Polymerase assay for detection of expression clones containing thermostable Pol activity from a boiling hot spring metagenomic library.

<p>Clones judged by sequence to encode complete <i>pol</i> genes were cultivated and thermostable proteins extracted as described in the Methods. Extension of a 37 nucleotide (nt) ROX-labeled primer <b>(peak 1)</b> on a 41 nt template oligonucleotide by a polymerase results in a shift from 37 to 41 nt <b>(peak 2)</b>. If a single nucleotide non-templated extension occurs as seen with Taq Pol, a peak at 42 nt results <b>(peak 3)</b>. Degradation of the ROX-labeled substrate by 3′ to 5′ exonuclease activity results in peaks of less than 37 nt <b>(peak 4)</b>. nt: size of standard markers in nucleotides.