DNA Technology

1. DNA Technology In vivo gene cloning

2. Learning Objectives: 1. Review knowledge of gene cloning so far. 2. Expand upon GCSE knowledge of genetic engineering to understand the concept of in vivo gene cloning.

3. DNA Technology • Isolation – of the DNA containing the required gene • Insertion – of the DNA into a vector • Transformation – Transfer of DNA into a suitable host • Identification – finding those host organisms containing the vector and DNA (by use of gene markers) • Growth/Cloning – of the successful host cells

4. Isolation of Required Gene • Reverse transcriptase – what does it do? • Restriction endonucleases – what do they do? Discuss with each other – 2 minutes! Key terms: cDNA, sticky ends, blunt ends, palindromic recognition sequence.

5. Isolation of Required Gene Reverse transcriptase – makes a cDNA copy of mRNA. Restriction endonucleases – cut DNA at a palindromic recognition sequence. May produce DNA fragments with blunt ends (non-overhanging bases) or sticky ends (overhanging bases).

6. The PCR (Polymerase Chain Reaction) An in vitro method of gene cloning. What ‘ingredients’ are needed to copy the target DNA? Primers, nucleotides, DNA polymerase.

7. The PCR (Polymerase Chain Reaction) What are the three stages of the PCR? • Heat to 95oC to break H-bonds • Cool to 60oC to allow primers to anneal • Heat to 72oC so DNA polymerase joins the sugar-phosphate backbone between new nucleotides as they H-bond to complementary bases.

8. DNA Technology • Isolation – of the DNA containing the required gene • Insertion – of the DNA into a vector • Transformation – Transfer of DNA into a suitable host • Identification – finding those host organisms containing the vector and DNA (by use of gene markers) • Growth/Cloning – of the successful host cells

9. Learning Objectives Stage 2 – Insertion in to a vector • What is the importance of “sticky ends”? • How can a DNA fragment be inserted into a vector?

10. Importance of “sticky ends” • DNA from different source can be joined together IF they have the same sticky ends – the same recognition site. • In order to have the same sticky ends they must have been cut using the same restriction endonuclease.

11. Sticky ends are joined together using DNA ligase to join the sugar-phosphate backbone together. • The new DNA molecule is called recombinant DNA.

12. Insertion of DNA into a vector • VECTOR – any molecule/substance used to transport DNA into a host cell. • PLASMID – the most commonly used vector. A circular piece of DNA found in bacteria.

13. Why? • In vivo gene cloning. • The plasmids will be reinserted into bacteria. As the bacteria reproduce, they will copy the gene as if it was a normal part of their DNA.

14. Example: The R Plasmid • One of the antibiotic resistant genes is disrupted when the restriction enzymes cuts open the plasmid. • The other antibiotic resistant gene is used in selection of the correct host cells.

15. Insertion into plasmids What combinations of plasmid and donor DNA could form?

16. DNA Technology • Isolation – of the DNA containing the required gene • Insertion – of the DNA into a vector • Transformation – Transfer of DNA into a suitable host • Identification – finding those host organisms containing the vector and DNA (by use of gene markers) • Growth/Cloning – of the successful host cells

17. Learning Objectives: Stage 3, 4 and 5 – Transformation, Identification and Cloning • How is the DNA of the vector introduced into host cells? • What are gene markers and how do they work?

18. Introduction of DNA into host cells – Transformation (stage 3) • Plasmids must be reintroduced into the host cell (bacteria). • This process is called transformation. • Bacteria and plasmids are heated to 42oC in a solution of calcium chloride. • Even this well-used method is far from perfect.

19. Few bacteria are transformed… • Most cells don’t take up any plasmids. • Some plasmids closed before the donor DNA was inserted. • Some of the DNA self-ligated.

20. Identification (stage 4) of bacteria containing the plasmid • Firstly, we must identify the bacteria containing the plasmids – we do this by growing the bacteria on a medium containing an antibiotic. • We know to which antibiotics the bacteria should be resistant. No plasmid = no resistance!

21. Identification (stage 4) of bacteria containing the plasmid with the DNA fragment • Gene markers are used to identify which plasmids have taken up the DNA fragment. • Gene markers can be: • Resistance to an antibiotic • A fluorescent protein • An enzyme whose action can be identified • Usually the gene marker is disrupted if the DNA fragment is present.

22. Fluorescent markers • GFP – green fluorescent protein – gene from jellyfish. • Spliced into plasmid. • The restriction site chosen is in the middle of the GFP gene.

23. Fluorescent markers • If the donor DNA is added correctly, the GFP gene is disrupted and cells don’t glow. • If the donor DNA doesn’t combine with the plasmid, the GFP gene will be intact and the cells will glow.

24. Enzyme Markers • Some plasmids contain a gene for lactase. • Lactase turns a colourless substance (X-gal) a blue colour. • If the gene has been disrupted by the incorporation of the gene fragment the substrate will remain colourless.

25. Example: The R Plasmid • One of the antibiotic resistance genes is disrupted when the restriction enzymes cuts open the plasmid. • The other antibiotic resistance gene is used in selection of the correct host cells.

26. Antibiotic-resistance Markers • E.g. donor DNA may be inserted at the Pst1 site. • Bacteria with the correct plasmid will be resistant to tetracycline, but not ampicillin. • In order to identify these bacteria we use a process called replica plating.

27. Replica Plating Yellow plate contains tetracycline Green plate contains tetracycline and ampicillin • Colonies are carefully transferred to the same places on a new plate. • The bacteria on the yellow plate have the plasmid. • The bacteria which do NOT grow on the green plate (containing ampicillin) contain a plasmid with a DNA fragment. Ampicillin sensitive bacteria – these have the DNA fragment

28. Cloning (stage 5) the bacteria • Following successful identification of the bacteria containing the plasmid AND the DNA fragment, the bacteria are cloned. • As the bacteria are cloned, so is the plasmid containing the DNA fragment. • This type of gene cloning is in vivo (cloned within a living organism).

29. Tasks • Without looking in the textbook, make a list of the similarities and differences between in vivo and in vitro gene cloning. (You can check your answers afterwards using p247!) • Answer the questions in the yellow box on p 245-6.