The polymerase chain reaction, known more commonly as PCR, is a lab technique used to amplify a sequence of DNA. The process takes advantage of the cellular system of DNA replication. Geneticists and medical researchers use PCR to determine if certain genes are present in a DNA sample, to create enough DNA to analyze the sequence, and for pulling out a sequence of interest for cloning and other manipulations.
PCR can be understood as a recipe. A certain amount of DNA is added to a measured mix of enzymes (protein) and primers (short sequences of DNA that determine the start and stop points along the sequence), along with extra nucleotides (building blocks of DNA) and a buffer, and then cycled through temperatures that activate and deactivate the enzyme in order to obtain the amplified sequence. The temperature gradients are determined by as much trial and error as calculation and provide optimal conditions for the two strands of DNA in a molecule to separate, the primers to anneal, and the new strand to elongate. The enzyme responsible for elongation is Taq polymerase. Polymerase is the enzyme cells use to copy DNA strands. Taq polymerase is a polymerase found in hot springs and withstands the temperatures needed to separate the double stranded DNA strands. The discovery of Taq polymerase freed researchers from having to add more enzyme to each cycle of PCR.
PCR machines now cycle small tubes of samples allowing the DNA strands to amplify via a chain reaction. As the program progresses through each cycle, the polymerase and primers are reused, making copies of each DNA strand present in the mixture, including the original DNA and all DNA already created. To amplify large portions of DNA many primers are used. To amplify portions of a single gene as few as two are used, depending on the size of the gene. By the end of 30-40 cycles, there is often enough DNA to run the PCR mixture on a gel and visualize the size of the DNA sequence amplified (called the PCR product). 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.
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 (called reverse transcription), but then PCR continues in the same manner (RT-PCR). Because RNA is converted directly to proteins, the presence of RNA in a blood or tissue sample can determine if a gene is turned on or off. In this way researchers have been able to discover the environmental influences of abnormalities and disease. They have also been able to determine the genes involved in certain disorders such as hypertension, diabetes, and enzyme deficiencies.
