PCR stands for Polymerase Chain Reaction.
The polymerase chain reaction is a technique for fast increasing the number of copies of specific regions of DNA and consists of 3 basic PCR steps and a relatively complex reaction mixture.
- DNA template
- Deoxynucleoside triphosphates (dNTPs)
- PCR buffer
- DNA primers
- Taq DNA polymerase
- Denaturation step
- Annealing step
- Extension step
PCR is one of the fundamental methods of molecular biology.
The product of the polymerase chain reaction acts as the means of further analysis. For instance, PCR is used along with gel electrophoresis to detect different DNA sequences.
PCR is a technique that raises the number of DNA fragments
PCR components have to include:
- a DNA template;
- two PCR primers;
- a DNA polymerase;
- deoxynucleoside triphosphates (dNTPs);
- a buffer solution.
In the lab, PCR reaction components are all mixed together.
DNA from a variety of sources may be used as the supplier of the DNA template.
PCR is a highly sensitive technique and requires only one or two molecules for successful amplification.
Deoxynucleoside triphosphates (dNTPs)
Deoxynucleoside triphosphates are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of an amplification.
A buffer serves as a chemical environment to maintain an activity and stability of the DNA polymerase.
The buffer concentration of magnesium ions is another crucial factor for the proper functioning of the DNA polymerase.
Magnesium ions serve as a cofactor for the enzyme.
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.
The PCR requires the knowledge of DNA sequences that flank the DNA template.
Primers are short nucleotide sequences 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.
Basic requirements to design PCR primers
Primers have to:
- be complementary to sequences flanking the target DNA;
- not be self-complementary;
- not bind each other to form dimers;
- have similar annealing temperatures and be matched in length and base composition;
- end (3') in a G or C, as this prevents loose ends and increases the efficiency of priming;
- be 17-28 bases in length.
If the primers are too short they might hybridize to non-target sites and give undesired amplification products.
If the primers are too long they hybridize at a slower rate, and the efficiency of the PCR might be reduced because complete hybridization to the template molecules cannot occur in the allotted time during the reaction cycle.
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 DNA polymerase in PCR
Taq DNA polymerase is the enzyme that replicates DNA.
PCR uses a special form of DNA polymerase.
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.
However, the availability of a 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 72oC 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 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 PCR may not be a completely precise copy of the original sequence.
Another imperfection of Taq polymerase is that Taq polymerase 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.
The polymerase chain reaction is a three step cycling process consisting of defined sets of times and temperatures.
3 basic PCR steps include:
- denaturation step;
- annealing step;
- extension (elongation) step.
Each of these steps is repeated 30–40 times (cycles).
In the course of each cycle, the PCR reaction mixture is transferred between three temperatures.
Profile of 3 basic PCR steps
During successive cycles of basic PCR steps (denaturation, annealing, and extension) all the new strands will act as DNA templates causing an exponential increase in the amount of DNA produced.
Each cycle doubles the number of DNA molecules amplified from the DNA template.
Polymerase chain reaction steps
PCR denaturation step
The first of 3 PCR steps is a denaturation step.
During the denaturation step, the hydrogen bonds that hold together the two strands of the double-stranded DNA are broken and the strands unwind from each other. This process releases single-stranded DNA to act as templates in the PCR elongation step.
The denaturation temperature is above 90°C (usually 94°C).
PCR annealing step
At the annealing step, the PCR reaction mixture is cooled down to 50–60°C.
Primers line up on exposed nucleotide sequences at the DNA target according to base-pairing rules. This is a typical temperature-dependent DNA: DNA hybridization reaction and has to be optimized.
At this PCR step, the annealed oligonucleotides provide a free 3’ hydroxyl group for Taq DNA polymerase and act as primers for DNA synthesis.
PCR annealing temperature
So, the exact annealing temperature is critical, because it can affect the specificity of the reaction.
If the temperature is too high the primers and DNA templates remain dissociated (no hybridization).
If the temperature is too low there are a lot of mismatched hybrids where not all the proper base pairs have formed.
The sequence and length of the primers bias the annealing temperature. Comparing to As and Ts, the more Gs and Cs in the primer the stronger it binds to the DNA template. So, the annealing temperature has to be higher.
There are computer algorithms to predict the optimal annealing temperature for a primer. Nevertheless, in practice, some trial and error are often needed.
One of the optimization techniques is touchdown PCR - a programmable thermal cycler is used to incrementally decrease the annealing temperature until the optimum is obtained.
The ideal annealing temperature must provide hybridization between primer and template, but prevent of forming mismatched hybrids.
PCR melting temperature
The annealing temperature can be estimated by determining the melting temperature (Tm) which is the temperature at which the correctly base-paired hybrid dissociates.
A temperature 1–2°C below the melting temperature might be good enough to form the correct primer–template hybrid, and high enough for mismatched hybrid not to be stable.
In addition, that the melting temperature can be determined experimentally, it can be calculated from the simple formula:
Tm = (4 × [G + C]) + (2 × [A + T])°C
[G + C] - the number of G and C nucleotides
[A + T] - the number of A and T nucleotides in the primer sequence.
The two primers should be designed so that they have identical melting temperatures.
PCR extension step
The last of 3 basic PCR steps is called extension or elongation step.
It is the DNA synthesis step and carried out by a thermostable DNA polymerase.
The temperature of the elongation step is usually set at 74°C. It is slightly below the optimum for Taq DNA polymerase. The Taq polymerase produces complementary DNA strands starting from the primers. The synthesis proceeds at approximately 1000 bases per minute.
The development of the programmable thermocycler helped spread the new PCR technology.
The programmable thermocycler is based on metal heating blocks with holes for the PCR tubes and designed to switch between the programmed series of temperatures of PCR steps.
References & further readings:
- Variants of PCR – Wikipedia
- PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer – New England Biolabs
- PCR Technique with its Application – RESEARCH & REVIEWS - INTERNATIONAL JOURNALS
- PCR Protocols & Applications – QIAGEN
- PCR Cycling Parameters—Six Key Considerations for Success – Thermo Fisher Scientific