Insulin and Genetic Engineering
- Diabetes mellitus is the inability of beta cells of pancreas to produce insulin
- Restriction enzymes/endonuclease cut DNA at specific recognition sites
- This produces either "sticky ends" or "blunt ends"
- DNA ligase can be used to re-join the ends
- Recombinant DNA technology combines the DNA from two different organisms
- Reverse transcriptase catalyses the formation of DNA from mRNA
- Vector is a gene carrier. It will carry a human gene into the cell of a bacterium or yeast that will be used to make human protein. Produces no benefit for viruses / carrier
- Plasmid, circular strand of DNA, are useful vectors to make human protein from bacteria
- Transgenic organisms contain another species DNA
[EXAM] Remove a particular gene from the DNA of an animal cell
- Locate with the use of a gene probe
- Use restriction enzymes
- Use endonucleases to cut at specific base sequence by hydrolysing
- Breaking sugar-phosphate bonds
[EXAM] Insert this gene into the genetic material of a bacterium
- Same restriction enzymes
- Cut at same base sequence in bacterial DNA
- Leaving sticky ends/hydrogen bonds break
- Join/splice with ligase
- Use of plasmid
Task to find and insert the gene into bacterium for Insulin production
- Isolate human gene, e.g. insulin, by using cytoplasmic mRNA (no introns)
- Reverse transcriptase, taken from a retrovirus, makes DNA from mRNA
- DNA is given "sticky ends" by using the enzyme restriction endonuclease
- Insert into a plasmid from a bacterium
- Dissolve cell walls using enzymes
- Centrifuge to separate bacterial chromosome ring from plasmids
- Cut open the plasmid
- Add sticky ends
- Mix plasmid and DNA gene together and use DNA ligase to stick them together
- Mix with bacteria //only ≈1% will take up the engineered plasmids
- Identify by using antibiotic resistance. Add gene for antibiotic resistance next to insulin gene in the plasmid. Add antibiotic to the culture / only bacteria surviving have insulin gene
- Grow transformed cells using industrial fermenters
- Isolate and purify human protein made by these cells
Moral and ethical issues associated with recombinant DNA technology
- Transgenic bacteria or viruses may mutate and may become pathogenic
- Genetically modified crops could "escape"
- Forms a genetically modified population in the environment
- Genetic modification may involve the resistance to herbicides
- Escaped crops may become "superweeds" that are difficult to kill and control
- Transgenic organisms could upset the balance of nature
- Population of transgenic salmon have been produced in which individuals grow rapidly
- These transgenic fish could compete for food with other fish species
