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Method of differential display of exposed mRNA by RT/PCR

  • US 5,599,672 A
  • Filed: 12/08/1994
  • Issued: 02/04/1997
  • Est. Priority Date: 03/11/1992
  • Status: Expired due to Term
First Claim
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1. A method of comparing the presence or level of individual mRNA molecules in two or more nucleic acid samples, comprising the steps of:

  • providing a first nucleic acid sample including mRNA molecules;

    providing a second nucleic acid sample including mRNA molecules;

    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;

    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;

    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 site includes NNNRNNATGN (SEQ ID NO;

    27), 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;

    extending said second primer to produce a third population of DNA strands that are complementary to the DNA strands in said first population to which said second primer hybridizes, and a fourth population of DNA strands that are complementary to the DNA strands in said second population to which said second primer hybridizes;

    amplifying portions of the DNA strands in said first and third populations of DNA strands with said first and second primers to produce a first population of amplification products;

    amplifying portions of the DNA strands in said second and fourth populations of DNA strands with said first and second primers to produce a second population of amplification products;

    comparing the presence or level of individual amplification products in said first and second populations of amplification products.

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