PCR
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.
Developed by: Kary Mullis (1983, Noble prize in 1993).
Applications:
DNA-based phylogeny
functional analysis of gene
the diagnosis of hereditary diseases
the identification of genetic fingerprints (used in forensic sciences and paternity testing)
and the detection and diagnosis of infectious diseases.
Amplification range:
100 to 5kb
10kb or 40 Kb sometimes
Requirements:
Basic PCR set up requires following components and reagents
- DNA template that contains the DNA region (target) to be amplified.
- Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
- Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
- Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously calleddeoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
- Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]
- Monovalent cation potassium ions.
Thermal cycler or PCR machine:
The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler.
The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration.
Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.
Procedure/process
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three:
*(Optimum temp: 95,55,75)
- Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA meltingof the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
- Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
- Synthesis/Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activitytemperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl groupat the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment
- Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.
PCR stages:
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
