8 - DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible

Abstract

Polymerase chain reaction (PCR) is a method for amplifying (copying) small amounts of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It can be used to isolate specific DNA fragments, end label DNA, clone complementary deoxyribonucleic acid (cDNA) and genomic DNA, sequence DNA, mutate specific DNA sequences, alter promoters, quantitate the amount of RNA or DNA, and identify molecular markers for taxonomic or ecological studies. The PCR requires a DNA polymerase, deoxynucleotide triphosphates (dNTPs), template DNA, and primers. Information about sequences at each end of the DNA to be amplified is needed in order to synthesize appropriate primers for a “standard” PCR. When two specific primers are used, amplification of DNA theoretically is geometric, producing large quantities of specific DNA suitable for sequencing, cloning, or probing. The PCR methods that use single primers, such as Random Amplified Polymorphic DNA-PCR (RAPD-PCR), can also result in DNA amplification. RAPD-PCR uses short, randomly chosen primers to amplify multiple DNA segments in a genome. The resulting banding patterns (similar to bar codes) provide information about a genetic variation within the entire genome of insects. The power of PCR to amplify DNA is dramatic; theoretically even a single molecule can be amplified, although efficiency is usually lower. This power creates formidable problems with contamination, and requires careful organization of PCR experiments and the use of adequate controls. The relative ease with which PCR can be used by novices in molecular biology has made it possible for a diverse group of biologists to use PCR to study molecular systematics, evolution, ecology, behavior, and development.