Drug Discovery 9 Flashcards
after neutralisation the DNA is able to re-anneal, why is this good for plasmids ?
small plasmids are able to anneal whereas chromosomal DNA is so large that it aggregates so it doesnt decontaminate the small plasmid DNA
how are plasmids removed from bacteria and how is the process carried out ?
ALKALINE LYSIS
cells are broken open- SDS ruptures the cells and NaOH denatures the DNA to single stranded, it also causes all the protein to aggregate so it can be removed and RNAase degrade all the RNA up
then neutralisation occurs using potassium acetate
this can then be centrifuged and the plasmids will be present in the solution at the top or a pipette with a filter can be used to extract the plasmids
how is the plasmid removed from the plasmid prep?
alcohol precipitation and centrifugation - precipitate the DNA out directly
or
silica purification- silica can bind DNA at a low salt concentration and then it is washed and then a high salt solution is used to wash the DNA off the silica
what configuration is most of the plasmid normally in ?
supercoiled
- this is done using DNA gyrase in bacteria
what can nicked and supercoiled configuration not tell us about the DNA?
they cannot indicate the size of the DNA, to do this the DNA has to be linearised
nicked moves too slowly through the gel
supercoiled moves too quickly through the gel
what is transfection ?
the introduction of foreign genetic material into eukaryotic cells
how can the genetic material be transfected into a eukaryotic cell?
using a virus or by injection or by electroporation or by lipofection
lipofection is the most commonly used
what are some examples of lipid reagents used in lipofection ?
lipofectamine
DOTAP
how does lipofection work ?
take DNA and mix with a lipid reagent to produce a complex
- the lipid forms a vesicle around the DNA
- the vesicle can merge with the membrane so DNA enters the cell
what is transient transfection ?
most of the transfected DNA that gets to the nucleus remain extrachromosomal- this is transcribed and translated during the lifetime of the cell
- assay 2-3 days after transfection
- this is quick and convenient but doesnt last as long
what is stable transfection ?
small fraction of DNA that gets integrated into the cells chromosome and these cells can be selected if the plasmid used has a resistance gene so this DNA survive mitosis and is passed onto daughter cells
- assay 4-6 weeks after transfection
- integrated into the cell line
what does PCR allow and how is it similar/different to quikchange?
enables the amplification of the DNA that you flank in an exponential amplification
process is similar to quikchange but the 2 primers are not complimentary and so they bind at different places on opposite strands with their 3’ end pointing towards each other
what is needed to carry out a PCR reaction ?
template DNA
dATP, dTTP, dGTP, dCTP
primers
DNA polymerase- thermostable from bacteria- Taq polymerase
what is the cycle process of PCR and why is it different t quikchange?
denature at 95 degrees
anneal primers at 30-65 degrees
extend at 72 degrees
the difference s that the product of each round becomes the template for the next round
what is wrong with the first round product ?
it has the incorrect 3’ end because DNA polymerase overshoots the desired termination point
