Quantitative real-time PCR (qPCR) is the most popular technique for quantifying nucleic acids. qPCR is simple to perform and offers excellent detection sensitivity, so scientists employ it in a diverse array of fields ranging from agricultural biology to molecular diagnostics.
Like conventional PCR, qPCR uses denaturation, annealing, and extension cycles to exponentially produce additional copies of nucleic acid sequences of interest. Unlike conventional PCR, qPCR attaches a signal-emitting agent — typically a dye or a probe — to these sequences of interest. Signal magnitude after each cycle is measured and compared against controls to quantitate DNA abundance.
This guide from LGC Biosearch Technologies discusses considerations for selecting probes and primers for qPCR including sample type, research aims, and logistical obstacles, and it outlines probe options and how to troubleshoot probe-based qPCR.
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