Nicking and extension amplification reaction for the exponential amplification of nucleic acids
The invention is in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature.
1. 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.
2. The system of claim 1 , wherein the amplification reagent mixture contains no oligonucleotides other than the first oligonucleotide and the second oligonucleotide.
3. The system of claim 1 , wherein said nucleic acid target sequence is present in a sample from an animal, plant, or food.
4. The system of claim 1 , wherein said nucleic acid target sequence is from a human pathogen present in a sample of mucus, sputum, or saliva obtained from a human.
5. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 10-15 nt and/or said recognition region of the second oligonucleotide is 10-15 nt.
6. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 11-14 nt and/or said recognition region of the second oligonucleotide is 11-14 nt.
7. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 12-13 nt and/or said recognition region of the second oligonucleotide is 12-13 nt.
8. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 10-15 nt and said recognition region of the second oligonucleotide is 10-15 nt.
9. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 11-14 nt and said recognition region of the second oligonucleotide is 11-14 nt.
10. The system of claim 1 , wherein said recognition region of the first oligonucleotide is 12-13 nt and said recognition region of the second oligonucleotide is 12-13 nt.