Question

Discuss how the technique of PCR allows a scientist to quickly clone a particular piece of DNA.  Be clear about the importance of 2 primers and Taq DNA polymerase.  Make sure you explain how you do a PCR – what is needed.

Answer 1

Answer: Polymerase chain reaction (PCR) is technique that is used to amplify a gene or pieces of DNA of interest using gene or DNA specific primers (Forward and Reverse primers) and Taq DNA polymerase in a thermal cycler. The amplified DNA is used in cloning experiments by scientist. A PCR reaction typically involves DNA template, dNTPs, forward and reverse primers, Taq DNA polymerase and buffer. Forward and reverse primers are designed using template sequence and will amplify full length of the DNA piece of interest. These two primers have complementary sequence to DNA template and binds to template during annealing step of the PCR. The Taq DNA polymerase amplifies the DNA by adding dNTPs complementary to the template DNA at the 3'-end of forward and reverse primers. Taq DNA polymerase have both polymerase and proof reading activity and hence, amplifies the DNA without any errors and is essential for cloning experiments. PCR setup involves three steps: Denaturation, annealing and extension. During denaturation, double stranded template DNA is denatured to single stranded DNA. In annealling step, forward primer attaches one strand of the template DNA and reverse primer attaches to other strand. In extension step, the primers are extended to full length DNA strand by Taq DNA polymerase. Once DNA is amplified it is cloned in to plasmid vector to create a clone.