Patent ID: 6197554
Filing Date: 2001-03-06
Classification: C12N

Abstract:
A method of generating a complete full-length cDNA library from single cells, comprising the steps of:a. providing a plurality of fixed cells, wherein said fixed cells inhibit intracellular messenger RNA degradation and also increase the permeabilisation of said cells for enzyme penetration;b. incubating said fixed cells in a reverse transcription reaction with a plurality of oligo(dT)n-promoter sequences, wherein said reverse transcription reaction is reverse transcription of a plurality of messenger RNAs by using said oligo(dT)n-promoter as primer, to form a plurality of complementary DNAs from said messenger RNAs;c. permitting said complementary DNAs in a cDNA tailing and double-stranding reaction to form a plurality of poly(N)-tailed cDNAs, wherein said cDNA tailing and double-stranding reaction is a DNA polymerase and terminal transferase reaction capable of adding multiple copies of the same nucleotide to the tails of said complementary DNAs and then double-stranding said complementary DNAs from the tails;d. incubating said poly(N)-tailed cDNAs in an in-vitro transcription reaction to generate a plurality of full-length aRNAs, wherein said in-vitro transcription reaction is RNA's polymerase reaction capable of synthesizing said full-length RNA's from said poly(N)-tailed cDNAs;e. incubating said full-length aRNAs in said reverse transcription reaction with a plurality of oligo (anti-poly(N))-promoter sequences to form a plurality of full-length cDNAs; wherein said oligo (anti-poly(N))-promoter sequences are complementary to the poly(N) tails of said poly(N)-tailed cDNAs andf. amplifying said full-length cDNAs with a template-dependent extension of specific primers attached to the poly (dA)-tail and complementary promoter regions of said full-length cDNAs, and thereby providing a complete library enriched in full-length cDNAs from said fixed cells.