Molecular Techniques

Question 1

Stages of Polymerase Chain Reaction (PCR)

Answer 1

1. Denaturation of DNA template
2. Annealing / Hybridisation
3. Extension / Elongation

Question 2

Describe the procedure for PCR - Denaturation of DNA template

Answer 2

• brief heat treatment at 94-95°C for 30 seconds

- hydrogen bonds between complementary bases of dsDNA broken so target DNA template is denatured into 2 separate strands

Question 3

Describe the procedure for PCR - Annealing / Hybridisation

Answer 3

• temperature reduced to 50-60°C for 30 seconds

Separated DNA strands do not reanneal as mixture contains excess of DNA oligonucleotide primers which anneal to opposite ends of DNA target sequence through complementary base pairing

Question 4

Describe the procedure for PCR - Extension / Elongation

Answer 4

• temperature raised to 72°C, optimum temperature of Taq polymerase, for 1 minute
• Taq polymerase binds to one end of each DNA oligonucloetide primer, adding deoxyribonucleotides to 3’ ends; new DNA strands synthesised in 5’→3’ direction, complementary to target DNA

Question 5

Describe the principle of PCR.

Answer 5

Involves selective amplification of chosen region of DNA molecule in vitro, from a trace starting sample, producing many copies of identical DNA

Question 6

Describe the procedure for gel electrophoresis - Preparation of agarose gel

Answer 6

• agarose gel powder mixed with electrophoresis buffer solution (maintain DNA in stable form) before mixture is heated until agarose dissolves
• gel tray with gel comb placed at one end before solution is poured and allowed to cool

Question 7

Describe the procedure for gel electrophoresis - Setting up of gel

Answer 7

• gel comb removed after gel has solidified; gel tray then put in electrophoresis chamber and covered with electrophoresis buffer solution which provides ions to support conductivity

Question 8

Describe the procedure for gel electrophoresis - Loading of DNA samples

Answer 8

• loading buffer added to DNA samples before they are pipetted into sample wells in the gel
• DNA molecular weight marker loaded into one of the wells to determine molecular size

Question 9

Describe the procedure for gel electrophoresis - Electric field

Answer 9

• safety cover placed over electrophoresis chamber and electric current is applied through electrodes at opposite ends of the gel
• done long enough for DNA molecules to be optimally separated

Question 10

Describe the procedure for gel electrophoresis - Staining

Answer 10

• visualisation of discrete bands by staining with DNA-binding dye

Question 11

Describe the principle of gel electrophoresis.

Answer 11

• DNA negatively charged due to phosphate groups in sugar-phosphate backbones; mixture of DNA fragments placed in well in porous gel with electric field applied across it
• as opposite charges attract, negatively charged DNA moves towards positive anode
• gel matrix consists of complex network of pores that DNA must past through, separating DNA molecules by size
• after gel electrophoresis, gel is removed and stained with DNA-binding dye (e.g ethidium bromide, GelRed) to allow visualisation of discrete bands

Question 12

Stages of gel electrophoresis

Answer 12

1. Preparation of agarose gel
2. Setting up of gel
3. Loading of DNA samples
4. Electric field
5. Staining