At GCSE you were taught that after smashing cells, the chromosomes were treated with restriction enzymes. What is the disadvantage of this blunder-bus approach?
What molecule is likely to be in high concentrations in the beta cells of the pancreas tissue which would contain the instructions for building insulin peptides?
How can the mRNA for making insulin be isolated from any other mRNA
Before this 'insulin' mRNA can be added to bacteria for cloning, it needs to be converted back into DNA. What molecule is added to achieve this?
The problem with using reverse transcriptase to make a DNA copy (cDNA), is that it is only a single stranded copy. What therefore has to be added to convert it into a useful double stranded helix that can be inserted into a bacterial plasmid?
What is a bacterial plasmid?
What must the plasmid be cut with in order to insert the 'insulin' cDNA
Because the 'insulin' cDNA was not generated by using restriction enzymes to cut the insulin gene from the DNA, the cDNA does not have 'sticky' ends. What must therefore be added to allow it to bind to the opened plasmid DNA?
What chemical is needed to seal the 'insulin' DNA into the bacterial DNA?
What is the name given to this hybrid DNA plasmid?
These hybrid plasmids are then mixed with plasmidless bacteria and given a mild electirc shock to make the bacterial membrane more permeable. But not all the bacteria will take up the plasmids. How are the bacteria which have taken them up identified?
How do scientist ensure that the cloned bacteria actually 'read' the insulin gene and produce the insulin peptides?
What is the one problem with using prokaryotes to build the insulin gene?