PCR & Gel Electrophoresis

Biology: IB Diploma Higher Level, topic 13.3.4

Polymerase Chain Reaction (PCR)

The polymerase chain reaction is used to amplify small amounts of DNA.

The polymerase chain reaction process enables us to take a small sample of DNA and produce many more copies of it, allowing for further analysis to take place.

Several ‘ingredients’ are required for the PCR reaction:
DNA primers (sections of DNA about 20 base pairs long which are complementary to the ends of the target sequence of DNA that you wish to amplify).
Free nucleotides adenine, thymine, guanine, and cytosine.
Taq polymerase (a special polymerase enzyme extracted from bacteria that live in very hot environments such as hot springs, so that the enzyme will not denature at the temperatures used in the PCR).
The target sequence of DNA that is to be amplified.

PCR is carried out in a thermocycler and involves a cycle of three steps at three different temperatures:
Denaturation at 95°C - DNA strands are separated by breaking the hydrogen bonds.
Annealing at 60°C - primers attach to complementary DNA by forming hydrogen bonds.
Elongation at 72°C - optimum temperature for Taq polymerase to add the free nucleotides to the exposed DNA strands.
These three steps are then repeated 20-30 times in a typical PCR reaction to potentially produce over 1,000,000,000 copies of the target sequence of DNA.

Gel electrophoresis is used to separate proteins or fragments of DNA according to size. DNA profiling is a common application of gel electrophoresis.

Gel electrophoresis involves adding samples of proteins or DNA fragments to a gel immersed in a chamber with an electric current running through it.
The current causes the charged particles to move through the gel as it is porous.
The smaller particles can move through the gel more easily and therefore move further.

A banding pattern is formed which when compared to a reference can allow for the identification of specific proteins or DNA fragments.

DNA profiling, also known as DNA fingerprinting, is used to compare samples of DNA.
DNA profiling is commonly applied in forensic investigations and paternity testing.
The process involves both PCR and gel electrophoresis.

Firstly, PCR is used to amplify what could be a very small sample of DNA.
The amplified samples are then subjected to gel electrophoresis to compare them.
The bars of DNA fragments that form in the gel are compared to another reference e.g. with other suspects or evidence.

Which of these is NOT an ingredient required for the PCR reaction?
Which of these is NOT a key step in the PCR reaction?
Which of the following DNA fragments will move furthest through the gel in gel electrophoresis? The fragment length in number of base pairs (bp) is given.