Biotechnology and Bioinformatics (1+1)

PCR principles and procedure

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. A basic PCR set up requires several components and reagents. These components include:

1. DNA template that contains the DNA region (target) to be amplified.

2. Two primers , which are complementary to the DNA regions at the 5' (five prime) or 3'

(three prime) ends of the DNA region.

3. Taq polymerase

4. Deoxynucleoside triphosphates (dNTPs), the building blocks from which the DNA

polymerases synthesizes a new DNA strand.

5. Buffer solution , providing a suitable chemical environment for optimum activity and

stability of the DNA polymerase.

6. 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.

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 therm al cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction. 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.

The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

1. Initialization step : This step consists of heating the reaction to a temperature of 94-96°C which is held for 1-9 min. It is only required for DNA polymerases that require heat activation by hot-start PCR .

2. Denaturation step : This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 secs. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

3. Annealing step : The reaction temperature is lowered to 50-65°C for 20-40 secs allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5ºC 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.

4. Extension/elongation step : The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, 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 group at 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.

5. Final elongation : This single step is occasionally performed at a temperature of 70-74°C for 5-15 min after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

6. Final hold : This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.