Analysts interested in quantifying and verifying the presence of DNA or RNA specific to an organism in a sample should consider using the Thermo Fisher QuantStudio 3 system. The QuantStudio system 3 uses a quantitative polymerase chain reaction (qPCR) technique to calculate how much DNA or RNA is present. With a qPCR assay specific to an organism’s DNA, the QuantStudio 3 system heats the sample to split, or denature, the DNA strand. Next the temperature is reduced and primers and sequence specific probes anneal to the newly created single stranded DNA. DNA polymerase binds to the primer and starts attaching complimentary nucleotides to the single stranded DNA making it double stranded. Once the polymerase reaches the target-specific probe, a fluorophore is cleaved from its quencher resulting in fluorescence that increases with each round of amplification. The instrument measures the fluorescence after each cycle. The qPCR cycle is repeated 35-40 times and an accurate concentration of DNA is determined based on the fluorescence of a previously run calibration curve for the amount of DNA specific to a particular organism. Typical qPCR work conducted on the QuantStudio 3 in the environmental engineering laboratory is for microorganisms in water, soil, and air.
