Polymerase chain reaction (PCR) is a technique used widely in molecular biology applications as a method capable of amplifying a single DNA molecule to generate millions of copies of a specific target region of the DNA.
Since its inception in 1983, PCR has become increasingly widespread and is now virtually indispensable, allowing analysis of DNA samples in cases where only tiny amounts of DNA were present in the original sample.
However, with a variety of techniques, applications and reagents available, it’s critical to know which approach is correct to achieve the desired outcome and results.
Which PCR technique?
Endpoint PCR is the ‘standard’ method of PCR, and is commonly used for applications such as genotyping and cloning. It is limited when it comes to quantitation, though, in which case Quantitative PCR (qPCR) is typically used.
qPCR allows more accurate quantitation and detection of a specific DNA sequence in real time, measuring concentration while the synthesis process is taking place.
Successful PCR reactions are dependent on five key ingredients – these are template DNA, PCR primers, Nucleotides (dNTPs), DNA polymerase and reaction buffer. Our focus for this blog post will be on DNA Polymerase.
A DNA polymerase has five key properties – thermostability, specificity, extension rate, fidelity and processivity – which define the best/most appropriate enzyme for each particular PCR method.
- Thermostability – PCR amplification is dependent on polymerases that are thermostable and which can perform consistently at the range of operating temperatures associated with each PCR cycle, without impairing PCR activity.
- Specificity – The capacity of the DNA polymerase enzyme to produce one amplification product that is the intended target sequence. Influenced by the highly active nature of DNA polymerase enzymes.
- Extension rate – the speed at which extension of the target region of DNA takes place, measured in nucleotides/second or kilobase (kb)/minute.
- Fidelity – essentially the accuracy of the DNA sequence after amplification. Fidelity refers to the ability of a polymerase to insert the correct nucleotide during PCR.
- Processivity – the efficiency of a polymerase, defined by the number of nucleotides that can be incorporated during template binding.
However, these five properties can vary between classes of DNA polymerase, and different classes can be useful for different applications, including:
Standard TAQ DNA Polymerases. These enzymes are typically suitable for use in routine Endpoint PCR applications where qualitative detection of a specific target region is required. This class of DNA polymerase enzyme encompasses flexible and commonly-used thermostable polymerases which can operate at a broad range of temperatures and amplify a single DNA target that is typically ≤ 5kb in size having a GC content between 40% – 60%.
Hot-Start DNA Polymerases. Nonspecific binding of the target region of DNA is a considerable issue in PCR since it can drastically impact yield and sensitivity of the amplified product. Even at room temperature, DNA polymerases are highly active and are prone to extension of mis-primed targets which can result in non-specific amplification. Setting the PCR up on ice helps to keep the activity of the DNA polymerase low, however “hot-start” polymerase enzymes were developed and are specifically used to provide increased product yields by preventing nonspecific primer binding during the reaction setup.
Hot-Start DNA polymerases allow for convenient room temperature setup and are the best choice of enzyme for applications such as multiplex PCR and Low-Copy PCR.
High Fidelity DNA Polymerases. DNA polymerases in this class possess proofreading capabilities and are able to “recognise” mis-incorporation of nucleotide bases, therefore minimising the introduction of amplification errors. The higher the fidelity of the polymerase, the more accurate the final/amplified DNA sequence.
High fidelity DNA polymerases are particularly useful for applications such as cloning/subcloning and DNA sequencing/Next Generation Sequencing where accuracy is paramount.