Isothermal amplification

A method of amplifying a target nucleic acid, comprising contacting a nucleic acid sample suspected of containing the target nucleic acid with six primers and a DNA polymerase with strand displacement activity, wherein

• the target nucleic acid comprises from 5′ to 3′, an F3 site, an F2 site, an F1 site, an R1 site, an R2 site, and an R3 site, and the complement to the target nucleic acid comprises, from 3′ to 5′, an F3c site, an F2c site, an F1c site, an R1c site, an R2c site, and an R3c site, and
• the six primers comprise
• a forward outer primer (FO) complementary to the F3c site,
• a reverse outer primer (RO) complementary to the R3 site,
• a forward inner primer (FI) comprising the F1c and F2 sites,
• a reverse inner primer (RI) comprising the R1 and R2c sites,
• a forward reverse pairing-competition primer (FC) complementary to the F1 site, and
• a reverse pairing-competition primer (RC) complementary to the R1c site,
• wherein the forward outer primer (FO), the reverse outer primer (RO), the forward reverse pairing-competition primer (FC), and the reverse pairing-competition primer (RC) are single site primers, and the forward inner primer (FI) and the reverse inner primer (RI) are double site primers;
• and
• amplifying the target nucleic acid with the DNA polymerase, wherein amplifying comprises
• producing a first basic structure comprising from 5′ to 3′, the R1 site, the R2c site, the R1c site, the F1c site, the F2c site, and the F1 site, wherein producing comprises
• (a) synthesizing DNA from (1) the forward outer primer (FO) annealing to the F3c site with the DNA polymerase and (2) the F2 region of the forward inner primer (FI) annealing to the F2c site with the DNA polymerase, wherein the strand elongated from the forward inner primer (FI) is displaced and released by the extension from the forward outer primer (FO), and
• (b) synthesizing DNA (1) with the DNA polymerase from the reverse outer primer (RO) annealing to the R3 site and (2) with the DNA polymerase from the R2c region of the reverse inner primer (RI) annealing the R2 site of the released nucleic acid strand, wherein the strand elongated from the reverse inner primer (RI) is displaced and released by the extension from the reverse outer primer (RO), thereby producing the first basic structure, and
• cyclically amplifying the first basic structure by contacting the first basic structure with the DNA polymerase, the forward inner primer (F1), the reverse pairing-competition primer (RC), the forward reverse pairing-competition primer (FC), and the reverse inner primer (RI) to provide amplified target nucleic acid.