| 主权项 |
1. A method for amplifying a target nucleic acid by an activation by polyphosphorolysis (APP) reaction comprising the steps of:
(a) annealing to a template strand of the target nucleic acid a complementary activatable oligonucleotide P* having a non-extendable nucleotide at its 3′ terminus and having no mismatched nucleotides at or near its 3′ terminus, so that the terminal nucleotide is hybridized to the template strand when the oligonucleotide P* is annealed, (b) polyphosphorolyzing the resulting duplex with:
1) one or more polyphosphorolyzing agents selected from the group consisting of a compound of the general formula I: wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;a compound of general formula II: wherein n and/or m are selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, with the proviso that n and m cannot both be 0, andX is selected from the group consisting of: and2) an enzyme having polyphosphorolysis activity that activates the oligonucleotide P* by removal of the hybridized terminal nucleotide, and (c) polymerizing a nucleic acid by extending the activated oligonucleotide P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase to synthesize the desired nucleic acid strand; and (d) repeating the steps (a)-(c) as necessary to amplify the target nucleic acid. |
