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Polymerase Chain Reaction

Designing Primers, A Typical Pcr Reaction, Contamination In Pcr Reactions, Pcr Applications And Variations

The polymerase chain reaction (PCR) is a laboratory technique for "amplifying" a specific DNA sequence. PCR is extremely efficient and sensitive; it can make millions or billions of copies of any specific sequence of DNA, even when the sequence is in a complex mixture. Because of this power, researchers can use it to amplify sequences even if they only have a minute amount of DNA. A single hair root, or a microscopic blood stain left at a crime scene, for example, contains ample DNA for PCR.

PCR has revolutionized the field of molecular biology. It has enabled researchers to perform experiments easily that previously had been unthinkable. Before the mid-1980s, when PCR was developed, molecular biologists had to use laborious and time-consuming methods to identify, clone, and purify DNA sequences they wanted to study. Kary Mullis was awarded the 1993 Nobel Prize in Chemistry for inventing PCR.

PCR is based on the way cells replicate their DNA. During DNA replication, the two strands of each DNA molecule separate, and DNA polymerase, an enzyme, assembles nucleotides to form two new partner strands PCR begins by separating complementary DNA strands, and ends by adding short primers that match opposite ends of complementary strands. Adapted from Alberts, 1995. for each of the original strands. The original strands serve as templates for the new strands. The new strands are assembled such that each nucleotide in the new strand is determined by the corresponding nucleotide in the template strand. The nucleotides adenine (A) and thymine (T) always lie opposite each other, as do cytosine (C) and guanine (G). Because of this base-pairing specificity, each newly synthesized partner strand has the same sequence as the original partner strand, and replication produces two identical copies of the original double-stranded DNA molecule.

In PCR, a DNA sequence that a researcher wants to amplify, called the "target" sequence, undergoes about thirty rounds of replication in a small reaction tube. During each replication cycle, the number of molecules of the target sequence doubles, because the products and templates of one round of replication all become the templates for the next round. After n rounds of replication, 2n copies of the target sequence are theoretically produced. After thirty cycles, PCR can produce 230 or more than ten billion copies of a single target DNA sequence. This is called a polymerase chain reaction because DNA polymerase catalyzes a chain reaction of replication.

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Medicine EncyclopediaGenetics in Medicine - Part 3