Patent ID: 6468749
Filing Date: 2002-10-22
Classification: C12N

Abstract:
A method of sorting genes comprising:(1) preparing ds cDNA molecules from mRNA molecules by reverse transcription, using a poly-T primer optionally having a general primer-template sequence upstream from the poly-T sequence, yielding ds cDNA molecules having the poly-T sequence, optionally having the general primer-template sequence; (2) digesting the ds cDNA molecules with a restriction enzyme that produces digested cDNA molecules with cohesive ends having overhanging ssDNA sequences of a constant number of arbitrary nucleotides; (3) ligating to the digested cDNA molecules a set of dsDNA oligonucleotide adaptors, each of which adaptors has at one of its ends a cohesive-end ssDNA adaptor sequence complementary to one of the possible overhanging ssDNA sequences of the digested cDNA, at the opposite end a specific primer-template sequence specific for the ssDNA adaptor complementary sequence, and in between primer-template sequence specific for the ssDNA adaptor completmentary sequence, and in between the ends a constant sequence that is the same for all of the different adaptors of the set; (4) amplifying by separate polymerase chain reactions the ligated cDNA molecules, utilizing for each separate polymerase chain reaction a primer that anneals to the cDNA poly-T sequence optionally having the cDNA general primer-template, and a primer from a set of different specific primers that anneal to the cDNA specific primer-template sequences; and (5) sorting the amplified cDNA molecules into non-redundant groups by collecting the amplification products after each separate polymerase chain reaction, each group of amplified cDNA molecules determined by the specific primer that annealed to the specific primer-template sequence and primed the polymerase chain reaction.