TspMI, a thermostable isoschizomer of XmaI (5'C/CCGGG3'): characterization and single molecule imaging with DNA.

摘要: TspMI, a thermostable isoschizomer of XmaI from Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin SP sephadex C50, and Mono-Q fast protein liquid chromatography found be homodimer 40 kDa. Restriction mapping run-off sequencing TspMI-cleaved DNA ends depicted that it cleaved at 5'C/CCGGG3' generate four-base, 5'-CCGG overhang. sensitive methylation second third cytosines in its recognition sequence. TspMI worked optimally 60 degrees C with 6 mM Mg(2+), no Na(+)/K(+), showed star activity the presence 25% glycerol. could efficiently digest labeled higher concentration YOYO-I (one dye molecule one nucleotide), making useful candidate for real-time imaging experiments. Single interaction between lambda studied total internal reflection fluorescence microscopy. survived 30 polymerase chain reaction (PCR) cycles 10% glycerol 0.5 M trehalose without any loss and, hence, is suitable incorporation restriction-endonuclease-mediated selective-PCR various applications.