Compositions and methods quantifying a nucleic acid sequence in a sample

A method of quantifying a specific product in a nicking and extension amplification reaction, the method comprising:

• (a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, a nicking enzyme, and a detectable polynucleotide probe, wherein each of the primer oligonucleotides comprises 5 contiguous 2′-O-methyl modified nucleotides are positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule;
• (b) generating amplicons comprising at least a portion of said target nucleic acid molecule;
• and
• (c) detecting a signal specific for oligonucleotide probe hybridization to the target nucleic acid molecule or amplicon thereof, wherein the signal indicates the quantity of the target nucleic acid molecule present in the sample or an amplicon thereof.

An isolated oligonucleotide comprising from 5′ to 3′,

• i) a first region, and
• ii) a second region,
• wherein the first region comprises a nicking enzyme recognition sequence;
• wherein the second region comprises at least 9 nucleotides that specifically bind a complementary sequence on a target nucleic acid molecule;
• and wherein the second region comprises one or more 2′ modified nucleotides.