Definition
Polymerase
chain reaction (PCR)
is a rapid technique that enables the copying of desired
deoxyribonucleic acid (DNA) molecules. This technique consists
of three steps: denaturation, annealing, and extension, repeated in a cyclical fashion.
In step one of PCR, the reaction is exposed to temperatures as high as 194° to 201° Fahrenheit (90° to 94° Celsius). The high temperature helps to separate the two strands of the DNA template by breaking the hydrogen bonds that hold the two strands together. This melting of the DNA duplex is called denaturation.
The second step in PCR is called annealing or hybridization and requires short single-stranded DNA sequences called primers. The reaction uses two primers, one complementary to each strand of the original DNA duplex. The primers bind to their complementary sequences (or anneals) on the template DNA and provide the free 3’OH (hydroxyl) group that DNA polymerase needs to copy the DNA. This sets the stage for the last step in PCR, extension. During extension, the DNA polymerase uses deoxyribonucleotides (dNTPs) to build the complementary strand on the template DNA.
The foregoing set of three processes (making up a cycle) is typically repeated several times, about twenty to twenty-five cycles, to allow for an exponential increase in copies of the target gene. A simple formula can be used to calculate the yield (number of copies of the DNA or gene template). According to this formula, the number of gene copies after n cycles of PCR is 2 n. For example, if a person starts with a single copy of the gene, the yield will be 225 at the end of a PCR reaction with twenty-five cycles.
Background
PCR was discovered by Kary Mullis in 1983 while trying to
develop a method that would allow the sequencing of single nucleotide
polymorphisms (SNPs). SNPs are variations produced in DNA by
alterations to a single nucleotide and, therefore, serve as the absolute genetic
marker. Specifically, Mullis was trying to devise a rapid clinical assay for
genetic disorders such as sickle cell disease that are caused by
a single nucleotide polymorphism. DNA research in the 1980’s faced two major
challenges: not enough DNA and no easy way to separate a gene’s DNA from the
genomic DNA (1 milligram of DNA contains about 200,000 copies of a person’s target
gene). PCR offered a solution to both of these problems; one could amplify DNA
rapidly and could obtain as many copies as needed of the target gene alone.
The PCR Reaction
A PCR reaction, which is typically around 50 milliliters, requires the following: DNA template or target gene (10 femtograms to 10 milligrams), primers (0.1-1.0 millimolars each), dNTPs (200 millimolars each), DNA polymerase (0.2-2 units) and Tris buffer (pH8.0). Also, a PCR reaction requires MgCl2 (magnesium chloride), which is a cofactor for DNA polymerase. The DNA polymerase used in PCR is unique in that it can withstand high temperatures such as those used for melting DNA during the denaturation step. Before thermostable polymerases such as Taq and Vent were discovered, fresh polymerase had to be added after each round of PCR.
DNA in the cell is typically bound to packaging proteins such as histones. For a successful PCR reaction, the DNA template must be free of any inhibitory molecule (such as these packaging proteins) and thus easily accessible to the primers and polymerase. Therefore, once the DNA has been extracted from the cell, it is typically purified using some kind of enzymatic, mechanical, or chemical purification technique.
Next in the PCR process is the design of primer (forward and reverse primers). PCR primers are chemically synthesized nucleotide segments called oligonucleotides (or oligos) and are typically 18 to 30 bases long. Because primers are short sequences, misalignment can occur, leading to erroneous amplification. To minimize nonspecific binding, primers are preferably 18 to 21 bases long. The length of the PCR product is determined by the distance between the annealing sites of the two primers. Also important in primer design are factors such as guanine-cytosine content, polypurine or polypyrimidine stretches (multiples of any one nucleotide), the secondary structure of the primer (mutually complementary sequences within the primer can form “hairpin” structures), and the probability of a complementary sequence on the DNA residing anywhere except on the intended target gene (or region of amplification).
Once the PCR reaction is completed, the next important step is analysis of the PCR products with electrophoresis and various DNA intercalating dyes; this is followed by sequencing the amplimer (the amplified PCR product).
Impact
It is widely believed that since the discovery of the DNA double helix in the
1950’s, few discoveries have transformed the field of biology as rapidly as PCR.
This technique that allows the copying of millions of desired DNA beginning with
minute DNA samples has accelerated progress in forensics and in medical
diagnostics and has revolutionized biotechnology and biomedical
research.
Several modifications of the original PCR technique have been developed to meet the diverse needs of biomedical research. One such modification, qPCR (quantitative PCR), is frequently used in drug discovery to look for and assess new putative drug target sites in humans. The qPCR method is also used to determine the number of working copies of a specific gene, and this measure of gene expression is important in analysis of genetic disorders. For example, in STEP (single target expression profiling), qPCR is used to compare active gene numbers in affected versus healthy (unaffected) persons, allowing for the study of the progression or development of genetic disorders.
Bibliography
McPherson, M., S. G. Møller. PCR (The Basics). New York: Taylor & Francis, 2006. This is an excellent resource for those learning to set up and run PCR experiments.
Rabinow, P. Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press, 1996. A great description of how the PCR technique has evolved since its development.
Van Pelt-Verkuil, E., A. van Belkum, and J. P. Hays. Principles and Technical Aspects of PCR Amplification. New York: Springer, 2008. A great resource for persons interested in furthering their understanding of PCR.
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