1. TOPICS IN (NANO) BIOTECHNOLOGY Lecture III 10th April PhD Course

2. Overview So we have looked at what is DNA and what is a gene. We also looked at DNA replication and protein synthesis, and the path from the gene to protein This week we will look at Recombinant DNA technology We will also look at the amplification of DNA and finally at its sequencing

3. History of Recombinant DNA technology Antibiotics such as penicillin, the sulfonamides and streptomycin gave much hope However, in the 50s theye starting to fight back, becoming increasingly resistant to antibiotics In just a few years 60-80% of bacteria showed resistance not just to one drug, but to multiple drugs The genes responsible for infectious drug resistance were plasmids, genetic elements that could replicate themselves independently. In different plasmids, the replication region encodes traits not essential to the bacterial host. Antiobiotic resistance is one of these traits.

4. History of Recombinant DNA technology In the late 60s, it was shown that CaCl 2 made the cells of E.coli permeable so that they could take up DNA, but could not grow E.coli cells with genetic property changes. In 1971 Cohen, exploited the antibiotic resistance of the plasmids to selectively enrich offspring that contained cell propogating plasmids. In late 1972, Berg reported on methods for joining fragments of DNA outside of cells. Endonucleases, or restrictions enzymes, would however, provide the tool for linking DNA.

5. Una cerveza y... In Nov. 1972, Herb Boyer and Cohen met up at a deli bar in Honololu, and discussed the endonuclease that Boyer was working on, and that night they dreamed of the collaborative project that would be the true start of recombinant DNA technology. In March 1973, the pair produced DNA fragments and joined them to plasmids using Boyer’s technique, and then introduced them into bacteria using Cohen’s technique. The first demonstration of DNA cloning had been achieved.

6. But let’s look at it in more detail....

7. Recombinant DNA technology

8. The two essential elements of recombinant DNA technology are: 1. Restriction endonucleases 2. Vectors for gene cloning

9. Restriction endonucleases

10. What is a restriction enzyme? There are two classes of restriction enzymes: Type I Cuts DNA on both strands but at non- specific location Random imprecise cuts Not very useful for rDNA applications Type II Cuts both strands of DNA within the particular sequence recognised by the restriction enzyme

11. What is a restriction enzyme? Restriction enzymes (or endonucleases) are bacterial enzymes that cut DNA at very specific sequences They generally cut in a ‘staggered’ manner, leaving sticky ends but some enzymes generate blunt ends (i.e. Cut DNA in the middle) Their biological function is to destroy invading foreign DNA

12. What is a restriction enzyme? Each bacteria has different restriction enzymes Enzymes from E.coli cells cut GAATTC/CTTAAG Enzymes from B. Amyloloquefaciens cut GGATCC/CCTAGG The restriction enzymes are named after the organism from which they were derived EcoRI from E.coli BamHI from B. Amyloloquefaciens

13. What is a restriction enzyme? Restriction enzymes are used to make recombinant DNA and gene cloning and genetic engineering were made possible by these enzymes Over 200 different restriction enzymes are commercially available (some are VERY expensive) DNA ligase ‘sticks’ the ends back together

14. What is a restriction enzyme?

15. Recombinant DNA technology can be used to isolate a genomic clone from DNA or for the isolation of human cDNA Isolating a genomic clone provides a piece of DNA identical in base sequence to the corresponding stretch of DNA in the cell and is often designed to contain a specific gene Isolating human cDNA is used for gene expression. Human cDNA (c=complementary) is double stranded DNA copy of mRNA but WITHOUT introns

16. Vectors for gene cloning

17. Vector requirements Dependent on design of experimental system Most vectors contain a prokaryotic origin of replication Antibiotic resistance genes and/or other selectable markers Examples of cloning vectors are plasmid bacteriophages yeast artificial chromosomes (YAC) bacterial artificial chromosomes (BAC) retrovirus

18. What is a plasmid? Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant They are ‘shuttle vectors’ to create, produce, and maintain recombinant DNA An example of one of the first plasmids is pBR322 Both Amp & Tet resistant, Several unique restriction sites pUC18 now the most commonly used Derivative of pBR322 Smaller, Higher copy number per cell, Multiple cloning sites

19. lacZ gene Gene encoding for enzyme  -galactosidase Polylinker resides in the middle Enzyme activity can be measured as marker of gene insertion WHITEDisrupted gene – nonfunctional – WHITE Intact gene – functional – BLUE Amp resistance gene still present, Tet resisitance gene omitted

20. What is a bacteriophage?

21. Lambda vector Bacteriophage lambda ( ) infects E.coli Double stranded linear DNA vector, suitable for library construction Can accomodate large segments of foreign DNA, central 1/3 is a ‘stuffer’ fragment Can be substituted with any DNA fragment of similar size Can accomodate  15kbp of foreign DNA

22. Recombinant DNA technology

24. Video 3a: Plasmid Cloning

25. Genes can be cloned in recombinant DNA vectors Cloning vector Procedure for cloning a eukaryotic gene in a bacterial plasmid 1. Isolation of vector and gene-source DNA 2. Insertion of DNA into the vector 3. Introduction of cloning vector into bacterial cells 4. Cloning of cells (and foreign gene) 5. Identification of cell clones carrying the gene of interest Nucleic acid hybridization Nucleic acid probe Genomic Clones

28. cDNA Clones Genes can be cloned in recombinant DNA vectors Cloning vector Procedure for cloning a eukaryotic gene in a bacterial plasmid Cloning and expression eukaryotic genes: problems and solutions 1.Difference in promoters Expression vector 2.Introns Complementary DNA (cDNA)

29. cDNA Clones

30. Cloned genes are stored in DNA libraries 1.genomic library – cloned set of rDNA fragments representing the entire genome of an organism 2.cDNA library - cloned set of rDNA fragments representing genes transcribed in a particular eukaryotic cell type (no introns, extrons etc) rDNA fragments generated, ligated & cloned The larger the fragments that are cloned, the smaller the size of the library Genomic and cDNA Libraries

31. Contains at least 1 copy of each fragment Screened using nucleic acid probes to identify specific genes Subcloning usually necessary for detailed analysis of genes N = ln (1-P)/ln (1-f) e.g. Human genome = 3.2 x 10 9 bp Lambda vector can accommodate 17kbp inserts N = ln(1-0.99)/ln(1-(1.7x10 4 bp insert/ 3.2 x 10 9 bp genome)) N = 8.22 x 10 5 plaques required in library Genomic Libraries

32. mRNA represents genes that are actively transcribed (or expressed) Eukaryotic mRNA – introns have been removed mRNA – converted into a DNA copy (cDNA) Size of library depends on number of ‘messages’ More complex than genomic library cDNA Libraries

33. Genomic Libraries

34. Libraries searched using specific probe Specificity extremely important Single-stranded nucleic acid fragments Radioactive vs non-radioactive Radioisotopes serve as tag - autoradiography Chemiluminescence, colorimetric, fluorescence Sources of probes Heterologous (other species) cDNA (genomic sequences with introns/promoter elements) Probe based on protein sequence 18-21 bases sufficient (ssDNA, RNA, antibodies) ID of specific DNA sequences

35. Expression Library Detect protein product of clone using antibodies Microarray technology ID of specific DNA sequences Chromosome walking If nearby sequences have been cloned, this can be used as starting point for isolation of adjacent genes

36. The PCR clones DNA entirely in vitro Polymerase chain reaction 1.Denaturation (heat to ~94 o C) 2.Annealing (37-72 o C) 3.Extension (72 o C) Polymerase Chain Reaction

37. Video

38. Separation of DNA fragments based on size, charge and shape differences Standardised MW markers run on the same gel for size comparison Agarose gel electrophoresis

39. Gel electrophoresis Video

40. DNA digested with restriction enzymes and separated by gel electrophoresis Gel treated with NaOH to denature DNA to ssDNA DNA transferred from gel to DNA binding filter DNA ‘fixed’by baking membranes/UV Incubate with ssDNA probe Autoradiography/chemiluminescence Southern blotting

41. Southern Blotting

42. DNA sequencing

43. Isolation, amplification & sequencing 3 videos

44. Exercises Describe a plasmid prep. What is meant by the term ‘sticky ends’? What is a genomic clone? What is a cDNA clone? What are the steps involved in making a genomic library? Explain in a few sentences the importance & principle of PCR. Explain in a few sentences the importance & principle of gel electrophoresis. Explain in a few sentences the importance & principle of southern blotting. Explain in a few sentences the importance & principle of DNA sequencing.