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 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. 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.
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, says Jeff Williams, Ph.D., president of Lucigen (Middleton, WI), but “these days temperature cycling instruments have fairly similar performance characteristics and should not be a factor in reagent selection.”
Recently, environmental scientists detected the presence of Asian carp 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.
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.”
PluriPCR™ qPCR Kit
- Designed as a quantitative, easy to use, and reliable assay of five genes strongly specific to pluripotency
- Ability to accurately and unambiguously define the pluripotency of a cell line
- Ideal for all aspects of pluripotent stem cell research
- Fully optimized for routine quality control
Precision Melt Supermix
- Delivers robust high resolution melt (HRM) performance for sensitive and accurate detection of single nucleotide polymorphisms (SNP) and CpG methylation for epigenetic studies
- Compatible with all HRM-capable thermal cyclers
- Discriminates all four SNP classes across a broad range of amplicons
PrimeTime® qPCr Assays
- Available in three different sizes (mini, standard, and XL)
- Provide the forward and reverse primers as well as a hydrolysis probe in a single tube
- Allow users to select from multiple dye-quencher combinations
- Pre-designed assays are now offered which stress accurate Tm prediction and protection against off-target amplification
Integrated DNA Technologies (IDT)
Multiplex PCR Plus Kit
- Allows parallel amplification of different targets in a single tube
- Provides rapid establishment of multiplex PCR assays, without optimization
- Features consistent and reliable results for advanced applications
- Convenient with short reaction times