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