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Polymerase Chain Reaction

Designing Primers

To replicate DNA, DNA polymerases require not only a template, but also a primer. A primer is a sequence of single-stranded DNA that "anneals," or binds, to the template by specific base-pairing. An automated apparatus called an oligonucleotide synthesizer, sometimes nickname a "gene machine," can produce primers of any chosen sequence.

Primers for PCR are typically short sequences, around twenty nucleotides long. It is the primers' sequences that are responsible for PCR's enormous specificity. Researchers design primers so they are likely to bind to sequences on either side of the target DNA. They do so by making the primers complementary to the appropriate sequences and by making them long enough that they are unlikely to bind elsewhere.

The longer the primer, the more likely it is that it will be complementary only to the target sequence. Because any single position in a DNA sequence can be occupied by either an A, T, C, or G, there is a one in four chance that any position will contain an A, for example. (This is an approximation, because the nucleotides are not distributed equally or randomly in DNA.) The odds that any specific DNA sequence that is n nucleotides long would be present at a given spot in a DNA sequence is therefore 1 in 4. The chance that a particular twenty-nucleotide sequence (a typical length for a PCR primer) would occur in a given spot at random is less than one in one trillion (10−12). The human genome has only about three billion (3 × 109) nucleotide pairs, so any twenty-nucleotide-long sequence is very unlikely to occur more than once by chance in the human genome.

Researchers design two primers that will bind to opposite strands of the DNA on either side of the target sequence. They design them to "point" the right way, so that the section of DNA between, not outside of them, is copied. Designing the primers to "point" in the right direction simply requires building them so that their 3′ ends lie toward the target DNA and their 5′ ends lie away from it. The ends of any segment of DNA, including the complete strand, are chemically different.

Multiple rounds of separation and copying can quickly produce billions of copies of the original DNA sequence. Adapted from Alberts, 1995.

One end is called the 5′ (pronounced "5-prime") end. The other is called the 3′ end. In DNA replication, nucleotides are always added to the 3′ end of a growing strand of DNA. DNA synthesis is said to proceed in a 5′ to 3′ direction. The two complementary strands of DNA are anti-parallel, which means that they run in opposite directions. The 5′ end of one strand lies next to the 3′ end of the other, as shown in the diagram.

Additional topics

Medicine EncyclopediaGenetics in Medicine - Part 3Polymerase Chain Reaction - Designing Primers, A Typical Pcr Reaction, Contamination In Pcr Reactions, Pcr Applications And Variations