Polymerase Chain Reaction (PCR)

There are three stages to PCR: denaturation, heating, and extension.

In the first stage, denaturation, the reaction tube in the thermal cycler is heated to 95 degrees Celsius. The high heat denatures DNA by breaking the hydrogen bonds; the double helix is broken into single strands. In the second stage, annealing, the temperature is cooled to 60 degrees Celsius to allow DNA primers to form H-bonds with their complementary bases at 3' end at both strands. In the third stage, extension, the temperature is increased to 72 degrees celsius. Taq DNA polymerase binds to DNA template where primer has annealed. Complementary nucleotides are added to 3' end of both primers, thus catalysing synthesis of a new daughter strand. Chain reaction occurs as the products of previous reactions are used as the reactants in the next cycle.

Q: List the advantages and limitations of PCR

Advantages:
PCR is highly sensitive as it is capable of amplifying sequences from minute sequences of target DNA. It also has high fidelity as it produces the sequences required with a low error rate. It is also rapid as it can generate many copies within a short period of time. It is a quick and reliable method for detecting all mutations. Moreover, amplification can be done from unusual sources e.g. Egyptian mummies where the DNA quality is not good due to degradation.

Limitations:
Some knowledge of DNA/amino acid sequence of desired gene/protein is needed for the synthesis of flanking primers.
Non-target DNA sequence may be amplified instead, as primers are short and there is the possibility of many sequences complementary to primers. Also, Taq polymerase does not perform proofreading so there is the possibility of errors

Q: Explain the functions of primers in PCR

Primers mark out a sequence of DNA to amplify. This sequence would be complementary to the target DNA strand which it attaches itself to. They also prevent the single stranded DNA from re-annealling by sticking to a part of the target DNA. They also allow Taq polymerase to work from a 5' to 3' direction, since the enzyme can only work with a free 3' end.

Q: Explain how a foreign DNA fragment can be introduced into a plasmid that produces blunt ends.

Cut the DNA fragment and plasmid using the same restriction enzyme. Add guanines to DNA fragment blunt ends and terminal transferase, to produce a single chain of guanines to both the blunt ends of the plasmid vector. Add cytosines to vector blunt ends, to produce a single chain of cytosines to both blunt ends of the DNA fragment.
Mix the cut plasmid and cut DNA fragment together for recombination to occur. Single chain guanine will bind to single chain cytosine by complementary base pairing. Add ATP and DNA ligase to seal the nicks by forming phosphodiester bonds in the sugar backbone.
Add recombinant DNA to bacteria culture for transformation to take place. Use heat shock and add calcium chloride to facilitate uptake of recombinant DNA.

From here on, answer is question specific...
Culture bacteria in agar plate containing ampicillin (assuming original plasmid had ampicillin resistant gene). Bacteria that has taken up the plasmid vector, whether reannealed or with the foreign DNA will be resistant to ampicillin and grow on the agar plate. ...