Drug Discovery 8

Question 0

what is the problem with using a single restriction endonuclease?

Answer 0

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

Question 1

what are restriction endonucleases?

Answer 1

enzymes that cut at a specific DNA sequence in which you want to insert a gene of interest

Question 2

what is the resolution for the adverse effects caused by using a single restriction endonuclease?

Answer 2

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

Question 3

what type of enzyme is DNA ligase ?

Answer 3

modification enzyme

Question 4

what is DNA ligase and what does it do ?

Answer 4

it is an ATP dependent modification enzyme which reforms phosphodiester bonds between the 5’ phosphate and 3’hydroxyl groups of the DNA strand

Question 5

when are the effects of DNA ligase less efficient ?

Answer 5

less efficient when rejoining blunt ends

with sticky ends there are already hydrogens so it is quite efficient

Question 6

what is a common use of DNA ligase ?

Answer 6

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

Question 7

what needs to be considered when making a DNA construct ?

Answer 7

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

Question 8

how is the re-ligating of the vector avoided ?

Answer 8

using another modification enzyme called alkaline phosphatase

Question 9

what happens to DNA when it is cut with restriction enzyme and what does this allow ?

Answer 9

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

Question 10

what does alkaline phosphatase do ?

Answer 10

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

Question 11

what is T4 PNK?

Answer 11

T4 polynucleotide kinase
it is a modification enzyme
it is able to add a phosphate to the 5’OH of a dephosphorylated DNA molecule

Question 12

when is the use of T4 PNK useful ?

Answer 12

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

Question 13

what happens in gel electrophoresis of DNA ?

Answer 13

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

Question 14

what does ethidium bromide do ?

Answer 14

it intercalates between the DNA bases

Question 15

what is the plasmid vector and what does it do ?

Answer 15

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

Question 16

what are plasmids and what is their purpose ?

Answer 16

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

Question 17

what properties do plasmids need and why ?

Answer 17

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

Question 18

what is transformation ?

Answer 18

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

Question 19

what is the easiest way to determine if transformation has occurred ?

Answer 19

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

Question 20

where does the cDNA need to be in the plasmid to ensure it is expressed ?

Answer 20

needs to be put downstream of a bacterial promotor

Question 21

what can be produced after overnight growth of the bacteria and how ?

Answer 21

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

Question 22

why are plasmids put into bacteria ?

Answer 22

primarily to amplify them - extract large quantities for further use

Question 23

why is site directed mutagenesis useful for drug discovery ?

Answer 23

• 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

Question 24

what is the main used of SDM in drug discovery ?

Answer 24

for interpreting and understanding the molecular nature of the drug target interaction

Question 25

what is the principle for disrupting the molecular interactions of a protein ?

Answer 25

you can observe a change in the pharmcology caused by this disruption indicating that it is an important site
if no change is observed then it probably isnt involved in the drug bindign/action

Question 26

how was SDM used for the advancements with beta 2 adrenoreceptor?

Answer 26

in 1986 it was discovered that it was 7 transmembrane receptor
presence of the 2 negatively charged aspartic acid residue is the 2nd and 3rd residues was unusual because transmembrane domains are using hydrophobic so they targetted this as a potential binding site for positive adrenaline

Question 27

what did SDM experiments show about beta-2 receptors and how was this determined ?

Answer 27

showed that asp-113 in TM was important for binding agonists and antagonists
they changed this amino acid to Glu (also -ve charged)- caused a 300 fold reduction in potency
they changed this amino acid to Asn- (not charged) - causedn an 8000 fold reduction in potency
they also determined that asp-79 was important for the ability to fully activate the receptor because when this was removed the Emax was reduced

Question 28

how is the altered protein target achieved ?

Answer 28

by altering the coding strand of the gene - normally the cDNA

Question 29

in recombinant DNA work in terms of codons how do we need to think about them ?

Answer 29

think of codons in terms of DNA seuence

Question 30

explain the codons in transcription ?

Answer 30

mRNA sequence is derived from a DNA template and has a complementary code
- coding strand of DNA is transcribed to give the codons of mRNA

Question 31

what is the 1st stage of SDM ?

Answer 31

expose template strand - the new DNA is made based on the template strand

Question 32

what does DNA polymerase do ?

Answer 32

binds to double stranded nucleic acids and adds new nucleotides to the 3’ end based on the sequence of the template strand

Question 33

how does DNA replication work ?

Answer 33

DNA is unwound by helicase to single stranded proteins - done in vitro with heat
DNA is made from dNTP building blocks by DNA polymerase
DNA polymerase binds to double stranded nucleic acids
DNA polymerase only makes DNA in the 5’ to 3’ direction
replication fork there are some problems- the leading strand is easy to make because the double helix unzips in the same direction in which DNA polymerase moves so it only binds once and then just slides along
but the lagging strand is more difficult because it is exposed in the wrong direction compared to the growing chain- therefore DNA polymerase has to be rebinding but it can only bind to double stranded DNA - therefore lagging strand requires continual priming
primers are made of RNA and are made by primase enzyme - these RNA fragmets are replaced by DNA later in the process

Question 34

what does DNA replication require ?

Answer 34

DNA polymerase 
DNA template
deoxynucleoside triphosphates 
unravelling of the double helix
priming 
extreme accuracy 
very rapid 

in vitro process used DNA primers instead of RNA and the strands are unravelled by heat

Question 35

how does the replication of DNA occur in vitro ?

Answer 35

DNA strands exposed to heat at 95 degrees to split the strands and then cooled in the presence of the primer to anneal it
then DNA polymerase can use dNTPs to make a complimentary chain
it is the 5’ end of the new chain that incorporates the synthetic DNA of the primer

Question 36

what happens if you make a single base change in the primer ?

Answer 36

as long as the primer is long enough (20-30) bases the primer should still bind to the specific designed region even if there is one mis-matched base pair
because the new DNA incorporates the DNA primer it will now have an altered base

Question 37

what is quikchange mutagenesis?

Answer 37

real situation you have 2 stranded DNA at the same time
therefore 2 primers are required that are complimentary and bind to the template strands at equivalent sites
then DNA polymerase extends the primers in the 5’ to 3’ direction
therefore the product DNA is made in the opposite directions
denaturation/annealing/extension cycle is repeated 20-30 times to make more and more mutated DNA

Question 38

why are the original primers not able to amplify more DNA using the product as a template ?

Answer 38

because even though they could bind to the product they are pointing in the wrong direction so therefore DNA polymerase couldnt extend them
ALL new DNA is made from the template and therefore amplification is linear

Question 39

what is the final product produced by SDM by quikchange ?

Answer 39

plasmid DNA with 2 ‘nicks’ where the DNA polyermase finished making the strand
these nicks are repaired once the plasmid is put into a cell
another difference between product and template is the product contains the DNA of the primers and therefore the altered base
another difference is the template was made in vivo in bacteria whereas the product was made in vitro

Question 40

what will have happened to the template DNA because it was made in vivo and why is this useful ?

Answer 40

the template will be methylated whereas the product isnt
this is useful because the restriction enzyme DpnI which recognises the specific sequence GATC only cleaves methylated DNA
this means if the reaction mixture is exposed to DnpI it will result in all the un mutated template being destroyed leaving only mutant DNA

Question 41

explain the stages in quikchange mutagenesis?

Answer 41

1- template is the plasmid containing the cDNA of interest
2- denaturation, annealing and extending cycle occurs repeatedly
3- original template is destroyed with DnpI
4- nicked product is put in to bacteria where it is repaired and amplified