The Three Steps of PCR

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The polymerase chain reaction, or PCR, is a way to make large amounts of a particular sequence of DNA using only a small amount of the original substance. This technique, the refinement of which earned Kary Mullis the 1993 Nobel Prize in Chemistry, includes three distinct steps.

Components of the Reaction

  • PCR is an amplification process. In order to generate many micrograms of a given nucleic acid, you must, of course, have the DNA of interest, called a template. It can come from any source -- animals, plants, viruses or bacteria. (RNA can be analyzed using PCR, but you have to first use the viral enzyme reverse transcriptase to generate a DNA copy of the RNA; then, you can use that DNA copy as the template.) For PCR, you must have a heat-resistance form of the enzyme DNA polymerase, generally Taq polymerase, which makes new strands of DNA. You also need primers; short sections of DNA that bind to specific spots on the template and touch off replication of that portion of the template. Finally, you need dNTPs, the nucleotides used to build the new strands, and a specific saline solution, called a buffer, in which to perform the PCR. This helps to stabilize the reagents and products of the reaction.

Denaturing the DNA

  • The first step in PCR is denaturing the DNA template, which means unzipping the two strands that, bound together, form the double helix structure of DNA. In the case of PCR, the template is briefly heated to between 92° to 95° C, hot enough to break the hydrogen bonds that keep DNA in its characteristic double-helix form and separate it into two single strands.

Annealing the primers to the DNA strands

  • After denaturing is complete, the mixture is cooled to a temperature as low as 45° C. This allows hydrogen bonds to form between complementary nucleotide base pairs of the DNA template and the DNA primers, with adenine bonding to thymine and cytosine to guanine. The annealing temperature is determined based on what is known about the template DNA and the primer sequence. For example, if amplifying a gene from a mosquito, if you have primers from a mosquito species in the same genus, you can have a higher annealing temperature; if the only sequence available is from a fruit fly, the annealing temperature will have to be lower because the sequences will match less perfectly. Annealing occurs quickly and efficiently because there is much more primer DNA than template DNA in the mixture, especially at first.

Extending the DNA Primers

  • Once annealing has occurred, a heat-stable form of DNA polymerase steps in to begin rapid, sequential addition of the nucleotides to the primer strands -- 50 to 100 nucleotides per second. To kick this into gear, the mixture is again heated, this time to a more modest 72° C.

    PCR is an exponential process. This means that, after one cycle, twice the original amount of template DNA, exists; after two cycles, four times the original amount exists; after three cycles, eight times, and so on.

References

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