Isothermal Amplification and Multimerization of DNA by Bst DNA Polymerase

Abstract

We have demonstrated the isothermal in vitro amplification and multimerization of several different linear DNA targets using only two primers and the strongly strand-displacing exonuclease-negative Bst DNA polymerase. This reaction has been termed linear target isothermal multimerization and amplification (LIMA). LIMA has been compared with cascade rolling-circle amplification and has been found to be less sensitive but to yield similar variable-length multimeric dsDNA molecules. Products from several different LIMA reactions were characterized by restriction analysis and partial sequence determination. They were found to be multimers of subsets of the target sequence and were not purely primer derived. The sensitivities with respect to target concentration of several different LIMA reactions were determined, and they varied from 0.01 amol to 1 fmol. The sensitivity and specificity of LIMA were further tested using E. coli genomic DNA, and the selective amplification of a transposon fragment was demonstrated. A successful strategy for reducing LIMA-dependent background DNA synthesis in rolling-circle amplification embodiments was devised. This entailed the affinity purification of circular DNA templates before amplification.

Loop-Mediated Isothermal Amplification for Detection of African Trypanosomes

“…The LAMP reaction relies mainly on autocycling strand displacement DNA synthesis that is similar to the cascade rolling-circle amplification reported by Hafner et al (12). However, there is a possibility that linear target isothermal multimerization and amplification also occurs during the LAMP reaction.…”

“…It was reported that Bst DNA polymerase has two distinct activities, termed linear target isothermal multimerization and amplification and cascade rolling-circle amplification (12). In the same manuscript, target DNA-specific amplification of both linear target isothermal multimerization and amplification and cascade rolling-circle amplification was also proved (12).…”

“…The mechanism of LAMP reaction was well explained by Notomi et al (23). In addition, Hafner et al reported that the isothermal in vitro amplification and multimerization of linear DNA targets (linear target isothermal multimerization and amplification) with two primers and Bst DNA polymerase (12).…”

“…Since the LAMP reaction is done under isothermal conditions (63 to 65°C), simple incubators, such as a water bath or block heater, are sufficient for the DNA amplification. Moreover, LAMP synthesizes 10 to 20 g of target DNA within 30 to 60 min, and the LAMP reaction appears to be limited only by amount of deoxynucleoside triphosphates and primers (12,23). In the process, a large amount of pyrophosphate ion is produced, which reacts with magnesium ions in the reaction to form magnesium pyrophosphate, a white precipitate by-product (20).…”

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…In this case, the LAMP product hybridized only with the panfungal U210 probe, as expected. We have no clear explanation for the formation of these odd banding patterns, but it appears to be the result of specific linear target isothermal multimerization and amplification of the template DNA (19), a property of the Bst DNA polymerase that has been demonstrated by Hafner et al (12).…”