Drug Discovery 7- gene to assay Flashcards

0
Q

how do we produce the protein ?

A

using the central dogma

  • recombinant systems make use of the replication, transcription and translation machinery inside the expression host to produce a protein
  • we just have to supply the cell with the right DNA and the right signals it needs to tell the cell what to do with that DNA
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1
Q

what is the first thing we need to assay our protein for drug binding ?

A

first of all we need our protein- the drug target
we also need to assay to assess efficacy - tissue based approaches are expensive and/or low throughput so generally the target needs to be expressed in a recombinant system

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2
Q

what does DNA look like ?

A

it is double stranded with the strands running in opposite directions to each other
- 2 hydrogen bonds between A and T and 3 hydrogen bonds between G and C

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3
Q

where is our DNA stored ?

A

within our chromosomes which are complex structures made up of DNA and proteins
all our cells contain the same chromosomal DNA

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4
Q

if all our cells contain the same chromosomal DNA why do they not all express the same genes?

A

only certain distinct regions of DNA are transcribed, different cell types transcribe a unique compliment of genes and they express a unique compliment of proteins

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5
Q

what does transcription require ?

A

it requires RNA polymerase to synthesise mRNA from the DNA template
- it is directed to do this by a promoter that is upstream of the gene and can be recognised by RNA polymerase

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6
Q

what are introns?

A

non- coding regions of DNA

they are removed during post translational modification by a process called splicing

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7
Q

other than splicing what is another process that occurs in post translational modification ?

A

the addition of a polyA tail to the mature mRNA

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8
Q

how do we obtain our protein for drug binding ?

A

obtain our protein by first finding the DNA encoding it and this this is cloned by isolate the mRNA encoding it

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9
Q

what is cDNA ?

A

complimentary DNA

DNA made in vitro from the mRNA isolated from cells expressing our target protein

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10
Q

why is cDNA different from the original genomic DNA ?

A

it doesnt contain any introns

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11
Q

how is cDNA made ?

A

made from mRNA using the enzyme reverse transcriptase which is derived from retrovirus

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12
Q

what is cloning ?

A

it is the process of obtaining the correct cDNA

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13
Q

if cells contain the same DNA why are they different ?

A

because it depends on the proteins in which they express which are needed for their function

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14
Q

what reflects the proteins that are expressed by a cell?

A

the mRNA content instead of the genomic DNA

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15
Q

what are the stages in cDNA isolation ?

A

1- start with tissue that expresses the protein of interest
2- extract and purify all the mRNA
3- make a cDNA library convert all the mRNA to DNA using the enzyme reverse transcriptase - this complementary DNA is called cDNA
4- isolate the cDNA encoding our protein of interest from the library using gene cloning

16
Q

What does reverse transcriptase do ?

A

it is derived from retroviruses which use it to convert their RNA into double stranded DNA prior to integration - we use the enzyme as a tool

17
Q

once the appropriate cDNA has been extracted what happens to it ?

A

cDNA is put into a vector so it can be expressed in a cell

  • it is a means of getting the cDNA into the host cell and provides enough info to cause it to be expressed
  • the vector contains a promoter which is a bit of DNA that causes RNA polymerase to transcribe the cDNA
18
Q

why are strong promotors put into the vector ?

A

because it will lead to more copies of mRNA being produced
- these promotors are often viral e.g CMV - they are normally viral because viruses have evolved to take over a cells expression machinery and so have very strong promotors

19
Q

what are the very basics required for expressing recombinant proteins ?

A

choose an appropriate cell line e.g. HEK293- one that will conviently express the protein and ideally one that doesnt already express the protein
choose compatible expression vector-one that recognises human RNA polymerase
inserting cDNA into vector
put this construct into the cell line= transfections
assay

20
Q

where is the cDNA inserted into the vector ?

A

it is inserted inbetween the promotor region and the polyA sequence in the multiple cloning site

21
Q

how is the cDNA inserted into the multiple cloning site ?

A

restriction endonucleases cut at sites in the MCS which have specific sequences that are unique to this site - they are not present elsewhere in the vector

22
Q

what do the restriction endonucleases do ?

A

one or more restriction endonucleases is used to cut the DNA plasmid in the mutliple cloning site - breaks the covalent bonds of both strands
cDNA is then ligated into the plasmid by ligation

23
Q

what are restriction endonucleases produced by ?

A

bacteria- named after the parent organism

24
Q

how long roughly are the strands of DNA which the restriction endonuclease cuts and what is characteristic of the DNA sequence being cut?

A

about 4-8bp long

the DNA sequences are palindromic

25
Q

what are the 2 different cleavage process that can occur when restriction endonucleases cut DNA ?

A

cleavage at opposite sides of the axis of symmetry produces sticky ends
cleavage at the axis generates blunt ends

26
Q

what is produced when restriction enzymes cut DNA ?

A

there is a phosphate group at the 5’ end and an -OH group at the 3’ end

27
Q

what did restriction enzymes require for activity ?

A

magnesium 2+ ions

28
Q

what bond do restriction enzymes cleave ?

A

phosphodiester bond

29
Q

give examples of restriction enzymes and their palindromic sequences

A

EcoR1- GAATTC
Pst1- CTGCAG
EcoRV- GATATC
Dpn1 - GATC

30
Q

What do palindromic sequences demonstrate ?

A

symmetry