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1. (WO2013145431) METHOD FOR AMPLIFYING cDNA DERIVED FROM TRACE AMOUNT OF SAMPLE

Pub. No.:    WO/2013/145431    International Application No.:    PCT/JP2012/080141
Publication Date: Fri Oct 04 01:59:59 CEST 2013 International Filing Date: Thu Nov 22 00:59:59 CET 2012
IPC: C12N 15/09
C12Q 1/68
Applicants: HITACHI, LTD.
株式会社日立製作所
Inventors: TSUNODA Hiroyuki
角田 弘之
HUANG Huan
フアン フアン
OHTA Mari
太田 真理
KAMBARA Hideki
神原 秀記
Title: METHOD FOR AMPLIFYING cDNA DERIVED FROM TRACE AMOUNT OF SAMPLE
Abstract:
The problem of the present invention is to provide a method for preparing a specimen for the comprehensive and accurate analysis of gene expression in one or few cells using a large-scale DNA sequencer. The present invention relates to a method for amplifying cDNA from mRNA in a cell, the method comprising (1) a step for, in one reactor, capturing the cell-derived mRNA using a first DNA probe formed from a first tag sequence and a poly T sequence and immobilized on a solid carrier and synthesizing a first strand cDNA from the mRNA by reverse transcription, (2) a step for removing the reaction reagent from the reactor vessel in which the first strand cDNA synthesized on the solid carrier remains, (3) a step for adding a polynucleotide sequence consisting of one nucleotide to the 3' terminal of the first strand cDNA on the solid carrier, (4) a step for hybridizing a second DNA probe containing a second tag sequence and a sequence complementary to the polynucleotide sequence with cDNA to which the polynucleotide sequence has been added and synthesizing a second strand cDNA, and (5) a step for performing a DNA amplification using the second strand cDNA synthesized on the solid carrier as the template.