1. Biotechnology Lecture Notes Outline Biol 201 – K. Marr - Fall 2006 1.Overview of Recombinant DNA technologies a.Injection of DNA or a nucleus into a cell b.Gene Therapy c.“Pharm” Animals d.Genetic Modification of Plants (e.g. GM foods) e.Use of Prokaryotes to produce Eukaryotic gene products 2.Overview of various techniques a.Use of Restriction Enzymes & DNA Ligase to make recombinant DNA molecules b.Use of Gel Electrophoresis... To separate restriction fragments For DNA fingerprinting c.PCR (Polymerase Chain Reaction) 3.Strategies used to Genetically Engineer Bacteria How to isolate specific genes using.. RNA Probes Reverse Transcriptase 4.Human Gene Therapy using... a.Retroviruses b.Adenoviruses c.Liposomes d.Naked DNA

2. 1. Overview of Recombinant DNA technologies a.Injection of DNA or a nucleus into a cell b.Gene Therapy c.“Pharm” Animals d.Genetic Modification of Plants (e.g. GM foods) e.Use of Prokaryotes to produce Eukaryotic gene products

3. Injection of DNA or a nucleus into Cell Potential Applications 1.Germ line Gene Therapy—inject therapeutic gene into an egg cell (affects future generations) 2.Somatic Gene Therapy—Inject therapeutic gene into a somatic cell, culture & reinsert into an individual 3.Cloning—inject nucleus into an enucleated egg, culture & implant into a surrogate mother. Drawback: Inefficient means of gene transfer

4. Use of a Retrovirus for Gene Therapy Applications Somatic Gene Therapy to treat Gaucher Disease SCID’s “Bubble Boy” (Severe Combined Immune Difficiency)

5. Transgenic “Pharm” animals Potential Applications Genetically modify mammals to produce therapeutic peptide drugs (e.g. insulin, ) Isolate and purify drug from the milk Potentially a more cost effective method to produce pharmaceuticals

6. Using the Ti plasmid as a vector for genetic engineering in plants Potential Applications Genetically modify plants to... produce vaccines in their fruit (e.g. polio vaccine) be resistant to disease and pests require less fertilizer, pesticides and herbicides have a higher nutritional value

7. “Golden” rice contrasted with ordinary rice Transgenic Rice Genetically modify plants to produce beta-carotene Beta Carotene is converted to vitamin A in humans Vitamin A deficiency leads to poor vision and high susceptibility to disease ~70% of children <5 years old in SE Asia suffer from vit. A deficiency

8. Figure 20.2 An overview of how bacterial plasmids are used to clone genes

9. 2. Overview of various techniques a.Use of Restriction Enzymes & DNA Ligase to make recombinant DNA molecules b.Use of Gel Electrophoresis... To separate restriction fragments For DNA fingerprinting c.PCR (Polymerase Chain Reaction)

10. Using a restriction enzyme and DNA ligase to make recombinant DNA Figure 20.3

11. Gel Electrophoresis 1.A method of separating mixtures of large molecules (such as DNA fragments or proteins) on the basis of molecular size and charge. 2.How it’s done An electric current is passed through a gel containing the mixture Molecules travel through the medium at a different rates according to size and electrical charge: Rate  size and charge Agarose and polyacrylamide gels are the media commonly used for electrophoresis of proteins and nucleic acids.

12. Figure 20.8 Gel electrophoresis of macromolecules

13. Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

14. DNA fingerprints from a murder case Whose blood is on the defendant’s clothing?

15. PCR—Polymerase Chain Reaction A very quick, easy, automated method used to make copies of a specific segment of DNA What’s needed…. 1.DNA primers that “bracket” the desired sequence to be cloned 2.Heat-resistant DNA polymerase 3.DNA nucleotides 4.Thermocycler

16. The polymerase chain reaction (PCR) Figure 20.7

17. 3. Strategies used to Genetically Engineer Bacteria See fig. 20.2. An overview of how bacterial plasmids are used to clone genes 1.Isolate the gene of interest (e.g. insulin gene) 2.Insert the gene of interest into a bacterial R-plasmid R-plasmids are circular DNA molecules found in some bacteria that provide resistance to up to 10 different antibiotics 3.Place the transgenic plasmid into bacterial cells Plasmid DNA reproduces each time the bacteria reproduce 4.Culture the bacteria and isolate the gene product (e.g. insulin)

18. 3. Overview of how bacterial plasmids are used to clone genes Figure 20.2

19. Step 1. How to Isolate the Gene of Interest Use Reverse Transcriptase to make the gene of Interest Method #1 (see figure on next slide) 1.Isolate mRNA for the gene product of interest (e.g. Insulin mRNA) 2.Use Reverse Transcriptase to produce cDNA (complementary DNA) 3.Use PCR to clone the cDNA 3.Separate the synthetic gene of interest by electrophoresis

20. Use of Reverse Transcriptase to make complementary DNA (cDNA) of a eukaryotic gene

21. Step 1. How to Isolate the Gene of Interest Use Reverse Transcriptase to make the gene of Interest Method #2 1.Determine the primary structure (i.e. the amino acid sequence) of the protein of interest (e.g. insulin) with an automated protein sequencer 2.Use table of codons to determine the mRNA sequence 3.Synthesize the mRNA in the lab 4.Use Reverse Transcriptase to produce cDNA and PCR to clone the cDNA (as before) 5.Separate the synthetic gene of interest by electrophoresis

22. 1. How to Isolate the Gene of Interest Use a labeled DNA Probe to Isolate Gene of Interest (Southern Blot Method  see next slide) 1.Extract and purify DNA from cells 2.Cut DNA with restriction enzyme (e.g. Eco R1)  What’s a restriction enzyme? (fig. 20.3)  Note: Must cut outside of gene w/o too much “excess baggage” 3.Separate DNA fragments by gel electrophoresis 4.Transfer DNA from the fragile gel to a nylon sheet and heat to sep. strands (fig. 20.10) 5.Hybridize gene of interest with a radio-labeled DNA* or mRNA* probe and expose w/ film to locate gene  How do these probes work? (fig. 20.10) 6.Use PCR to clone the isolated gene of interest.

23. Figure 20.10 Restriction fragment analysis by Southern blotting

24. Steps 2 & 3. How to Insert the Gene of Interest into the R-Plasmid See next 3 figures and animation Lyse bacteria with detergent to release the R-plasmid (e.g. ampicillin resistance plasmid) Cut the plasmid with the same restriction enzyme used to isolate the gene of interest 3.Mix plasmid with gene of interest and join the two with DNA ligase  How does this work? 4.Add the recombinant plasmid to a bacterial culture  Induce bacteria to take up plasmid (transformation) 5.Grow bacteria on agar plate containing an antibiotic (e.g. ampicillin) 6.Isolate those bacterial colonies that contain the recombinant plasmid  How?  Only some of the bacteria take up a plasmid—How do you know which ones did?  Not all plasmids are recombinant plasmids—How do you find those that are?  Only some of plasmids contain the gene of interest—How do you identify these?

25. Using Plasmids to Create Recombinant DNA

26. 1.Digest a plasmid vector with a restriction enzyme (e.g. EcoRI) at a single site to produce two sticky ends. 2.Digest human DNA with EcoRI to produce pieces with the same sticky ends Use Human DNA or cDNA copied from mRNA using reverse transcriptase from retroviruses. 3.Mix the two samples and allow to hybridize. Some plasmids will hybridize with pieces of human DNA at the EcoRI site. 4.Use DNA ligase is used to covalently link the fragments.

27. Insertion of Recombinant Plasmids into Prokaryotic Cells 1.Only some of the bacteria take up a plasmid—How do you know which ones did? 2.Not all plasmids are recombinant plasmids—How do you find those that are? 3.Only some of plasmids contain the gene of interest—How do you identify these?

28. Identification of cells containing plasmids Cells containing plasmids contain the ampicillin resistance gene Grow cells on medium containing ampicillin How do you know which colonies contain the gene of interest? Use a DNA probe (see fig. 20.5)

29. Figure 20.5 Using a DNA probe to identify a cloned gene in a population of bacteria

30. Step 4. Culture Bacteria and Isolate Gene Product Grow the recombinant bacteria in nutrient broth and isolate/purify the gene product from the broth Expensive to do, therefore mammals (e.g. cows and goats) are now being genetically modified to produce desired gene products in their milk!!

31. Human Gene Therapy using... a.Retroviruses b.Adenoviruses c.Liposomes d.Naked DNA

32. Use of a Retrovirus for Gene Therapy Applications Somatic Gene Therapy to treat Gaucher Disease SCID’s “Bubble Boy” (Severe Combined Immune Difficiency)

33. Basic Strategies of Human Gene Therapy (1 of 2) 1.Isolate and then clone the normal allele by PCR 2.Insert normal allele into a disabled virus Retroviruses and adenoviruses are the most common vectors Retroviruses are much more efficient at forming a provirus, but have a greater chance of mutating to cause disease Adenoviruses are safer, but are relatively inefficient as a vector Liposomes (lipid spheres) are also used as vectors  e.g. Gene therapy for Cystic Fibrosis involves using an inhaler to bring liposomes containing the CFTR gene to the cells lining the lungs) 3.Infect host cells with recombinant virus

34. a.Add recombinant virus directly to individual e.g. Jesse Gelsinger—  Had Ornithine Transcarbamylase Deficiency; Causes build up of ammonia in liver cells since they cannot convert the ammonia (toxic) produced by amino acid metabolism to urea (less toxic)  Died in Sept.’99 due to a severe immune response to the genetically modified adenovirus containing the OTC gene b.Isolate host cells from body and then add recombinant virus (e.g. blood stem cells in gene therapy for Gaucher disease) Inject genetically engineered cells back into the body Basic Strategies of Human Gene Therapy (2 of 2)

35. Figure 20.6 Genomic libraries

36. Figure 20.11 Chromosome walking

37. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 1)

38. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 2)

39. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 3)

40. Figure 20.12 Sequencing of DNA by the Sanger method (Layer 4)

41. Figure 20.13 Alternative strategies for sequencing an entire genome

42. Table 20.1 Genome Sizes and Numbers of Genes

43. Figure 20.14a DNA microarray assay for gene expression

44. Figure 20.14b DNA microarray assay for gene expression

45. Figure 20.15 RFLP markers close to a gene