Real time polymerase chain reaction, also known as RT-PCR, is a new take on an established genetic research technique. The polymerase chain reaction, PCR, is the amplification of a sequence of DNA using the cell’s own system of DNA replication. Geneticists and Medical Researchers use PCR to determine if certain genes are present in a DNA sample and to create enough DNA to measure it. RT-PCR is a computerized way of measuring the reactions as they progress, thus adding a quantitative capacity to the previously qualitative technique.
PCR is setup like a cooking recipe. A portion of DNA is added to a mix of enzymes and primers, small pieces of DNA in the sequence of interest, and then cycled through temperatures that activate and deactivate the enzyme allowing the DNA pieces to separate, anneal, and elongate. The enzyme is Taq polymerase. Polymerase is the enzyme cells use to replicate, or copy, DNA strands. Taq polymerase is a polymerase found in hot springs and withstands the temperatures needed to separate the double stranded DNA strands. This freed up researchers from having to add more enzyme each cycle of PCR. Machines now cycle small tubes of samples through the heat and cold allowing the DNA strands to build up. As the program progresses through each cycle the polymerase and primers are reused, making copies of the DNA strands starting at the sequence where the primers bind. To amplify large portions of DNA many primers are used. To amplify portions of a single gene as few as two are sometimes used. By the end of 30-40 cycles there should be enough DNA to run the PCR mixture on a gel and visualize the size of DNA amplified. Because the sequence of the primers was known the researcher can determine if the result is positive or negative. If the researcher is aiming to determine differences in the samples, the DNA can then be used in further testing and purification procedures.
RT-PCR has changed this procedure by adding fluorescent probes to the primers. These probes have to be within a certain distance from one another on the DNA strand to be detected. As they alter their distance from one another a computer measures the luminescence and calculates this in correlation with the cycle. This measurement adds a quantitative measurement to the procedure. Not only can a researcher know that amplification is occurring, and therefore the gene or particular sequence is present, but they can measure how much of that particular sequence is in the sample based on its rate of amplification.
This same process can be used with RNA, which aids in determining the activity of a gene as well as discerning the genomes of certain infectious agents such as HIV. An additional step is needed to convert RNA to DNA, but then continues in the same manner. RT-PCR has simplified the RNA process, previously the conversion and purification steps added days to research time. Computerized results also decreases the time needed for calculations and visualization.