Polymerase Chain Reaction

PCR is such a powerful, easy, and relatively inexpensive technique that it seems that molecular biologists are always looking for ways to use PCR in their research. Every month, scientific journals describe modifications to tailor the basic PCR approach to new applications.

One variation that has proved very fruitful in gene identification is the use of "degenerate primers." Many genes tend to be highly conserved among different species. Homologues, which are genes from different organisms whose protein products have similar functions, tend to have very similar, but not necessarily identical, sequences. The differences in sequence make it challenging to design standard PCR primers to search for homologues.

However, by comparing the DNA sequences of the gene as it occurs in many different species and finding portions of the sequence that are the same in all the species, a researcher can make an educated guess regarding which nucleotides in an unidentified homologue are likely to be identical to those in a known homologue.

The researcher can design a set of "degenerate" PCR primers, which are primers whose nucleotide sequence is fixed only in those positions where the nucleotides are presumed to be known. In the other positions, nucleotides are allowed to incorporate at random. This makes it likely that at least one of the primers will amplify the unknown target. By conducting PCR with degenerate primer sets, and by using primer annealing temperatures that are lower than normal to allow for less-than-perfect base-pairing, a researcher can often amplify a gene in a single experiment, thus isolating the new homologue and allowing it to be sequenced and studied.

Another important variation on PCR is reverse-transcription PCR. This technique involves first copying RNA into DNA molecules, using the enzyme reverse transcriptase, and subsequently using the standard PCR technique to amplify this complementary DNA (cDNA). Because the messenger RNA content of a cell or tissue represents only the genes that are actively being expressed, this technique provides a powerful method of analyzing gene expression.