1. GENE TECHNOLOGY Objectives: To describe how sections of DNA containing a desired gene can be extracted from a donor organism using enzymes. To explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase. To state other vectors into which fragments of DNA may be incorporated.

2. What is genetic engineering? The processes used to obtain a specific gene and place it in another organism recipient transgenicThe recipient organism expresses the gene and is transgenic (or GMO) recombinant DNAThe new DNA is said to be recombinant DNA because it contains DNA from another organism or source

3. Why do we want to genetically engineer organisms? Improving the recipient organism e.g. herbicide resistant crops, growth promotion (the myostatin gene for muscle growth) in animals Creating organisms to make useful products e.g. insulin or GH production, production of pharmaceuticals through ewe milk, beta-carotene production in rice

4. An overview of the process 1.Obtain copy of gene (from a donor) 2.Place gene in a vector 3.Introduce vector to host cell 4.Select cells containing donor DNA 5.Clone the gene

5. Obtaining a copy of the Gene Three main methods: reverse transcriptase 1.make a copy of the gene from its mRNA using reverse transcriptase 2.Artificially synthesise the gene restrictions endonucleases 3.Cut up the DNA with restrictions endonucleases and search the DNA containing the gene – shot gun approach or use a DNA probe to identify the DNA before cutting it

6. polymeraseReverse transcriptase is a polymerase enzyme transcriptionIt performs the reverse of transcription It synthesises DNA from an mRNA template Complementary DNA (cDNA)Complementary DNA (cDNA) is DNA made from mRNA Reverse transcriptase

7. Making cDNA from mRNA

8. The principle mRNA is complimentary to the DNA in a specific gene Reverse transcriptase is able to make a strand of DNA that is complimentary to the mRNA If the mRNA for a specific gene is isolated then the gene can be synthesised using reverse transcriptase The DNA formed is called complimentary or cDNA Animation of the Formation of cDNA

10. b) Synthesising a gene The base sequence of the DNA can be worked out from the aa sequence of the required protein. The gene can then be constructed using free nucleotides and joining them together in the right order by a polynucleotide sequencer This is only possible for short genes at present.

11. C) Restriction Endonucleases Enzymes that cut DNA at specific sites Cut the bonds in the middle of the polynucleotide chain sticky ends which are ssDNAMost enzymes make a staggered cut in the two strands, forming sticky ends which are ssDNA blunt endsSome restriction enzymes cut straight across both chains, forming blunt ends Artificial sticky ends need to be added to blunt ends Restriction Enzymes Animation Steps in Cloning a Gene Animation

12. Inserting a gene into a vector

14. Placing the Gene in the vector The most common vectors are plasmids (other e.g.s are certain virus genomes or yeast cell chromosomes). Plasmids are extracted from bacterial cells by centrifugation Restriction endonuclease enzymes are used to cut the plasmid DNA and the gene from the donor DNA DNA ligaseOnce cut the plasmids and cDNA are mixed with DNA ligase This allows the sticky ends to form complimentary base pairs by forming covalent phosphodiester bonds Recombinant DNARecombinant DNA is produced

15. DNA ligase catalyses the formation of phosphodiester bridges, splicing the DNA into the plasmid DNA.

16. Introducing the Vector DNA into the Host Cell The plasmid is now introduced into a bacterial host cell to multiply up A mutant harmless form of E. coli is commonly used because it has a doubling time of 30 minutes E. coli, the plasmids and ca 2+ ions are given a brief heat shock which temporarily makes the CSM permeable to DNA This process of adding new DNA to a host is called transformation and produces transformed or transgenic bacteria (there are a range of alternative methods on p174 – make a list)

17. Genetic Engineering of Human Insulin as an example

18. There are 2 major problems with this technique. 1.Not all bacteria will take up the plasmids. 2.Not all the plasmids will have taken up the foreign DNA i.e. Be recombinant plasmids.

19. Solution to Problem 1 Use plasmids with antibiotic resistance genes E.g. ampicillin Grow the bacteria on agar containing that antibiotic then only those that have taken up the antibiotic resistant plasmids will survive. BUT: You know the bacteria have taken up the plasmid but you still don’t know if they took up RECOMBINANT plasmids. How could we tell if the bacterium had taken up a recombinant plasmid?

20. Solution to Problem 2 Use a restriction enzyme will cut through a different antibiotic resistance gene e.g. tetracycline. The recombinant plasmids will have the human insulin gene inserted in the middle of the tetracycline resistance gene, meaning that it no longer has resistance. Use replica plating to identify the recombinant colonies

21. Replica Plating The colonies that can grow on the tetracycline are NOT transformed, so the transformed colonies can be identified from the original ampicillin plate

22. Homework Make notes on the advantage of bacterial conjugation for taking up recombinant DNA and the experiments on pneumonia in mice and their findings (p176-177) (note conjugation animation in scheme)