5 PCR components play crucial roles in DNA amplification.
PCR reaction mixture has to include:
- DNA template;
- two PCR primers;
- DNA polymerase;
- deoxynucleoside triphosphates (dNTPs);
- buffer solution.
DNA template in PCR amplification
DNA from a variety of sources may be used as the supplier of the DNA template for 3 basic steps of the polymerase chain reaction.
PCR is a highly sensitive technique and requires only one or two DNA templates for successful amplification. Nevertheless, the optimal quantity of DNA for PCR amplification depends on the composition of the DNA and utilized DNA polymerase.
It is important to optimize DNA input, because high amounts lead to nonspecific amplification whereas low amounts reduce the production of amplicons.
Usually, 0.1–1 ng of plasmid DNA or 5–50 ng of genomic DNA is sufficient for a 50 µL PCR reaction mixture.
Function of dNTPs in PCR
Deoxynucleoside triphosphates (dNTPs) are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of PCR amplification.
dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP.
For optimum base inclusion, dNTPs are usually added to the PCR mixture in equimolar amounts.
As a rule, the final concentration of each deoxynucleoside triphosphate is 0.2 mM.
PCR buffer composition
A buffer of the PCR reaction mixture serves as a chemical environment to maintain an activity and stability of the DNA polymerase. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HСl.
A common component of the Taq polymerase buffer is potassium ion from KCl, which promotes primer annealing.
The buffer concentration of magnesium ions (Mg2+ from MgCl2) is another crucial factor for the proper functioning of the DNA polymerase. Mg2+ ions serve as a cofactor for the enzyme.
In addition, magnesium ions facilitate formation of the complex between the primers and DNA templates by stabilizing negative charges on phosphate backbones.
Generally, a magnesium concentration is 1.5 mM.
A higher magnesium concentration is linked with a higher output, but a lower specificity, while a lower magnesium concentration gives decreased enzyme activity and increased specificity.
Role of DNA primers in PCR
The PCR requires the knowledge of DNA sequences that flank the DNA template.
Primers are short nucleotide sequences (approximately 15–30 bases) that base pair to a specific portion of the DNA being replicated.
In order for hybridization to occur, the primer nucleotides must have a sequence that is complementary to the 3′ end of each strand of the DNA target sequence, and the 3′ ends of the hybridized primers should point toward one another.
The sequences of the primers are very important for the polymerase chain reaction because the reaction cycle has the specific temperatures used in the heating and cooling stages.
Besides, a great excess of the primers in the PCR reaction mixture cause them more likely to encounter a partially complementary primer than a perfectly complementary DNA template. So, primer complementarity has to be avoided.
Two oligonucleotide primers have to be designed and chemically synthesized.
The increasing use of bioinformatics resources in the design of primers makes the design and the selection of reaction conditions much more straightforward.
These resources allow inputting the sequences to be amplified, primer length, and another design details. After analysis, they provide a choice of matched primer sequences.
Using design specifications, DNA primers can be synthesized by a specialist supplier within a couple of days.
Function of Taq polymerase in PCR amplification of DNA
Taq DNA polymerase is the most commonly used enzyme for standard PCR amplification.
The function of Taq polymerase is to replicate the target DNA. The DNA polymerases recognize primers as start tags.
One drawback of early PCR reactions was the temperature needed to denature the DNA, as it also denatures the DNA polymerase. Nowadays, the polymerase chain reaction utilizes a special form of the DNA polymerase - a thermostable DNA polymerase.
The availability of the thermostable DNA polymerase provided the means to automate the reaction. It was originally isolated from Thermophilus aquaticus that thrives in hot springs.
Taq DNA polymerase has a temperature optimum of 75–80oC and survives prolonged exposure to temperatures as high as 96oC. So, it can stay active after each of the denaturation steps.
Due to the ability to automate the PCR reaction, thermal cyclers have been produced giving the possibility to set the temperatures and times for a particular PCR reaction.
Besides of this thermostable feature, Taq polymerase is a normal DNA polymerase. It synthesizes a new DNA strand complementary to a singlestranded DNA template, and, like other DNA polymerases, it requires a primer to start its synthesis from.
Taq polymerase lacks proofreading activity and unable to correct wrong incorporated nucleotide bases. On average, these errors occur approximately once per 9000 nucleotides.
This might be important for some applications, such as cloning, because the product of the polymerase chain reaction may not be a completely precise copy of the original sequence.
Another imperfection of Taq polymerase is that it can only efficiently amplify fragments of a few thousand base pairs.
The DNA fragment to be amplified should not be greater than about 3 kb in length. Usually, they are less than 1 kb.
Amplification of very long fragments (up to 40 kb) requires special methods.
These problems can be solved by using other thermostable polymerases, such as Pfu and Pwo DNA polymerases. These enzymes have proofreading activity.
To improve PCR performance, new generations of DNA polymerases have been engineered. They are effective to generate lower-error PCR products for cloning, long amplification, and GC-rich PCR.
Additional PCR reaction components
To improve PCR results, there are various PCR enhancers. These components of polymerase chain reaction can relieve secondary DNA structure, lower temperature of template denaturation or stabilize DNA polymerases.
Commonly used PCR additives include dimethyl sulfoxide (DMSO), ammonium sulfate, polyethylene glycol, bovine serum albumin (BSA), gelatin, N,N,N-trimethylglycine, and glycerol.
References & further readings:
- Lorenz TC. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. Journal of Visualized Experiments : JoVE. 2012;(63):3998. doi:10.3791/3998.