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
PCR primers are
short 20bp single stranded DNA
-OLIGONUCLEOTIDES
for expression of eukaryotic genes
normally need to make cDNA copy of mRNA
dna moves through gel depending
size and shape -smaller the faster due to less reissitance
dye used for staining
ethidium bromide
genetic engineering basic
involves the introduction of foreign in DNA into cells. The the cell will replicate and express the dna e.g. in plasmids by inserting a transformed plasmid
gene cloning
- introduce rDNA (recombinant plasmids0 into bacterial cell
- will replicate when cells divide
- dna amplified
- recover dna for analysis (purify)
reporter genes
proteins tagged with green fluorescent protein (GFP)
-from bioluminescent jellyfish
-autcatalytic, fluorescent protein
0in vivo reporter, high signal to to noise, no enzyme activity
-many spectral mutant available.
in vivo cloning
insertion of dna into bacteria
-plasmid is cut with a restriction endonuclease (bacterias defence mechanism)–> specific enzymes t a specific recognition sequence
-enzyme will leave staggered cut–> by cleaving phosphodietser linkages
DNA ligase will jin the DNA together by reforming phosphodieter bonds (requires ATP)
e.g. of calculating recognition sites
sequence occurs every 1/5096bp–> 4.6x10^6/4x10^3=1150 sites
what is a gene library
a collection of recombinant clones
what can a gene library be used for
-can screen for clones containing gene of interest
what is plasmid cut with and then rejoined with
restriction endonuclease
ligase–> requires ATP
where is cloning a very large DNA possible
yeast artificial chromosomes (YACs)
e.g. artemisinin production- produced in yeast- treatment against malaria–> originally found in plant, however gene recovered and inserted into yeast, grows much more effectively (by feb 2013 40 million treatments produced
