Patent ID: 6045998
Filing Date: 2000-04-04
Classification: C12N,C12Q

Abstract:
A method of obtaining a purified DNA complementary to an mRNA that is differentially expressed in two nucleic acid samples comprising the steps of:(a) providing a first nucleic acid sample including mRNA molecules;(b) providing a second nucleic acid sample including mRNA molecules;(c) contacting each of said first nucleic acid sample and said second nucleic acid sample with a first oligodeoxynucleotide primer that hybridizes to a first site in mRNAs in said first and second nucleic acid samples;(d) reverse transcribing said mRNAs to which said first primer hybridizes to produce a first population of DNA strands that are complementary to said mRNAs in said first nucleic acid sample to which said first primer hybridizes, and a second population of DNA strands that are complementary to said mRNAs in said second nucleic acid sample to which said first primer hybridizes;(e) quantifying the amount of complementary DNA strands produced as a result of the reverse transcription step;(f) titrating said complementary DNA strands by running at least two different concentrations of the complementary DNA through a polymerase chain reactor and simultaneously contacting said first and second populations of DNA strands with a second oligodeoxynucleotide primer that hybridizes to a second site in said first and second populations of DNA strands, which second primer comprising T.sub.12 MNN, wherein M is A, G or C and N is A, G, C or T, said contacting being performed under conditions in which said second primer hybridizes with at least some of the DNA strands in said first and second populations;(g) amplifying portions of the DNA strands in said first and second populations of DNA strands with said second primer to produce a first and a second population of amplification products;(h) comparing the presence or level of individual amplification products in said first and second populations of amplification products; and(i) subtracting contaminating cDNAs from the amplification products, thereby resulting in a purified DNA complementary to mRNA that is differentially expressed in the samples.