Enhanced Diagnostic Tools
The quenching of pyrrolo-dC allows local structural changes to be probed with great sensitivity. Using pyrrolo-dC, Liu and Martin8 have characterized the transcription bubble in elongation complexes of T7 RNA Polymerase to single-base resolution by observing roughly a two-fold increase in fluorescence as the polymerase induces melting. By starving the T7 RNA Polymerase of specific nucleoside triphosphates, the enzyme could be stalled at specific sites, producing 'fluorescence snapshots' of the complex, and yielding detailed information on the nature of the transcription bubble and heteroduplex.
Work is still progressing in evaluating the effect of this modified fluorescent nucleoside in biological systems and will be reported. However, a few comments on our findings to date may be of interest. Oligonucleotides containing pyrrolo-dC act as efficient primers and the PCR products appear to be identical for primers with 0 to 5 pyrrolo-dC residues replacing dC. Preliminary data indicate that pyrrolo-dC codes as dC in PCR experiments. And very preliminary evidence indicates that pyrrolo-dC triphosphate is incorporated efficiently by Taq polymerase and is incorporated specifically opposite dG.
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