×

Microassay for serial analysis of gene expression and applications thereof

  • US 20030165910A1
  • Filed: 07/16/2002
  • Published: 09/04/2003
  • Est. Priority Date: 01/27/1999
  • Status: Abandoned Application
First Claim
Patent Images

1. A method of obtaining a library of tags capable of defining a specific state of a biological sample, comprising:

  • (1) extracting mRNA from the biological sample by contacting the biological sample with an oligo(dT) covalently bound to paramagnetic beads, (2) generating a double-strand cDNA library from the extracted mRNA according to a process comprising;

    (a) synthesizing the 1st strand of the cDNA by reverse transcription of the mRNA template into a 1st complementary single-strand cDNA, using a reverse transcriptase lacking Rnase H activity, and (b) synthesising the 2nd strand of the cDNA by nick translation of the mRNA in the mRNA-cDNA hybrid form by an E. coli DNA polymerase, (3) cleaving the cDNAs with an anchoring enzyme, wherein the anchoring enzyme is a restriction endonuclease having a 4-bp recognition site, (4) separating the cleaved cDNAs in two aliquots, (5) linking the cDNA contained in each of the two aliquots with two different oligonucleotide linkers comprising a type IIS recognition site, (6) digesting the products obtained in (5) with a type IIS restriction enzyme, to obtain two different tags, (7) blunt-ending the tags with a DNA polymerase, and mixing the tags ligated with the different linkers, (8) ligating the tags obtained in (7) with a DNA ligase, to form ditags, and (9) determining the nucleotide sequence of at least one tag of the ditags, to detect gene expression.

View all claims
  • 0 Assignments
Timeline View
Assignment View
    ×
    ×