Nicking and extension amplification reaction for the exponential amplification of nucleic acids

A system configured to amplify a double-stranded nucleic acid target sequence having a sense strand and an antisense strand under essentially isothermal conditions and to detect the amplified target nucleotide sequence without first subjecting the target nucleic acid to a thermal denaturation step associated with amplification of the target polynucleotide sequence, the system comprising an amplification reagent mixture comprising:

• a) a DNA polymerase;
• b) a first oligonucleotide comprising a recognition region at its 3′ end;
• a nicking enzyme binding site and a nicking site upstream of said recognition region;
• and a stabilizing region upstream of said nicking site,
• wherein the recognition region is complementary to the 3′ end of the target antisense strand and is 8-15 nucleotides in length;
• and
• wherein when the recognition region of the first oligonucleotide is hybridized to the target sequence, the stabilizing region of the first oligonucleotide is not hybridized to the target sequence,
• c) a second oligonucleotide comprising recognition region at its 3′ end;
• a nicking enzyme binding site and a nicking site upstream of said recognition region;
• and a stabilizing region upstream of said nicking site, wherein the recognition region is complementary to the 3′ end of the target sense strand and is 8-15 nucleotides in length;
• and
• wherein when the recognition region of the second oligonucleotide is hybridized to the target sequence, the stabilizing region of the second oligonucleotide is not hybridized to the target sequence;
• and
• d) a first nicking enzyme that is capable of nicking at the nicking site of said first oligonucleotide and does not nick within said target sequence;
• and a second nicking enzyme that is capable of nicking at the nicking site of said second oligonucleotide and does not nick within said target sequence.