VPB 321: Animal Biotechnology (2+1)

COMPONENTS OF PCR 

Template

• PCR can amplify as little as one molecule of starting template. Therefore, any source of DNA that provides one or more target molecules can in principle be used as a template for PCR.
• This includes DNA prepared from blood, sperm or any other tissue, from older forensic specimens, from ancient biological samples or in the laboratory from bacterial colonies or plaques as well as purified DNA.

Primers

• Oligonucleotides used for priming, should be atleast 16 nts and preferably 20-24 nts in length.
• They should have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.
• They are designed to anneal on opposite strands of the target sequence so that they will be extended towards each other by addition of nucleotides to their 3’ ends.
• If the DNA sequence being amplified is known, then primer design is relatively easy.

dNTPs

• The 4 dNTPs, dATP, dGTP, dCTP and dTTP, used at saturating concentration (200 m M each).

Enzymes

• Thermostable DNA polymerases from a number of thermophilic bacteria are used for PCR.
• The most common is Taq polymerase from Thermus aquaticus. It survives the denaturation step of 95^ºC for 1-2 min, having a half-life of more than 2hr at this temperature.
• It carries a 5’-3’ polymerization dependant exonuclease activity, but lack in 3’-5’ exonuclease activity (proof reading).
• Hence, it is more prone for introducing errors. There are high-fiedality thermostable enzymes with 3’-5’ exonuclease activity. e.g., Vent polymerase, pfu polymerase.

Buffer

• The standard buffer for PCR contains 50 mM KCl, 10 mM Tris.Cl and 1.5 mM MgCl2. pH is approximately 7.2. The presence of divalent cations is critical (Mg2+).