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Molecular Basis of Heredity: Part 4. Gene Identification and Tests

Author(s): Raye L. Alford, PhD

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) was invented in 1985 by Kary Mullis, PhD. Dr. Mullis received the Nobel Prize in Chemistry in 1993 for his work.

PCR is the enzymatic amplification of DNA, creating millions of copies of a segment of DNA from only a small amount of starting material. To begin, DNA primers are manufactured that are complementary to a portion of the target DNA sequence. The primers and the target DNA are mixed and denatured (separated into single strands) by high temperature. The mixture is then cooled, allowing the primers to hybridize (bind) to the target DNA. After hybridization, a special DNA polymerase enzyme that is resistant to heat inactivation synthesizes new DNA strands from the primers, using the target DNA as a template. The process is repeated for 30-50 cycles of heat denaturation, primer binding, and DNA synthesis. After many cycles of PCR, millions of copies of the target DNA are created where only a few existed before. PCR is also fast. Most PCR reactions take only a few hours to perform in the laboratory.

DNA fragments generated by PCR can be subjected to a variety of different analyses, depending on the type of mutation or polymorphism sought. For example, gel-based methods that separate fragments based on size are ideal for detecting small expansions or deletions such as occur in repetitive elements, such as trinucleotide repeats, while DNA sequencing will detect changes, such as single nucleotide substitutions, in the sequence of a gene.

PCR has several advantages over methods like Southern analysis. First, PCR uses very small amounts of DNA as starting material, while Southern analysis and other methods require large amounts of input DNA. PCR also can use degraded DNA as starting material because even a degraded DNA sample (DNA that is broken into pieces by decay) is likely to have pieces still large enough to be bound by the primers and amplified while Southern analysis tyipcally examines much larger pieces of DNA. These properties make PCR useful for forensic DNA analyses where evidentiary DNA is frequently scarce and/or compromised by environmental or other exposures.

In the early days of PCR, the methodology could only amplify DNA fragments a few hundred to two or three thousand base pairs in length. Recent improvements in PCR technology allow today’s scientists to amplify DNA fragments that are up to several thousand base pairs in length, but even today PCR cannot amplify very long target DNA sequences.