Within a decade of its discovery in 1983, polymerase chain reaction (PCR) evolved into one of biology’s most useful tools. PCR amplifies specific segments of genes so accurately that an early National Institutes of Health publication dubbed the technique “Xeroxing DNA.” Today, PCR is used routinely in dozens of approved medical tests in diagnostic labs and physician offices, and by millions of scientists worldwide.
Understanding PCR reagents requires a bit of knowledge about the reaction itself. The steps involved in PCR are:
- Denaturation: Heating the sample to separate the target DNA’s double helix into individual strands. Denaturation takes place in specialized buffers.
- Annealing: Cooling the sample in the presence of an “antisense” (or complementary) DNA sequence, known as a primer (there are two primers, one for each strand separated in the first step).
- Elongation: Addition of an enzyme, polymerase, that builds the new DNA strands, and deoxynucleotide triphosphates (dNTPs), which are building blocks for the new DNA copies.
These steps repeat 30 or 40 times in a thermocyling instrument. Each step doubles the quantity of DNA produced.
Each of the steps involves specific reagents that are general for all PCR experiments: TRIS or some other buffer, enzyme, magnesium chloride (necessary for enzyme activity) and the dNTPs. Primers and templates are unique for every amplification. Since the patents on Taq polymerase expired, vendors have been free to develop and sell their own versions, resulting in downward price pressures.
“PCR reagents have become commoditized,” observes Laura Mason, PCR product manager for Agilent’s Strategene division (La Jolla, CA).
Almost every variant of PCR uses some type of Taq polymerase, an enzyme first isolated from the thermophilic bacterium T. aquaticus, which thrives at 70 to 80º C. Early PCR employed an E. coli polymerase, which had to be replenished before each cycle due to its temperature instability. Taq eliminates that problem because it easily survives the denaturation step, which occurs above 90º C.
PCR reagents are sold individually or as “master mixes.” The separate purchase route allows end users to fine-tune PCR reactions with favored (or less expensive) reagents, but this requires a fair degree of expertise. “For example, some users prefer to optimize their magnesium chloride levels,” says Jeff Williams, Ph.D., president of Lucigen (Middleton, WI). Master mixes contain all PCR reagents but the primers and, of course, the template. “Master mixes are gaining in popularity among nonexpert users,” Dr. Williams tells Lab Manager.
PCR is used wherever gene amplification is of interest, for example in foods/beverages, agriculture, forensics (e.g., blood, hair, skin), historical research, anthropology, and of course in numerous biomedical areas. Recently, environmental scientists detected the presence of Asian carp, an invasive species, in the Great Lakes—not by capturing a fish but by amplifying their genes with PCR. Similarly, beer brewers use PCR to uncover bacterial contamination in their process. PCR may also be used at relatively large scale to manufacture genes for gene therapy or vaccine work.
Purchase decisions for PCR reagents are based on performance (speed of analysis, fidelity of DNA-copying) and price. Instruments used to play a role in choice of reagents, Williams says, but “these days temperature cycling instruments have fairly similar performance characteristics and should not be a factor in reagent selection.”
Early PCR reactions were “endpoint” tests, to see if a specific strand of DNA was present or not. The debut of “real-time” or “quantitative” PCR (qPCR) allows investigators to quantify the number of copies made and, by backtracking, the concentration of the template in the sample.
Improvements in PCR have been mainly on the reagent side, in the way of providing greater speed (more amplification in less time), specificity (to the target DNA sequence) and fidelity (precise copying). One drawback of Taq is that it frequently makes copying mistakes. Since the 1980s polymerase enzymes from other thermophilic organisms have been isolated and commercialized, for example Pfu DNA polymerase, from the archaeon Pyrococcus furiosus. Pfu possesses “proofreading” capability, which enables it to check its work as it elongates the gene. Another improvement has been the introduction of hybrid polymerases that are instantly activated at high temperatures, thus reducing the amount of nonspecific binding.
One instrument-related issue that still concerns analysts is temperature. Specialized experiments may require reaching precise temperatures for exact times. “The wrong temperature can cause amplification of the wrong DNA segment,” notes Ms. Mason of Agilent, who believes that instrumentation is “lagging behind” reagents in terms of speed and quantity of DNA produced, particularly with multiplexing (several samples in one run) becoming more common. “Screening experiments don’t have to be as precise as quantitation. It depends on what you’re trying to achieve with a particular amplification.”