Lambda exonuclease mediates efficient MCT hybrid synthesis.
A) Bioanalyzer DNA 1000 capillary electrophoresis analysis of a MC-PCR sample subjected to lambda exonuclease and/or 418 amu MCT probes during hybrid synthesis reactions. Alien DNA served as template for MC-PCR resulting in amplicons (209 bp) encoded on one end by a 352 amu MCT derived from forward priming and modified on the other end by phosphate derived from reverse priming. Aliquots of pooled amplicons served as sample input for hybrid synthesis reactions for lanes 1–4 under the conditions specified. Aliquots of a pooled MC-PCR without Alien DNA served as no template controls (NTC) for each hybrid synthesis reaction condition. The molarity in nanomolar of the Alien amplicons present at the end of the hybrid synthesis process is shown under each band. Alien hybrids possess a 103 bp double stranded segment and a single stranded segment of 106 bases. They cannot be accurately sized or quantified by CE. B) MCT detection from the molecular species formed during the hybrid synthesis reactions described in panel A. Results from NTC samples (a) were compared to those of samples containing DNA (b) for each condition tested. Gray bars, measuring forward primer binding/extension, show the response levels detected by the mass analyzer at 353 amu [M+H]+; red bars, measuring probe binding/extension, show the 419 amu [M+H]+ response levels. L, DNA ladder.