METHOD FOR AMPLIFYING cDNA DERIVED FROM TRACE AMOUNT OF SAMPLE

摘要

The problem to be solved by the present invention is to provide a method for preparing a sample for comprehensively and accurately analyzing gene expression in a single cell or a few cells, for example, by a large-scale DNA sequencer. The present invention relates to a method for amplifying cDNA from mRNA in a cell, comprising: (1) capturing mRNA derived from the cell by a first DNA probe containing a first tag sequence and a poly T sequence and being immobilized to a solid carrier in a single reaction vessel, and synthesizing a first strand cDNA from the mRNA by a reverse transcription reaction; (2) removing a reaction reagent from the reaction vessel while keeping the first strand cDNA synthesized onto the solid carrier in the reaction vessel; (3) adding a polynucleotide sequence consisting of one type of nucleotides to 3′ terminal of the first strand cDNA on the solid carrier; (4) hybridizing a second DNA probe containing a second tag sequence and a complementary sequence to the polynucleotide sequence with the cDNA to which the polynucleotide sequence is added, and synthesizing a second strand cDNA; and (5) performing a DNA amplification reaction using the second strand cDNA synthesized on the solid carrier as a template.

主权项

1. A method for amplifying cDNA from mRNA in a cell, comprising:
(1) capturing mRNA derived from the cell by a first DNA probe comprising a first tag sequence and a poly T sequence and being immobilized to a solid carrier in a single reaction vessel, and synthesizing a first strand cDNA from the mRNA by a reverse transcription reaction; (2) removing a reaction reagent from the reaction vessel while keeping the first strand cDNA synthesized onto the solid carrier in the reaction vessel; (3) adding a polynucleotide sequence consisting of one type of nucleotides to 3′ terminal of the first strand cDNA on the solid carrier; (4) hybridizing a second DNA probe comprising a second tag sequence and a complementary sequence to the polynucleotide sequence with the cDNA to which the polynucleotide sequence is added, and synthesizing a second strand cDNA; and (5) performing a DNA amplification reaction using the second strand cDNA synthesized on the solid carrier as a template.