1. Review from last week

2. The Making of a Plasmid

3. Plasmid: - a small circular piece of extra-chromosomal bacterial DNA, able to replicate - bacteria exchange these plasmids to share DNA - E.g. antibiotic resistance genes

4. Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA Specific DNA sequences can be recognized by enzymes called restriction endonucleases

5. Restriction Enzymes—restriction endonucleases enzymes that are able to cut double- stranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’

7. Example plasmid: Origin of Replication: the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate where the plasmid starts to duplicate itself

8. Restriction enzymes can create “sticky ends or “blunt ends”

9. Sticky Ends fragment end of a DNA molecule with a short single-stranded overhang Blunt Ends fragment end of a DNA molecule with no overhang Once made, the ends can be re-joined together by other enzymes ("enzyme glue")

10. To Make a Recombinant Plasmid: 1.Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. Insert 2.Combine the sticky ends using ligase. ligase: enzyme used to join DNA together 3. Introduce the recombinant plasmid into bacteria.

11. Making a Recombinant Plasmid

12. Ways to make recombinant DNA - Bacterial Transformation - - - - - - - - + + + + + - ++ -++ - - ++ + + + + + +-++-+ phospholipid bilayer plasmid Ca 2+ ions introduction of foreign DNA into a bacterial cell plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell

14. Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

15. Part 2: Where do we get our insert sequence? From another organism’s DNA –ex. fish gene in strawberries, –jellyfish gene in plants

16. In order to do these things, we need a way to make many copies of the genes we want Use bacterial “factories” –easy to grow lots –no ethical issues –small genome is easy to manipulate

17. Using Bacteria as Production Factories

18. Common uses of biotechnology: 1.Making "stuff” proteins, enzymes, medication, etc. can be produced by engineered bacteria! 2.Genetic screening crime cases, relationship, genetic screening, etc.

19. First there was…..RFLP analysis Restriction fragment length polymorphism is a variation in a DNA sequence that is detectable through gel electrophoresis Was used to map the human genome (in part), for genetic fingerprinting, etc. before PCR (polymerase chain reaction) came along

20. Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

21. A pioneering method but….. It takes about one month for the process It requires a large amount of DNA Along came………

22. A quick and easy way to make lots of copies of the DNA we want Polymerase Chain Reaction (PCR)

23. Why PCR? Often a gene of interest is not in large enough quantities to analyze Much faster than cloning a certain gene or piece of DNA in bacteria

26. All happens in a test tube: 1st step: Denaturation – the break down of double-stranded DNA using heat 2 nd step: Annealing – primers attach to either end; lower temperature 3rd step: Extension - DNA polymerase extends the strand using nucleotides that have been added, medium temperature

27. And the test tubes are in this……

28. Application DNA fingerprinting for forensics and paternity Genetic testing for hereditary and infectious diseases

29. Move aside PCR Constant T PCR has been developed Recombinase polymerase amplification No need for sophisticated PCR equipment Video clip on PCR

30. Agarose Electrophoresis of DNA used to separate fragments of DNA DNA is negatively charged, charge is proportional to size agarose can be used as a molecular strainer (sieve) to separate the pieces of DNA by size

31. Gel electrophoresis A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

32. Gel Electrophoresis

34. DNA sequencing Used to determine the bases along a particular stretch of DNA Was developed during the time of the Human Genome Project – allowed it to proceed much faster

35. What do you need? First we need: DNA of interest, with known sequences at the ends DNA polymerase ATCG primers, modified A, T, C and G in each test tube. Party ice

36. How does it work? 1st. Double-stranded DNA is denatured 2 nd. Primers attach to known ends 3rd. DNA polymerase adds nucleotides 4th. All four nucleotides are mixed into testtubes with the DNA of interest

37. continued 5th. One of the four nucleotides mixtures has an ‘A’ that has been modied – the terminator. Then separate by running an agarose gell (electrophoriii