PCR stands for Polymerase Chain Reaction which is one of the fundamental methods of molecular biology.
Substantially, the primary purpose of polymerase chain reaction is to rapidly increase the number of copies of specific DNA regions.
It consists of 3 basic PCR steps and a relatively complex reaction mixture.
Today, different types of PCR technique, combined with other technologies, find numerous applications in such fields as research, forensic science, agricultural sciences, medicine, etc. It is used to diagnose diseases, clone and sequence genes.
Polymerase chain reaction can be performed using DNA from a variety of sources. It is very sensitive and needs only trace amounts of nucleic acids to produce enough copies for conventional laboratory analysis.
The polymerase chain reaction process serves to raise the number of DNA fragments
3 basic steps of PCR process
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 polymerase chain reaction 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 (amplicons) amplified from the DNA template.
First polymerase chain reaction step – DNA denaturation
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 nucleic acids are broken and the strands unwind from each other.
This process releases single-stranded DNA to act as templates in the final PCR extension step.
The denaturation temperature is above 90°C (usually 94°C) and the time is up to one minute (usually 30 seconds).
Polymerase chain reaction steps
Second polymerase chain reaction step – DNA Primer annealing
At the annealing step, DNA 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.
This is the only temperature in a PCR cycle steps that can be widely varied.
The temperature depends on the exact sequence and length of the primers.
Usually, the PCR reaction mixture is cooled down to 40–60°C. However, annealing temperatures for DNA templates with a high GC content can be as high as 72°C (the normal temperature of the extension step).
The temperature for this PCR step is chosen for the optimum binding of the DNA primers to the correct DNA template and depends on primer’s melting temperature. The wrong annealing temperature can result in false products, or in no detectable products at all.
Since the primers are relatively short, and at high molar concentrations, duration of the annealing step is around 30 seconds.
At this step, the annealed oligonucleotides provide a free 3’ hydroxyl group for Taq polymerase and act as primers for synthesis of nucleic acids.
Final polymerase chain reaction step – DNA synthesis
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 (usually Taq polymerase).
The temperature of the elongation step is usually set at 72°C. It is slightly below the optimum for Taq polymerase.
The Taq polymerase produces complementary DNA strands starting from the primers.
The synthesis proceeds at approximately 1000 bases per minute.
Therefore, to amplify a DNA template that is 500 bases in length, under normal conditions a time of the PCR extension step should be at least 30 seconds.
Usually, PCR extension time is 30 seconds for every 500 bp (base pair) of product.
DNA amplification process during PCR cycles
During the very first PCR cycle the only templates available for primer annealing are the target nucleic acids.
Because the initial template is many times larger than the length of the desired amplicon, the polymerization of the first cycle will proceed until it is interrupted at the denaturation step of the second cycle.
At the end of the first PCR cycle, there are two double-stranded nucleic acid molecules for each one that the reaction started with.
Each nucleic acid molecule contains one strand of the original template, and one novel strand, which is bounded at one end by the oligonucleotide primer and at the other end by how far polymerization was able to proceed during the extension step.
These PCR products form DNA templates that are bounded on only one end (semi-bounded DNAs).
In the second cycle, both the original nucleic acid targets and the semi-bounded DNAs will serve as templates.
Original DNA templates will continue to make semi-bounded products in every cycle of the polymerase chain reaction.
Starting with the second cycle of PCR amplification, semi-bounded DNAs will form the PCR amplicons.
In every subsequent cycle, the DNA templates, the semi-bounded DNAs, and the amplicons will serve as templates for the PCR primers.
In a PCR reaction mixture:
- the amount of template DNA does not change;
- the number of semi-bounded DNA templates increases arithmetically every cycle;
- every cycle starting with cycle 2, the number of amplicons increases geometrically.
At the end of 35 PCR cycles there are more than 34 billion copies of the DNA amplicons for every copy of the original template DNA sequence.
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 polymerase chain reaction 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