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 loopmediated 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 0 -and 5 0 -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/.

Point of Care Technologies for HIV

“…LAMP is a rapid amplification method employing a strand displacing Bst DNA polymerase and 4–6 primers, two of which are “fold back” primers [74] which form stem-loops motifs with self-priming capability. This results in an amplification scheme where the priming sequence is copied with each round of replication and remains tethered to the previous amplicon resulting in a concatenated product of alternating sense/antisense repeats of varied length.…”

“…Among the isothermal nucleic acid methods currently available, loop-mediated isothermal amplification [ 73 ] (LAMP) is the most widely researched and has been well characterized offering significant support during the development process. LAMP is a rapid amplification method employing a strand displacing Bst DNA polymerase and 4–6 primers, two of which are “fold back” primers [ 74 ] which form stem-loops motifs with self-priming capability. This results in an amplification scheme where the priming sequence is copied with each round of replication and remains tethered to the previous amplicon resulting in a concatenated product of alternating sense/antisense repeats of varied length.…”

“…Whilst a 95°C initial strand separation step is not essential [ 79 ], it has been shown to increase analytical sensitivity [ 80 ]. As with some other isothermal methods (SMAP, NEAR, and SDA), LAMP is highly dependent on the careful design of multiple complex primers and this can be overcome by the use of appropriate software [ 74 ].…”

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

“…Increasing the number of primers in a reaction heightens the chance of primer-primer interaction. Coupled with certain auxiliary activities of the Bst DNA polymerase (terminal transferase, template switching, extension from 3ʹ-mismatches), primer structures can lead to nontemplate amplification [23][24][25]. Primer sets should be screened in advance for potential primer-primer interaction, but nevertheless, false-positives can still occur during experimentation.…”