DNA amplification and genetic engineering Flashcards
what components are needed in PCR
- template DNA (single stranded)
- primers
- dNTPS- nucleotides
- buffers (mg2+)
- Taq polymerase
Uses of PCR
-amplification od DNA
-sequencing
-detection of pathogens in water or blood
-genetic fingerprinting
forensic analysis
-diagnosis of genetic disorders
-prenatal diagnosis
-analysis of ancient DNA
limitations of PCR
- two specific primers needs to be made
- limit on length of amplified frag
- high mutation rate- taq does not have a proof reading mechanism
- sensitive to exact reaction conditions
- tiny amounts of contaminating DNA will also be amplified
PCR process
carried out in a thermocycler- repeats itself 30-40 times
- denturaton- 95 degrees- DNA becomes single stranded
- annealing- 50 degrees. primers bind to dna and polymerase attaches and starts the synthesis of DNA
- extension- 72 degrees- optimum temp for TAQ pol- extension fragment
TAQ polymerase is..
thermostable–> extremophile
power of genetic engineering
- genes can be shared between species
- geentic code is universal so expression is possible
useful products produced using genetic engineering
human insulin blood clotting factor human growth hormone bovine chymosin hepatitis B vaccine artemisini (anti-malarial)
gel electrophoresis
fractionation of dna depending on size
- potential difference applied along a gel
- dna moves to +ve electrode through gel depending on: conformation (shape) and size
- dna is stained with fluorescent dye for detection by UV exposure e.g. ethidium bride
- moves towards positive end due to negative phosphate group1
why is there a signifiant mutation rate in PCR
Taq pol does not have checking mechanism
denaturation
95
annealing
50
extension
72
only dna between.. is amplified
primers- specific sequences can be amplified from a complex mixture of dna
ends of the amplified frag are defined by ..
2 primers
after 30 cycles of PCR…
10^6 fold amplification–> enough for analysis on gel
