| 主权项 |
1. A method of identifying the presence of a target sequence in a nucleic acid sample, the method comprising:
a. mixing in a reaction mixture ingredients comprising:
i. a nucleic acid sample;ii. a first primer binding at a first binding site of a first strand of a nucleic acid target sequence having a 5′ end and a 3′ end;iii. a second primer binding at a second binding site to a complementary strand to the target sequence 5′ to the first binding site with respect to the first strand;iv. one or more additional primers selected from:
(a) an internal primer pair comprising a third primer binding to the first strand at a third binding site between the first binding site and the second binding site and a fourth primer binding to the complementary strand at a fourth binding site between the first binding site and the third binding site; or(b) one or more spiral primers binding the target sequence between the first binding site and the second binding site, selected from:
(i) a reverse internal spiral primer having a 5′ portion and a 3′ portion in which the 5′ portion binds to the first strand and the 3′ portion binds to the complementary strand;(ii) a forward internal spiral primer having a 5′ portion and a 3′ portion in which the 5′ portion binds to the complementary strand and the 3′ portion binds to the first strand;(iii) a reverse internal triplex spiral primer having a 5′ portion, a 3′ portion and a middle portion in which the 5′ portion and the 3′ portion binds the first strand and the middle portion binds the complementary strand; and(iv) a forward internal triplex spiral primer having a 5′ portion, a 3′ portion and a middle portion in which the 5′ portion and the 3′ portion binds the complementary strand and the middle portion binds the first strand.v. a DNA polymerase; and b. incubating the reaction mixture at a temperature or combination of temperatures effective to produce one or more amplification products in a reaction mixture containing a nucleic acid comprising the target sequence. |
