Drug Discovery 8 Flashcards
what is the problem with using a single restriction endonuclease?
it can cause the plasmid to ligate in the wrong direction causing the formation of the wrong product
- this can theoretically happen 50% of the time
what are restriction endonucleases?
enzymes that cut at a specific DNA sequence in which you want to insert a gene of interest
what is the resolution for the adverse effects caused by using a single restriction endonuclease?
using 2 different restriction endonucleases
these enzymes cut at different sites and therefore this prevents re-annealing
this ensures the insert goes into the vector in the correct orientation
what type of enzyme is DNA ligase ?
modification enzyme
what is DNA ligase and what does it do ?
it is an ATP dependent modification enzyme which reforms phosphodiester bonds between the 5’ phosphate and 3’hydroxyl groups of the DNA strand
when are the effects of DNA ligase less efficient ?
less efficient when rejoining blunt ends
with sticky ends there are already hydrogens so it is quite efficient
what is a common use of DNA ligase ?
used to add ‘adaptors’ to cDNA inserts if they dont have suitable ends for ligating into the vector
after the complementary ends have been added the DNA ligase can then be used to form new phosphodiester bonds
what needs to be considered when making a DNA construct ?
the ability of the DNA ligase to re ligate the vector - this happens more readily compared to the insertion of DNA because there is more chnace of vector ends adhering to each other via hydrogen bonding as they are so closely located
how is the re-ligating of the vector avoided ?
using another modification enzyme called alkaline phosphatase
what happens to DNA when it is cut with restriction enzyme and what does this allow ?
a phosphorylated 5’ end is produced which is critical for the ability of DNA ligase to ligate this to the 3’ end
but if only one strand is phosphorylated the DNA can still be ligated because still one phosphodiester bond can form
the hydrogen bonding in the other strand will maintain the structure but it will be ‘nicked’ but this can be repaired once the DNA is in the cell
what does alkaline phosphatase do ?
it removes the 5’ phosphate to prevent DNA re-annealing
however an insert will still have 5’ ends so it will be able to ligate into the vector
this approach is only really necessary if the vector is cut with one restriction enzyme
what is T4 PNK?
T4 polynucleotide kinase
it is a modification enzyme
it is able to add a phosphate to the 5’OH of a dephosphorylated DNA molecule
when is the use of T4 PNK useful ?
useful when a DNA insert is made using PCR because the ends of a PCR product are not phosphorylated
or the primers used in PCR can first be phosphorylated with PNK to result in a phosphorylated product
what happens in gel electrophoresis of DNA ?
DNA is electrophoresed in alkaline buffer -pH8
because DNA is negatively charged it migrates towards the positive end
the smaller fragments will move further/faster
it is stained with ethidium bromide so it can be viewed under uv light
what does ethidium bromide do ?
it intercalates between the DNA bases
what is the plasmid vector and what does it do ?
cDNA for the target protein needs to be replicated, transcribed and translated by the machinery of a cell
it is expressed in either a mammalian cell or yeast, insect or bacterial cell
construct needs to be prepared and amplified in the bacteria- it is the vector that enables this
what are plasmids and what is their purpose ?
vectors used in recombinant DNA technology derived from naturally occuring DNA plasmids
- naturally occuring to confer resistance
- closed circles of double stranded DNA
- extra-chromosomal
- stably inherited so they can be replicated
- replicate via host proteins
- exist as multiple copies- recombinant DNA technology plasmids have high copy numbers
what properties do plasmids need and why ?
antibiotic resistance gene- need to be stored and amplified in bacteria
bacterial origin of replication- bacteria can replicate the plasmid and pass it onto daughter cells
multiple cloning site- so cDNA is inserted into the correct place
promoter- this must match RNA polymerase of the expression host
for replication in mammalian cells- need a polyadenylation signal and an origin of replication so it can be replicated
what is transformation ?
bacteria need to be made competent - puncturing of their cell wall to encourage them to take up the plasmid
to make the cell wall competent a heat shock is done- 42 degress for 90 seconds and then put on ice
once the new DNA has been taken up they are referred to as transformed
what is the easiest way to determine if transformation has occurred ?
use a plasmid containing an antibiotic resistance gene - if the plasmid has this then it will grow in a dish containing ampicillin but it willl prevent the growth of bacteria which dont contain the plasmid
where does the cDNA need to be in the plasmid to ensure it is expressed ?
needs to be put downstream of a bacterial promotor
what can be produced after overnight growth of the bacteria and how ?
amplification of up to 5x10 to the power of 8 plasmid copies
- because each colony can contain 10 to the power of 6 cells and each can contain about 500 copies of the plasmid
why are plasmids put into bacteria ?
primarily to amplify them - extract large quantities for further use
why is site directed mutagenesis useful for drug discovery ?
- drugs bind to their targets via specific molecular interactions
- understanding these interactions can aid drug discovery
- interaction can be identified by disrupting them and then analysing the consequences
