Molecular Cloning/PCR

PCR is used to amplify a specific region of a DNA strand (the DNA target). 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: 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. Deoxynucleotide triphosphates (dNTPs), 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 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 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.

Protocol

PCR for DNA amplification was done as per the method of describe here. The templates were 10 ng of plasmid DNA for routine PCR, 0.5 to 1 μg of genomic DNA for genomic PCR, 1 μl of RT reaction for RT-PCR or a part of the bacterial colony for bacterial colony PCR in a 25 to 50 l reaction. PCR was done in a reaction mix containing 1X PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% w/v gelatin), 250 M each of the dNTPs, 250 ng each of primers and 1.5 units of Taq DNA polymerase. When using Pfu DNA polymerase 500 ng of each primer and a hot start for Pfu reaction was carried out. The samples were then overlaid with mineral oil to prevent evaporation during cycling, 35 step cycles were carried out after initial denaturation at 94C for 15 min. Each step cycle consisted of denaturation, 94C for 30 sec, annealing (4C below Tm of primer) for 30 sec or 1 min and extension, 72C for 1 min. A final extension was given at 72C for 7 min for completion of truncated products. For preparing radiolabelled probe by PCR, high specific activity was ensured by adding only 2.5 M each of the dNTPs and primer concentration was reduced to100 ng each. 25Ci (2.5 l) of -32P dATP was generally used for incorporation. A 50 l reaction volume was used and all other conditions of step-cycles were identical to cold PCR.

Inverse PCR was carried similar to conventional PCR, except that the divergent primers would be used instead of the conventional convergent primers used for routine amplification. Also the DNA polymerase like pfu or pfu Turbo, which give blunt end amplification products should be used. Inverse PCR is used for generating insertion, deletion or point mutations on any desired DNA fragment. Multiplex PCR was carried out by mixing multiple primer sets for different genes in the same reaction mix and PCR was carried out at as a single reaction.