BCM302 Food and Beverage Biotechnology

1. BCM302 Food and Beverage Biotechnology Topic 3: Basic Principles of Recombinant DNA technology

2. Learning objectives 1. Know the function of restriction endonucleases, how they work to cut DNA, and why they are important in biotechnology. Compare blunt ends with sticky ends. 2. Know the mechanism by which electrophoresis separates pieces of DNA. 3. List and know the steps of DNA cloning. 4. Know how vectors are used to transform bacteria, and know the methods of selecting for successfully transformed bacteria. Compare the types of vectors in terms of the sizes of DNA that can be carried by the vector into the bacteria. 5. List the types of vectors that can be used to transform yeast mammalian cells and plants, and why they are effective in those organisms. 6. List the methods of transformation of cells.

3. Learning objectives 7. Compare genomic libraries, cDNA libraries, and expression libraries in terms of how they are constructed, what the libraries are looking for, and how they are screened. 8. List the various types of reporter genes used in research. 9. Compare Northern and Southern blot hybridization in terms of how they are constructed and what each type of hybridization is looking for. 10. Know the function of PCR, the steps of PCR, and what PCR allows researchers to accomplish. 11. Compare the two methods of DNA sequencing: the chemical method and the Sanger method, and know which method is more widely used. How does automation impact DNA sequencing? 12. List and define the various methods of analyzing proteins. Are any of these methods similar to DNA methods? 13. Know the types of microarrays, and how DNA and protein microarrays work. 14. List the applications of recombinant DNA technology.

4. Recombinant DNA Technology: Promise and Controversy • What is DNA cloning? • Applications: • New medical diagnostics and treatments • Better vaccines • Stress-resistant crops • More nutritious food • Healthier livestock • Cleaner environment

5. Recombinant DNA Technology: Promise and Controversy • Criticisms: • Where to draw the line (human cloning, organs for donation, selecting characteristics in engineered children) • Effect on health, hidden dangers, environmental pollution (eg escaping genes and organisms) • Tampering with the “natural world” • Solutions?: • Open communication

6. Cutting and Joining DNA • Recombinant DNA: When two pieces of DNA are joined together to form a new DNA molecule • Cloning:Insertion of DNA molecules in bacteria so that many identicalcopies of the DNA are made • Gene expression: DNA can be transcribed and protein can be translated within cell

7. Restriction enzymes • Also called restriction endonucleases • Cut DNA at “restriction sites or “restriction sequences” • Cut across the sugar-phosphate backbone of DNA

8. Restriction enzymes • Palandromic, Four-six bases in length

9. Restriction enzymes • Derived from bacteria • Natural role: defence against bacteriophage • Bacterial DNA protected by methyl groups on adenine or cytosine

10. Restriction enzymes • Blunt ends: enzyme cuts directly across the two strands • Sticky ends: enzyme cuts strands in different places, leaving a short single stranded piece of DNA hanging over the end of the molecule

11. Restriction enzymes • Blunt vs Sticky

12. Separating restriction fragments • Digestion of DNA with Restriction enzymes may result in fragments of different sizes which need to be separated • DNA passed through a “molecular sieve” to separate molecules of different sizes

13. Agarose gel • Agarose: polysaccharide derived from seaweed • Powder mixed with buffer, heated and forms a solid gel when cooled • “Wells” formed in gel during cooling for sample loading

14. Electrophoeresis • Electric charge applied to allow DNA to migrate (towards +ve electrode) • DNA slightly negative due to sugar-phosphate backbone • Large molecules migrate slower • Small molecules migrate faster • Size of pores determined by Agarose concentration • Lower % = larger pores = better separation for larger molecules • Higher % = smaller pores = better separation for smaller molecules

15. Visualizing DNA • Ethidium bromide:binds to DNA and fluoresces when exposed to UV light • Size of DNA fragments determined by comparing with fragments of know sizes • DNA fragments migrate at a rate that is inversely proportional to the logarithm of the size of the fragment (in base pairs)

16. Agarose gel Electrophoresis

17. Acrylamide gel electrophoreis • Gel formed using polyacrylamide • Vertical • Used for smaller molecules (eg DNA sequencing)

18. DNA cloning • Isolation of DNA • Ligation of the DNA to a vector • Transformation of a host cell with the recombinant DNA • Selection of host cells containing the DNA

19. Isolation of DNA • Cleaved from a larger piece of DNA • Generated using the Polymerase Chain Reaction (see later)

20. Ligation • DNA ligated to a cloning vector • Enzyme: Ligase • Requires compatible ends

21. Cloning Vectors • Transport molecule so DNA can be replicated in cell: • Features: • Origin of replication (Ori) • Small • Multiple cloning site (MCS) with unique RE sites • Selectable marker (to determine which bacteria contain the vectors with the inserted DNA)

22. Bacterial vectors (Plasmids) • Extrachromosomal pieces of DNA not necessarily needed by bacteria • Natural roles eg antibiotic resistance pigment production • Engineered to accept DNA fragments up to 10 kilobases in length • High copy number and low copy number

23. Plasmid: pBR322 • Contains unique REs • Antibioticresistance (to determine which bacteria contain vectors)

24. Plasmid: pUC18/19

25. Alpha complementation • lacZ gene codes for beta-galactosidase • Beta-galactosidase breaks down X-gal to produce a blue product • If molecule cloned into MCS (in lacZ gene) X-gal will not be broken down and colony will be white

26. Colony selection

27. Other vectors • Bacteriophage • virus that infects bacteria • Cosmids • Has aspects of both plasmid and bacteriophage • Yeast Artificial Chromosome (YAC) • Contains centromeere, telomere, autonomously replicating sequence (ARS), selectable marker gene • Bacterial Artificial Chromosomes (BAC) • Created using a small plasmid with a F (fertility) factor that allows the vector to accommodate larger pieces of DNA (up to 25% of the size of the bacterial Chromosomes)

28. Plant Cloning Vector • Tumor inducing (Ti) plasmid • Replicates in bothE.coli. and Agrobacterium • Facilitates plant transformation

29. Cell transformation • Transformation: insertion of DNA into a cell • Methods: • Calcium Chloride/heatshock (Bacteria) • Electroporation (Bacteria, mammalian, protoplasts) • Microinjection (Mammalian) • Biolistics (Plant)

30. Plant Transformation • DNA coated onto small particles (eg gold or tungsten) • Accelerated using a particle gun

31. DNA Libraries • Used to help map genomes • Can be screened to find specific genes • Different types: • Genomic library • cDNA library

32. Genomic Library • Digest genomic DNA and plasmid • Ligate DNA to vector • Transform bacteria with recombinant vectors • Screen

33. cDNA Library • A DNA library constructed from cDNA • cDNA (complementary DNA): a DNA copy of messenger RNA (mRNA)

34. cDNA synthesis • Pre-mRNA is transcribed by cell • Introns are removed • A single stranded cDNA copy is made using reverse transcriptase

35. cDNA synthesis (con’t) • mRNA is degraded • Second DNA strand synthesised using DNA polymerase

36. cDNA Library • cDNA can be derived from RNA isolated from specific organism, tissue or treatment • cDNA ligated to vector as per genomic DNA

37. Screening libraries • Bacterial colonies can be screened for the presences of specific: • DNA (southern hybridisation) • RNA (northern hybridisation) • Proteins (western hybridisations)

38. Colony Hybridisaton • DNA transferred to membrane • Membrane probed with complementary DNA

39. Probe hybridisation • DNA probe with complementary sequence will bind to DNA from colony

40. Reporter genes • Connected to gene of interest to study expression pattern • Eg: Luciferase, GFP, GUS

41. Southern Hybridisation • Invented by Edward Southern in the mid-1970s • Allows the detection of a DNA fragment in a large population of molecules

42. Southern Hybridisation • Steps: • DNA fragments can be separated on a gel and denatured • Fragments transferred to a nylon or nitrocellulose filter • Radioactive DNA probeshybridised to membrane • Position of radioactivity (detected by autoradiography) indicates position of DNA of interest

43. Southern Hybridisation

44. Southern Hybridisation

45. Southern Hybridisation

46. Northern Hybridisation • Similar to Southern hybridisation except RNA used instead of DNA • Used to assess levels of gene expression

47. Western hybridisation • Similar to Southern and Northern Hybridisation except it involves detection of proteins with antibodies

48. Polymerase chain reaction (PCR) • Amplification of specific pieces of DNA • Three steps (cycles) repeated 25-40 times: • Denaturation (Double stranded melted) • Annealing (primers bind to specific seq.) • DNA synthesis (DNA replicated)

49. Polymerase chain reaction

50. Polymerase chain reaction • Primers: • Short pieces of single stranded DNA (oligonucleotides) complementary to sequence to be amplified • DNA polymerase: • Enzyme that can synthesise DNA suing a DNA template • Requires double stranded molecule to initiate synthesis