Patent ID: 6506561
Filing Date: 2003-01-14
Classification: C12Q

Abstract:
A method of obtained a library of tags capable of defining a specific state of a biological sample, comprising:(1) extracting mRNA from a biological sample comprising &lE;5Ã—106 cells, corresponding to at most 50 &mgr;g of total RNA or 1 &mgr;g of poly(A) RNA, 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) synthesizing 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 the restriction endonuclease Sau3A I as an anchoring enzyme, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of the two aliquots via the Sau3A I restriction site two different hybrid DNA molecules composed from linkers 1A and 1B or from linkers 2A and 2B, wherein the linkers have the following formulas: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, (6) digesting the products obtained in step (5) with a BsmF I 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 step (7) with a DNA ligase, to form ditags, and (9) determining the nucleotide sequence of at least one tag of the ditags.