B. Frey

HEAT-LABILE URACIL-DNA GLYCOSYLASE : PURIFICATION AND CHARACTERIZATION

A uracil‐DNA glycosylase (UNG) from a psychrophilic marine bacterium (BMTU 3346) has been purified to apparent homogeneity. The enzyme has a molecular weight of 23 400 Da. It is stable in complex buffers (containing glycerol/BSA), whereas it is heat‐labile in dilute buffers (free of stabilizers) with a half‐life of 2 min at 40°C. Due to the thermolability, uracil‐DNA glycosylase is suitable for application in the carryover prevention technique showing less residual activity and/or a slower reactivation rate than the usually applied UNG from Escherichia coli.

Amplification of genomic DNA by PCR.

The polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two regions of known sequence (1-3). It requires two oligonucleotide primers that flank the DNA fragment to be amplified and employs repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with a thermostable DNA polymerase (4). These primers typically have different sequences, are complementary to sequences that lie on opposite strands of the template DNA, and flank the segment of DNA that is to be amplified. Since the extension products themselves are also complementary to and capable of binding primers, successive cycles of amplification essentially double the amount of target DNA synthesized in the previous cycle.