Optimization of turn-back primers in isothermal amplification

The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as pathogen detection and SNP genotyping. 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. The evaluation showed that our approach yields a superior primer design in isothermal amplification and is robust against variations in the experimental setup. Our computer script is freely available at: http://gerg.gsc.riken.jp/TP_optimization/.