Polymerase Chain Reaction (PCR) amplification is a critical application in modern life sciences labs. PCR allows the rapid specific amplification of target DNAs either as they occur in situ or with specific modifications such as restriction sites or sequence tags. While the technique was first developed in the mid-1980s, improvements have been made over time to make the technique more efficient and specific.
In essence, all variations on the technique rely on the ability of thermostable polymerases to produce a copy of a single stranded DNA template using supplied nucleotides and a short DNA oligonucleotide primer. Where the thermostability of the enzyme becomes critical is in the ability to repeat this process over and over to turn a few copies of the sequence of interest into thousands or millions of copies. This occurs by dissociating the polymerase from the DNA to be amplified using high temperatures, usually in the 92-95°C range. This denaturation step is then followed by a low temperature annealing step where the oligonucleotide primer is able to bind to its complementary sequence in the target DNA template. Once this binding has occurred, a final elongation step follows which allows the polymerase to specifically copy the target sequence.
The beauty of the PCR reaction is that this sequence of events can occur over and over until either the nucleotide building blocks or the oligonucleotide primers are exhausted. This is possible because the thermostable enzyme is not damaged by repeated rounds of heating. Typical DNA polymerases would be permanently disabled by the high heat of denaturation and would require replacement at each cycle. Taq is stable at denaturation and can extend the new DNA strand at relatively high temperatures which prevents non-specific binding of the primer sequence to spurious targets thus increasing the specificity of the amplification.
One drawback to the technique, however, is that during reaction set-up, the primers may bind to less specific targets allowing for the generation of non-specific amplification products. In order to prevent this, reactions are normally assembled on ice, minus the polymerase, and then moved to the thermocycler. Once in the thermocycler, they are given a “hot start” usually 3 to 5 minutes at 93-95°C to allow full denaturation of the templates and primers. Once denaturation has occurred, the polymerase is then added and the reaction cycled normally through multiple rounds of denaturation, annealing and elongation. While performing this step is helpful in improving the quality and quantity of the resulting products, it can be very difficult if multiple samples are being amplified and almost precludes any type of high throughput analysis.
To address this issue, several companies have developed special hot-start DNA polymerases. The iTaq from Bio-Rad relies on antibodies to selectively inactivate the polymerase until after the initial hot start period. This improves the specificity and sensitivity of the reactions. Functionally, the antibodies are bound to the enzyme at low temperatures, but become irreversibly denatured by the initial hot-start conditions allowing the enzyme to function normally.
In our laboratory, we found that the iTaq enzyme worked quite well when compared to standard Taq polymerases from other vendors. The iTaq provided excellent yield when amplifying products from as small as 200-300 bp up to >2Kb. One definite advantage that we found when using this product was that at least in our hands, it required less optimization of reaction conditions than typical Taq polymerase to get acceptable amplification. While there are certainly less expensive options, iTaq provided us with an invaluable commodity in this sense, more time to devote to other experiments.