|There are a few basic tools commonly used in the world
of molecular biology. All together and in various combinations, they make
up the bulk of our toolbox for studying genes and gene expression.
One of these tools is the polymerase chain reaction.
When only very small amounts of a DNA to be examined are present, we need a method to copy and produce more of the sequence. Especially if there is a need to do this quickly, as in a test for a disease, for example, it may take too long to prepare a DNA fragment for ligation into a vector, cloning, and amplification to generate enough to analyze. The polymerase chain reaction, or PCR, is a way to amplify DNA fragments directly. Pieces of DNA called primers that can anneal to specific locations on a target gene are used to provide specific start sites for DNA polymerase, an enzyme that copies DNA, to produce a new copy of each complementary strand. These strands are separated, and the process repeated. Each time the PCR occurs, it results in a doubling of the DNA that is present. Repeating the PCR 40 times results in an amplification of the target DNA sequence equal to 240 or 1,000,000,000,000 times.
PCR can be used to amplify specific regions of the DNA, or to detect polymorphisms in the DNA similar to RFLPs. If the sequence complementary to the primer has a mutation, then the PCR will be blocked, and this can be tested for. PCR can also be used to amplify specific RNA molecules, to detect their presence. First the RNA must be converted to a DNA molecule using reverse transcriptase. This complementary DNA, or cDNA, molecule is a copy of the RNA molecule that can be used for PCR, ligation into vectors and cloning, sequencing, etc.