Isothermal amplification and multimerization of DNA by bst DNA polymerase

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