Success criteria - PCR

1. Success criteria - PCR • By the end of this lesson we will be able to: • The polymerase chain reaction (PCR) is a technique for the amplification ( making many millions of copies of the original ) of DNA in vitro i.e. in the lab. • In PCR, primers are complementary to specific target sequences at the two ends of the region to be amplified. • The stages of PCR involve • DNA is heated to separate the strands. • Cooling allows primers to bind to target sequences. • Heat tolerant DNA polymerase then replicates the region of DNA. • Repeated cycles of heating and cooling amplify this region of DNA. • Case study on the use and application of PCR including practical using thermal cycler or water baths.

2. Producing copies by PCR In order to sequence DNA or carry out DNA fingerprinting it is necessary to produce a huge number of exact copies of the original stands. The technique used to do this is known as PCR or ‘Polymerase Chain Reaction’. Once the copies of DNA have been produced they can be analysed. Note – This is the technique used by forensics to amplify tiny samples of DNA for ‘fingerprinting’

3. The Polymerase Chain Reaction (PCR) 5’ 3’ 3’ 5’ DNA strands unzip Primers attach 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ Taq 5’ Taq 3’ = Taq polymerase Taq

4. Understanding - PCR 1.PCR can amplify any DNA sequence hundreds of millions of times in just a few hours. It is especially useful because it is highly specific, easily automated and capable of amplifying minute amounts of sample 2.The whole process is only possible because of a special heat-stable enzyme called Taqpolymerase, isolated from thermophilic bacteria. 3.The enzyme Tac polymerase is able to tolerate temperatures of 95C and has a temperature optimum of 72C. 4.This enzyme can synthesise the complementary strand of a given DNA strand in a mixture containing the four DNA nucleotide bases and two short DNA fragments called primers. Each primer is usually about 20 base pairs (bp) long. The primers are designed to bind to the DNA at either side of the target sequence.

5. Procedure: • Step 1 – The DNA is heated to 950 C breaking the hydrogen bonds and separating the strands. • Step 2 – The strands are cooled to between 55 – 700C and the primers added. • Step 3 – The strands are heated to between 70 – 72 0C so that Taq Polymerase can copy each strand from the point of the primer. • Summary • PCR requires the following:- • Template DNA • Primers – starting points for the construction of new strands • Taq Polymerase – a polymerase enzyme which works at high temperatures • Supply of nucleotides • PCR can amplify a single strand of DNA by a factor of millions

6. This is a thermocycler which carries out the process of PCR automatically by adjusting the temperature and adding the ‘ingredients’ when required