The EnzChek® Reverse Transcriptase Assay Kit is a convenient, efficient and inexpensive assay for measuring reverse transcriptase activity. The key to this method is the PicoGreen® dsDNA quantitation reagent, which preferentially detects dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic acids or free nucleotides. In the assay, reverse transcriptase activity in a biological sample generates long RNA-DNA heteroduplexes from a mixture of a long poly(A) template, an oligo-dT primer and dTTP. The RNA-DNA heteroduplexes formed are then detected by the PicoGreen reagent. In less than an hour, samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluorescein (FITC). The assay is sensitive, detecting as little as 0.02 units of HIV-1 reverse transcriptase, and has about a 50-fold linear range
Because it is much more rapid and less expensive than standard isotopic assay or immunoassays, it is suitable for testing large numbers of biological samples. Its simplicity also makes it useful for automated high-throughput screening of reverse transcriptase inhibitors.
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