title: Optimization of turn-back primers in isothermal amplification.
abstract: The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as infectious disease, genetic disorder and drug dosage adjustment. Meanwhile, many of such technologies have complex reaction processes and often require a fine-tuned primer set where existing primer design tools are not sufficient. We have developed a primer selection system for one important primer, the turn-back primer (TP), which is commonly used in loop-mediated amplification (LAMP) and smart amplification process (SmartAmp). We chose 78 parameters related to the primer and target sequence, and explored their relationship to amplification speed using experimental data for 1344 primer combinations. We employed the least absolute shrinkage and selection operator (LASSO) method for parameter selection and estimation of their numerical coefficients. We subsequently evaluated our prediction model using additional independent experiments and compared to the LAMP primer design tool, Primer Explorer version4 (PE4). The evaluation showed that our approach yields a superior primer design in isothermal amplification and is robust against variations in the experimental setup. Our LASSO regression analysis revealed that availability of the 3'- and 5'-end of the primer are particularly important factors for efficient isothermal amplification. Our computer script is freely available at: http://gerg.gsc.riken.jp/TP_optimization/.
authors.affiliation: RIKEN Omics Science Center (OSC), RIKEN Yokohama Institute, Yokohama, Kanagawa, 230-0045, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Kimura
authors.forename: Michiel J L
authors.initials: MJ
authors.lastname: de Hoon
authors.forename: Shintaro
authors.initials: S
authors.lastname: Aoki
authors.forename: Yuri
authors.initials: Y
authors.lastname: Ishizu
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawai
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kogo
authors.forename: Carsten O
authors.initials: CO
authors.lastname: Daub
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.forename: Erik
authors.initials: E
authors.lastname: Arner
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D017931:DNA Primers / Q000737:chemistry*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012984:Software; D013696:Temperature
chemical_list: D017931:DNA Primers