Drug Discovery 7- gene to assay Flashcards
how do we produce the protein ?
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
what is the first thing we need to assay our protein for drug binding ?
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
what does DNA look like ?
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
where is our DNA stored ?
within our chromosomes which are complex structures made up of DNA and proteins
all our cells contain the same chromosomal DNA
if all our cells contain the same chromosomal DNA why do they not all express the same genes?
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
what does transcription require ?
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
what are introns?
non- coding regions of DNA
they are removed during post translational modification by a process called splicing
other than splicing what is another process that occurs in post translational modification ?
the addition of a polyA tail to the mature mRNA
how do we obtain our protein for drug binding ?
obtain our protein by first finding the DNA encoding it and this this is cloned by isolate the mRNA encoding it
what is cDNA ?
complimentary DNA
DNA made in vitro from the mRNA isolated from cells expressing our target protein
why is cDNA different from the original genomic DNA ?
it doesnt contain any introns
how is cDNA made ?
made from mRNA using the enzyme reverse transcriptase which is derived from retrovirus
what is cloning ?
it is the process of obtaining the correct cDNA
if cells contain the same DNA why are they different ?
because it depends on the proteins in which they express which are needed for their function
what reflects the proteins that are expressed by a cell?
the mRNA content instead of the genomic DNA