Optimization of turn-back primers in isothermal amplification

Yasumasa Kimura, Michiel J. L. de Hoon, Shintaro Aoki, Yuri Ishizu, Yuki Kawai, Yasushi Kogo, Carsten O. Daub, Alexander Lezhava, Erik Arner, Yoshihide Hayashizaki
2011 Nucleic Acids Research  
Since the amplification pathway of each strand is symmetric, here we show an example derived from one particular strand. i. First, a reverse trun-back primer (TPr1) hybridizes to target DNA sequence (1) and followed DNA extension mediated by DNA polymerase creates DNA fragment flanked by TPr1 (2). Both ends of newly synthesized strand can be denatured partly and forming a stem-loop structure with thermal fluctuation. Such condition allows another reverse TP (TPr2) to hybridize to the exposed
more » ... get sequence and extends new strand which peels the existing strand off by strand displacement activity of the DNA polymerase (3). ii. The released DNA strand becomes a template for the next step as a forward TP (TPf1) can hybridize there (4). Extension from TPf1 creates DNA fragment flanked by TPf1 and complement of TPr1 (cTPr1) (5), which can be peeled off by the same mechanism as described above for TPr1 (6). iii. We named this peeled fragment flanked by TPf1 and cTPr1 as Intermediate product (IM1) (7). The 3' end of the IM1, cTPr1, is designed to form a stem-loop structure and DNA extension can be initiated at the 3' end by self-priming mechanism (8). iv. This event generates in total two different pathways (9). The one is self-priming from the complement of TPf1 (cTPf1) after stem-loop formation with thermal fluctuation (10). The other one occurs at the cTPr1 loop in the middle of the IM1 where another reverse TP (TPr4) can hybridize there (11) . v. Products are continuously amplified by the same mechanism as described above.
doi:10.1093/nar/gkr041 pmid:21310714 pmcid:PMC3089485 fatcat:li4vvddyvzf43pjlq7ltdwc6pq