2006 Volume 43 Pages 143-148
Applicability and reliability of two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with PCR generated polydeoxyribonucleotide probes, specific for chromosomal encoded gene, methyl coenzyme M reductase (mcr) in Methanococcus vannielli, was tested and evaluated. The probes were labeled with dinitrophenol (DNP) and the efficiency of probe-labeling was improved by optimizing the concentrations of DNP-labeled nucleotide and Mg2+ in PCR mixture. The target mcr gene was successfully detected, which was again verified by the disappearance of the signals after treating the target with DNase prior to hybridization or washing with high stringency buffer. However, a few nonspecific signals were observed when the method was applied to pure cultures of Methanoculleus bourgensis and Escherichia coli.