Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification

We present eLAMP, a PERL script, with Tk graphical interface, that electronically simulates Loop-mediated AMPlification (LAMP) allowing users to efficiently test putative LAMP primers on a set of target sequences. eLAMP can match primers to templates using either exact (via builtin PERL regular expressions) or approximate matching (via the tre-agrep library). Performance was tested on 40 whole genome sequences of Staphylococcus. eLAMP correctly predicted that the two tested primer sets would amplify from S. aureus genomes and not amplify from other Staphylococcus species. Open source (GNU Public License) PERL scripts are available for download from the New York Botanical Garden's website. 1. Introduction Several highly efficient methods of DNA amplification have been proposed. Although the Polymerase Chain Reaction (PCR; [1, 2]) is the most widely used method, newer more sensitive techniques are favored for some applications—particularly diagnostic testing. Loop–mediated isothermal AMPlification (LAMP; [3]) is perhaps the most promising of these new methods [4]. LAMP can quickly generate large quantities of amplicon from low abundance template without temperature cycling—thereby lowering the cost and complexity of necessary laboratory equipment. In addition, LAMP amplicons, unlike PCR amplicons, can be directly detected via a colorimetric chemical reaction [5, 6]. Unlike PCR which requires one pair of primers, LAMP requires a minimum of two nested primer pairs. Generally, primers should be 15–25 bases long with 40–60% GC content (annealing 55–65°C). The amplicon defined by the outer primers should be ≤280 base pairs and the spacing between inner and outer primers including the outer primers ≥40 base pairs [5, 7]. Primers are synthesized such that the reverse complements of the inner primers are connected to the outer primers by a linker (usually five thymine residues). Thus, interconnected loops are produced during amplification [3]. Optionally, additional sets of primers can be used to increase amplification efficiency [3, 7–9]. The quality of LAMP primer/template match (i.e., the percent mismatched bases and the distribution of mismatches) necessary for efficient amplification has not been studied, but there are studies of PCR primer/template match quality that suggest an exact 3′ match of 2-3(4) bases is required for Taq polymerase to extend the primer along the template [10–14] and mismatches outside of the 3′ end of the primer have little effect on amplification efficiency [10, 14]. Although PCR and LAMP have many similarities, different