Genetics
Polymerase Chain Reaction
Unlock FRCOphth Part 1 Study Notes to access this content.
Get access- Allows small amounts of DNA to be amplified for detection and analysis
- Enables sequence detection without blotting or cloning
- Two flanking sequences are needed adjacent to the sequence of interest
- Heat-resistant DNA polymerase is added to the target DNA and two DNA primers
The mixture is denaturated with heat at 100 degrees Centigrade to break the strands to single stranded DNA
Cooling then occurs allowing hybridisation of the primers to opposite strands of the target sequence and synthesis of complementary DNA by the DNA polymerase: synthesised in a 5’ to 3’ direction
- The two new strands are identical replicas
- The cycle is repeated up to 50 times with doubling of molecules with each cycle
- The DNA is amplified 105-106 times
- Useful where the amount of starting DNA is very small
- Neonatal diagnosis
- Identification of microorganisms in tissue samples
- Research
- RNA can also be amplified
Even the slightest contamination can produce positive results so PCR suffers from high false positives