•What is PCR? Describe the technique in detail. •What enzyme is used in this p

   PCR (polymerase chain reaction) is a method, developed by Kary Mullis in the 1980s, to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA  (Cowan, 2012, pg. 15).  PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide  (Cowan, 2012, pg. 278)  This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

   PCR is a three-step process that is carried out in repeated cycles. The initial step is the denaturation, or separation, of the two strands of the DNA molecule (Suchman, 2013). This is accomplished by heating the starting material to temperatures of about 95° C (203° F). Each strand is a template on which a new strand is built. In the second step the temperature is reduced to about 55° C (131° F) so that the primers can anneal to the template(Suchman, 2013). In the third step the temperature is raised to about 72° C (162° F), and the DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about five minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA (Suchman, 2013).


   A DNA polymerase is a type of enzyme that synthesizes new strands of DNA complementary to the target sequence (Suchman, 2013). The first and most commonly used of these enzymes is Taq DNA polymerase (from Thermis aquaticus), whereas Pfu DNA polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA  (Suchman, 2013). Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant  (Suchman, 2013).


   The enzyme Taq polymerase is found in the bacterial species Thermus aquaticus. It is a class 4 enzyme, which is the Lyase class of enzymes. A lyase is an enzyme that catalyzes the lyses of a substrate generating a double bond. Taq polymerase is a DNA polymerase that allows the bacteria to replicate at the high temperatures of its environment due to its thermo stability (Suchman, 2013). It is because of this property that makes Taq polymerase such an important commodity. Every organism contains DNA polymerase enzymes vital to their replication, but most will be denatured at high temperatures due to their protein composition (Suchman, 2013).
   Pfu DNA polymerase enzyme is found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivoto replicate the organism's DNA (Suchman, 2013). In vitro, Pfu is used to swiftly amplify DNAin the Polymerase Chain Reaction, where the enzyme serves the central function of copying a new strand of DNA during each extension step (Suchman, 2013).


   PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections (Cowan, 2012, p. 276)  This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods.  PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made  PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases.

References:
1.   Cowan, M.K. (2012) Microbiology: A Systems Approach (3rd ed.) New York, NY: McGraw-Hill
2.   Suchman, Erica (2013) Polymerase Chain Reaction Protocol, Retrieved on April 5th, 2014 from:http://www.microbelibrary.org/library/laboratory-test/3656-polymerase-chain-reaction