How is PCR Done?
PCR is a 3 stage cycle;
- The first stage is the Denaturing of DNA which is carried out at 94-96°C. At this stage, the high temperature breaks up the hydrogen bonds which hold the double stranded DNA together and the strands separate into two single strands.
- The second stage includes the primers finding the complementary sequences in the DNA and combine with them at a temperature between 50-65°C. It is called the annealing of primers.
- At 72°C, the unattached DNA nucleotides bond (Taq polymerase allows the bonding to take place and it shows optimum activity at 72°C) to the original DNA strand through complementary base pairing in order to form new strands.
Result
Overall, there are 30 cycles and each cycle doubles the number of DNA molecules, resulting with a billion copies of DNA.
DNA Fragmentation
Restriction enzymes (specifically known as restriction endonucleases) function to cut strands of DNA at certain sites. These sites are called restriction sites and they are palindromic; meaning, that the nucleotides are read the same on the forward strand as it is read on the reverse strand. Genetic Engineering- Recombinant DNA Technology is based on the enjoinment of the sticky ends cut by different restriction enzymes when the base pairs are complementary to each other.
How do Restriction Enzymes Work?
- Initially, it cuts from two areas; one cut is through each strand, resulting in the separation of the sugar phosphate backbones and the other cut breaks the phosphodiester bond.
- Some restriction enzymes form blunt ends by cutting across both the two strands. However most enzymes form sticky ends by cutting the two strands staggered. Sticky ends are the areas located on the DNA which there is an overhang, meaning bases are exposed among the forward and backward ends of the DNA sequence which had been cut by the restriction enzyme.
-
These sticky overhangs are named cohesive ends and they enable the pieces of DNA to recombine with the other cohesive ends when the base pairs on the cohesive ends are complementary to each other; phosphodiester bonds are reformed by DNA ligase.
Where do Restriction Enzymes Come from?
The restriction enzymes used are obtained from bacteria and they are named accordingly with its name. For example, PstI- Providencia stuartii.
How is this stage useful?
Due to DNA being cut into fragments of various sizes by restriction enzymes it has an important role in DNA profiling. After these fragments are visualised with the help of gel electrophoresis, the pattern that occurs could be viewed to identify the guilty suspect in a criminal case.
Gel Electrophoresis
Why is Gel Electrophoresis Used?
The gel electrophoresis technique is used in order to separate the DNA molecules which have been cut into fragments according to their size; it is done to confirm the size of the DNA sequence which was copied during PCR. In DNA profiling, the visual layout of the DNA fragments for each individual could be compared with the others and could be useful to identify hereditary relations between individuals.
What is used in the set-up of Gel Electrophoresis?
- Agarose is the specific gel matrix, it is generally produced between the concentrations of 0.7% and 2%; 0.7 % gel will allow the large DNA fragments to be observed well; 2% gel will allow the small DNA fragments to be observed well. The gel is put inside a buffer as it conducts the electricity which is needed for the electric field to carry out its process without being interrupted; and it controls the pH which is an important factor towards the stability of the DNA molecules.
- The buffer can be either the TAE (Tris-acetate-EDTA) or TBE (Tris-borate-EDTA); TAE easily becomes exhausted due to its lower buffer capacity than TBE, however the double stranded DNA is able to run much quicker in TAE.
- An electrophoresis chamber and power supply is essential for the procedure to take place as it aids in the process of separating the DNA sample; giving a low voltage is the best to use because it allows a slower and more precise separation of the DNA sample.
- The loading buffers used to inject the DNA fragments into the wells are made of a dense liquid solution– e.g glycerol- in order to keep the sample inside the wells and not float away.
- It also contains a tracking dye that helps with the monitoring of how far the sample has travelled in the gel.
