A technology of content rate and gene, applied in the field of cDNA library preparation, can solve problems such as difficulty in obtaining full-length cDNA clones effectively, loss of low-expression gene cDNA clones, etc.

Active Publication Date: 2011-07-13
HITACHI HIGH-TECH CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During this process, the loss of cDNA clones of low-expressed genes will occur, so it is difficult to efficiently obtain full-length cDNA clones of low-expressed genes

Method used

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  • Method for production of cDNA library having reduced content of cDNA clone derived from highly expressed gene
  • Method for production of cDNA library having reduced content of cDNA clone derived from highly expressed gene
  • Method for production of cDNA library having reduced content of cDNA clone derived from highly expressed gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Inhibition of cDNA extension reactions by probe binding

[0072] (1) Determination of the target gene

[0073] The following experiment was performed in order to select a gene with a large expression level, which is easy to confirm the experimental effect, as the target gene. Using 10 μg of total RNA each obtained from the livers of male and female mice, and 300 ng of pGCAP10 vector primers (International Publication No. WO2004 / 087916 pamphlet), cDNA synthesis was performed to obtain cDNA clones. The preparation method follows the description in the pamphlet of International Publication No. WO2004 / 087916. The base sequence of the 5' end of the obtained cDNA clone was analyzed. For 3,217 clones derived from male liver and 2,325 clones derived from female liver that could be sequenced, a BLAST search was performed using the nucleic acid database of GenBank. As a result, among the cDNA clones derived from the male liver, the most repetitive gene was the major urinary pr...

Embodiment 2

[0085] Preparation of cDNA library with reduced content of highly expressed genes

[0086] (1) First strand cDNA synthesis

[0087] Using the above probes, cDNA synthesis was performed from total RNA. The probes used were Alb1-5, Mup2-3. 10 µg each of total RNA obtained from the liver of a male mouse, 150 ng of pGCAP10 carrier primer (JP-A-4-108385), 25 nmol of dNTP, and 100 pmol of probe were mixed to prepare a total of 8 µl of the mixture. After heating this mixed solution at 65 degreeC for 5 minutes, it cooled with ice for 2 minutes. 4 μl of buffer solution attached to SuperScript II RNase H-reverse transcriptase (manufactured by Invitrogen Corporation) was added, and incubated at 57° C. for 30 minutes. Then, after cooling with ice for 2 minutes, 100 nmol of DTT, 40 U of ribonuclease inhibitor, and 200 U of SuperScript II RNase H-reverse transcriptase (manufactured by Invitrogen Corporation) were mixed to adjust the total amount to 20 μl. This mixed solution was reacted...

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Abstract

Disclosed is a method for producing a cDNA library having a reduced content of a cDNA clone derived from a highly expressed gene with high efficiency. In a method for producing a cDNA library by using a double-stranded DNA primer containing an oligo-dT and also using mRNA as a template, the content of a cDNA clone derived from a highly expressed gene in the cDNA library can be reduced by allowing a probe to coexist, wherein the probe has such a property that the probe can bind to mRNA of a highly expressed target gene to inhibit the occurrence of a cDNA extension reaction with a reverse transcriptase.

Description

technical field [0001] The present invention relates to a method for preparing a cDNA library, and relates to a method for preparing a cDNA library in which the content rate of cDNA clones derived from genes with high expression frequencies is reduced. Background technique [0002] Through genome projects, the full-length sequences of genomic DNA of various organisms such as human, mouse, dog, nematode, and yeast have basically been determined. In today's post-genome era, in order to grasp the role of each gene in life phenomena macroscopically, a technology that promotes the investigation of gene group dynamics and comprehensively analyzes all genes is required. [0003] It is generally considered that cells constituting the human body have a genome encoding about 22,000 genes, and tens of thousands of genes are expressed depending on the cell type (see Non-Patent Document 1). Extensive investigation of such gene expression is becoming increasingly important not only for e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C40B40/06
CPCC40B50/06C12N15/1096C40B40/06C12N15/1093C12Q2537/159
Inventor 大床国世杉山雅英加藤诚志
Owner HITACHI HIGH-TECH CORP
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