| Polymerase chain reaction | - Invitro
- DNA amplification
- Application = amplifying DNA of crime scene
- It allows thousands of copies of DNA to be cloned in a short period of time |
| DNA polymerase (taq polymerase) | - The enzyme that joins nucleotides to form double stranded DNA
- Converts single-stranded DNA into double-stranded |
| Primer | - A short piece of complementary DNA that is required for DNA polymerase to attach |
| Thermocycler | - A machine which automates the process by heating then cycling through different temperatures (95, 55, 72) |
| Why not use human DNA polymerase? | - Denatures
- Breaks bonds in 3o structure, change its shape, no longer complementary, can't bind |
| How would the 3o structure of this enzyme be different to human DNA polymerase? | - More disulphide bridges in 3o structures therefore less likely to break |
| Why is the DNA heated to 95oC? | - Breaks H bonds, separate the strands
- To allow primers to attach |
| Why are primers required? | - To allow DNA to be double stranded so DNA polymerase can attach
- Identifies specific base sequence we can copy
- (Enzyme) needs starting strand onto which to attach nucleotides |
| Why is there excess primer added? | - Stop original DNA strands from reattaching |
| equation | 2^n
n = number of cycles |
| Why 2 different primers? | - DNA mol. anti parallel so different base sequences
- So primers will be complementary to the specific base sequence on either strand |
| Process | - Denaturation – DNA is heated to 95°C which breaks the h bonds, strands seperate
- Annealing - the temperature is decreased to 52°C so that primers can join to their complementary bases at the end of the DNA fragment
- Elongation / Extension – the temperature is increased to 72°C, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules |
