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Preferential amplification of mRNA over DNA using chemically modified primers

  • US 8,614,071 B2
  • Filed: 12/08/2010
  • Issued: 12/24/2013
  • Est. Priority Date: 12/11/2009
  • Status: Active Grant
First Claim
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1. A method of selective amplification of a messenger RNA (mRNA) target in the presence of corresponding genomic DNA contaminant comprising the steps of:

  • a. providing a sample that comprises said mRNA target and said corresponding genomic DNA contaminantb. hybridizing a first oligonucleotide to said mRNA target and performing RNA-directed DNA synthesis using at least one enzyme capable of RNA-directed synthesis, wherein said first oligonucleotide;

    i. comprises at least one nucleotide with a base covalently modified at the exocyclic amino group,ii. is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant, and,iii. spans an exon-exon junction in the target;

    c. amplifying the product of step b) using said first oligonucleotide and a second oligonucleotide with at least one enzyme capable of DNA-directed DNA synthesis;

    wherein said second oligonucleotide is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant;

    wherein said mRNA target is selectively amplified over said corresponding genomic DNA contaminant when compared to amplifying with an unmodified first oligonucleotide.

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