Since we have been discussing various diagnostic methods I think it is a good idea to have a short information about what those methods actually do, so here is the first of the technical posts. I will be posting those to help in explaining what the different methods and terms mean. This first installment is about PCR and some of the basics of how the DNA gets amplified as not all fragments of the genome are easy to work with and methods may require modification.
What is PCR?
Polymerase chain reaction, or PCR, that was developed in 1983 by Kary Mullis, has become an essential tool for molecular biology. It allows logarithmical amplification of deoxyribonucleic acid (DNA) from a very small amount of starting material. PCR amplification is used for a variety of applications, including amplifying small fragments for cloning, genotyping, whole genome amplification, Sanger sequencing, and next-generation sequencing. PCR is used by specialists in academia or clinical laboratories as well as more recently in point-of-care (POC) tests.
The process would not be possible without a thermostable polymerase. Historically, the frst one used was derived from Thermus aquaticus (known as Taq polymerase). With time, more polymerases were discovered, created, or modifed and now there are a wide variety of enzymes available for PCR.
Standard PCR protocol
The initial and still widely used PCR method consists of the following steps:
- Thermal cycling, where the number of cycles is most commonly 25 to 35 cycles and each cycle is made up of these 3 steps.
- Denaturation of the template, or DNA strand separation, occurs at 94°C to 98°C for 20 to 30 seconds
- Binding or annealing of primers to the DNA template for 20 to 40 seconds at a temperature specifc to the primers used, most commonly ranging between 50°C and 65°C
- Extension of primers along the template to create copies of the original template. This step most commonly takes place at 72°C for a time period dependent on the length of the fragment being amplified and the speed of the polymerase, typically one to three minutes
- Final extension – an optional step to ensure full elongation of any remaining single-stranded PCR products into double-stranded.
- Final hold – an optional step maintaining a temperature of 4°C to 10°C for short-term storage of the PCR products immediately after the reaction.
PCR amplification requires a DNA template, a pair of primers flanking the sequence of interest, reaction buffer and a heat-stable polymerase. Specificity of the process is driven by primer sequences and their melting temperature (Tm). The Tm sets the upper limit of the annealing temperature, as only very specific binding will occur at this temperature.
Hot-start PCR
As the use of PCR amplification was increasing, various problems were appearing. One of the issues was early activation of Taq polymerase resulting in non-specific amplification of the template. This problem was resolved by using polymerases where the activity was blocked at a lower temperature by using antibodies, chemical modifications or aptamer binding, commonly termed hot-start. Depending on the modification, an activation step, or initialization time before the thermal cycling steps, is necessary, ranging from two to fifteen minutes, most commonly at 95° C. Using a hot-start polymerase can reduce the formation of primer dimers and often increase yield.
Touchdown PCR
Another technique used for lowering non-specific background by eliminating non-specific primer binding is to use a touchdown PCR protocol. This method relies on performing a few initial thermal cycles at the upper limit of annealing temperature followed by cycles using a decreased annealing temperature. Specificity of amplification is driven by the first-round high annealing temperature. Lowering the annealing temperature over the following cycles maximizes the yield of specific PCR product.
Touchup PCR
A reverse technique to a touchdown is not as frequently used but can reduce the need for optimization of PCR amplification mostly in multiplex reactions, as well as bisulfite PCR. The initial annealing temperature is at the lower limit of the annealing temperature or even slightly below to enable priming of all targets. The annealing temperature is then increased at a set amount for a number of cycles, reaching the optimal value.
Two-step PCR
Certain PCR reactions require a relatively high annealing temperature of 70° C making a two-step protocol a viable option. In this case, annealing and extension steps are carried out at the same time at 70° C. This particular approach can be used with GC-rich templates. The main concern with this approach is non-optimal extension process due to the temperature being lower than optimal for the polymerase used.
Leave a comment