| 主权项 |
1. A method of amplifying a target polynucleotide sequence, the method comprising:
(a) obtaining, from an animal, a sample comprising a target nucleic acid, the target nucleic acid comprising the target polynucleotide sequence, (b) without first subjecting the target nucleic acid to a thermal denaturation step associated with amplification of the target polynucleotide sequence, combining, in a single step, the obtained sample directly with an amplification reagent mixture or diluting the obtained sample and combining, in a single step, the diluted sample with an amplification reagent mixture, in either case, the amplification reagent mixture being free of bumper primers and comprising: (i) a polymerase, (ii) a nicking enzyme, (iii) a first oligonucleotide comprising a 5′ portion that comprises a nicking enzyme binding site that is non-complementary to the target polynucleotide sequence and a 3′ portion that hybridizes to the target polynucleotide sequence, and (iv) a second oligonucleotide comprising a 5′ portion that comprises a nicking enzyme binding site that is non-complementary to the target polynucleotide sequence and a 3′ portion that hybridizes to the target polynucleotide sequence, (c) amplifying the target polynucleotide sequence, without the assistance of bumper primers, by subjecting the reaction mixture formed by the step of combining to essentially isothermal conditions, from steps comprising: (i) forming a first duplex comprising the target polynucleotide sequence and the first oligonucleotide; (ii) extending, using the polymerase, the first oligonucleotide along the target polynucleotide sequence to form an extended first oligonucleotide comprising a sequence complementary to the second oligonucleotide; (iii) forming a second duplex comprising the second oligonucleotide and the extended first oligonucleotide; (iv) extending, using the polymerase, the second oligonucleotide along the extended first oligonucleotide to form a third duplex comprising an extended second oligonucleotide comprising a sequence complementary to the first oligonucleotide and a first double-stranded nicking enzyme binding site; (v) nicking, with the nicking enzyme, the first nicking site on the third duplex to produce a fourth duplex comprising the extended second oligonucleotide and a fragment of the extended first oligonucleotide; and (vi) extending, using the polymerase, the fragment of the extended first oligonucleotide along the extended second oligonucleotide of the fourth duplex to produce a double-stranded nucleic acid product containing a second double-stranded nicking enzyme binding site, and (d) detecting the amplified target polynucleotide sequence in real time within 10 minutes of subjecting the reaction mixture to essentially isothermal conditions. |
