DNA polymerase profiling.
We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alteration is reported by fluorescence resonance energy transfer between two dyes introduced into the molecular beacon stem. We describe real-time reaction profiling of two model DNA polymerases. We demonstrate kinetic characterization, rapid optimization of reaction conditions, and inhibitor profiling using the presented assay. Furthermore, to supersede purification steps in screening procedures of DNA polymerase mutant libraries, detection of enzymatic activity in bacterial expression lysates is described.
Summerer
Daniel
D
The Scripps Research Institute, La Jolla, CA, USA.
D004256:DNA Polymerase I / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes; D018894:Reverse Transcriptase Inhibitors / Q000032:analysis*
D015335:Molecular Probes; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D004256:DNA Polymerase I