1. Bi430/530 Theory of Recombinant DNA Techniques First part of course: Technical aspects of molecular biology work--Molecular Cloning Second part of course: 1) Applications of molecular biology techniques 2) Emerging science 3) Bi530 student presentations Prerequisite: Molecular Biology (Bi 338) FRIDAY February 8: Midterm exam THURSDAY March 20: Final exam

2. Syllabus

3. Syllabus--first half The basics of DNA manipulation (and the Molecular Cloning Manual)

4. “Molecular Cloning” (2001), Sambrook and Russel (3rd ed.) See also Course Reading #2 (detailed table of contents) in course packet

5. Syllabus--second half Using DNA manipulation techniques

8. Recombinant DNA

9. Information flow in the cell DNARNAprotein

10. DNARNAprotein Replication/mutation Natural selection Evolution: a dialog between the genome and its environment

11. environment DNARNAprotein Dynamic, immediate, transient modification of DNA program Organisms respond to their environment via information from sensory input and changes in gene expression

12. DNARNAprotein evolution --Stable (“permanent”) --reflects effect of environment over large time scales environment Species respond to environment over long time frames via mutations in the DNA program

13. environment DNARNAprotein Human intervention: genetics (indirect), rDNA (direct) Human activity: transient modifications of environment, permanent modifications of DNA program

14. 2006: 53 years of DNA structure Rosalind Franklin and Maurice Wilkins: X-Ray fiber diffraction pattern of pure B-form DNA (1953) James Watson and Francis Crick: Proposed two antiparallel, helical strands forming a stable duplex with DNA bases on interior of the molecule, joined by hydrogen bonds (1953) But DNA was not discovered in 1953--it had been known as the element of genetic transmission at least since 1947, when Avery showed that DNA could “transform” bacterial colony morphology Why was the structure so important?

15. Structure of DNA To Watson and Crick, the structure suggested: --Mechanism for replication --Stability for information storage, yet accessing the information not difficult The DNA structure provided a new template for hypotheses regarding biological phenomena (amenability to study)

16. DNA is easy to work with… Readily isolated--plasmid isolation, PCR Stable--not chemically reactive like RNA (even archaeologically stable!) Easy to propagate and move from cell to cell Easy to make specific constructs Easy to make specific mutations Very easy to sequence (record-keeping) Predictable behavior Sequence lends itself to analysis--genome projects

17. DNA is very easy to sequence Etc….

18. http://www.genomesonline.org/

19. The behavior of DNA (genes) is predictable Gene sequence conservation often indicates functional similarity Non-protein coding information sequences can often be detected by homology (promoters for transcription initiation, transcription terminators, ribosome binding sites, DNA binding protein binding sites) the genetic code

20. The genetic code and the roots of biotechnology 1961 Marshall Nirenberg and Heinrich J. Matthaei: polyU mRNA encodes poly-phenylalanine 1966 Nirenberg and colleagues had deciphered the 61 codons (and 3 nonsense codons) for all 20 amino acids 1968 Nobel prize for Nirenberg, Holley, and Khorana

21. 1966 George and Muriel Beadle write: “The deciphering of the DNA code has revealed our possession of a language much older than hieroglyphics, a language as old as life itself, a language that is the most living language of all-- even if its letters are invisible and its words are buried deep in the cells of our bodies.”

22. The public reaction to the deciphering of the genetic code Wow “…just as big a breakthrough in biology as [Newton's discovery of gravitation in the seventeenth century] was in physics.” --John Pfeiffer, journalist, 1961 Optimism “No stronger proof of the universality of all life has been developed since Charles Darwin's 'The Origin of Species' demonstrated that all life is descended from one beginning. In the far future, the hope is that the hereditary lineup will be so well known that science may deal with the aberrations of DNA arrangements that produce cancer, aging, and other weaknesses of the flesh.” Chicago Sun-Times, 1962

23. Caution …knowledge gained from the genetic code “might well lead in the foreseeable future to a means of directing mutations and changing genes at will.” 1961, A. G. Steinberg of Case Western Reserve University …knowledge of the genetic code could “lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions.”1961, Arne Wilhelm Kaurin Tiselius, 1948 Nobel Laureate in Chemistry

24. 1967 “Will Society Be Prepared?” Marshall Nirenberg, editorial in Science (see WebCT)

25. "When man becomes capable of instructing his own cells, he must refrain from doing so until he has sufficient wisdom to use this knowledge for the benefit of mankind.... [D]ecisions concerning the application of this knowledge must ultimately be made by society, and only an informed society can make such decisions wisely." Nirenberg, 1967

26. Response from Joshua Lederberg, 1967 (see letter on WebCT): (paraphrased) -- We need to be particularly careful with manipulation of the germ cell lines (heritable changes). -- Considerations governing control of our biology are equally important to considerations governing control of our cultural institutions (given that culture is mutable and heritable)

27. http://nobelprize.org/nobel_prizes/chemistry/arti cles/berg/index.html 1975: The Asilomar Conference on Recombinant DNA 1974: moratorium on recombinant DNA research “…new technology created extraordinary novel avenues for genetics and could ultimately provide exceptional opportunities for medicine, agriculture and industry…. …concerns that unfettered pursuit of this research might engender unforeseen and damaging consequences for human health and the Earth's ecosystems”

28. 1975: The Asilomar Conference on Recombinant DNA Conference included internationally prominent scientists, government officials, doctors, lawyers, members of the press Conclusion: “… recombinant DNA research should proceed but under strict guidelines.” The moratorium was lifted, and “… guidelines were subsequently promulgated by the National Institutes of Health and by comparable bodies in other countries. ” http://nobelprize.org/nobel_prizes/chemistry/arti cles/berg/index.html

29. The Asilomar principles: 1)containment should be made an essential consideration in the experimental design 2)the effectiveness of the containment should match the estimated risk as closely as possible. Additional suggestions: Use biological barriers to limit the spread of recombinant DNA fastidious bacterial hosts that are unable to survive in natural environments nontransmissible and equally fastidious vectors (plasmids, bacteriophages, or other viruses) that are able to grow in only specified hosts

30. The Asilomar principles: Safety factors physical containment, exemplified by the use of hoods or where applicable, limited access or negative pressure laboratories strict adherence to good microbiological practices, which would limit the escape of organisms from the experimental situation education and training of all personnel involved in the experiments would be essential to effective containment measures.

31. Regulation of biotechnology: US National Institutes of Health (NIH) Guidelines stipulations of biosafety and containment measures for recombinant DNA research delineations of critical ethical principles and safety reporting requirements for human gene transfer research See http://www4.od.nih.gov/oba/Rdna.htm http://www4.od.nih.gov/oba/Rdna.htm (see also CR #3 in course packet)

32. “Unnatural Selection” (first reading in the course packet) By Allison Snow Is the process for altering genes (evolution vs. human): irrelevant? Product (transgenic organism): is the phenotype the only thing that is important? Reverberations from introduction of modified organisms? Spread of gene? Effects of spread? Success of organism? Effects on other organisms? “The technology’s main hazards are probably yet to manifest themselves”

33. What can we do with recombinant DNA technology? begin to learn how cells, tissues, organisms, communities work, interact, respond to the environment (gain scientific knowledge) improve human health industrial production of useful enzymes, metabolic products improve industrial process raise agricultural productivity investigate problems of geneology, paternity, anthropology, archaeology investigate criminal cases etc….

34. How is recombinant DNA technology useful in medicine?  Diagnosis of disease  Animal models for human diseases  Therapies  nucleic acids: gene therapy  pharmacologically active proteins  small molecule design and testing  Antimicrobials  Vaccines  Microbicides

35. The biotechnology industry is very new Case in point: Genentech (S. San Francisco) 2006: Genentech to open production facility in Portland (2010)

36. Day 1 summary: 1) The simplicity of a DNA-based information system makes genetic manipulation possible 2) This represents an unprecedented level of interaction with living systems 3) Benefits and costs of technology require continuous assessment

37. Visualizing DNA (and RNA, protein): non-specific detection methods I. Quantitation of DNA (Course Reading 4) II. Electrophoresis (Course Reading 5 ) III. Visualizing DNA (& protein) in gels (Course Reading 6)

38. Quantitation of DNA by UV absorbance Measure absorbance of UV light by sample (the aromatic bases have a characteristic absorbance maximum at around 260 nanometers) 1.0 A 260 (1 cm light path) = DNA concentration of 50 micrograms per ml (double stranded DNA) or 38 micrograms per ml (single-stranded DNA or RNA) the effective range for accurate measurement is rather narrow: A260 from 0.05 to 2.0 (DNA concentrations from 2.5 to 100 micrograms/ml) Sample must be very pure for accurate measurements (RNA, EDTA and phenol all absorb at 260 nm)

39. How can concentration be determined by absorbance? DNA has a characteristic “molar extinction coefficient”  The Beer-Lambert law: I = I o 10 -  dc I = intensity of transmitted light I o = intensity of incident light  = molar extinction coefficient d = optical path length c = concentration of absorbing material How much light gets through a solution depends on what’s in it and how much of it there is

40. The Beer-Lambert law: I = I o 10 -  dc Absorbance A measured by a spec is log I/I o When path length d = 1 cm, A is called the optical density OD If you know the , the absorbance of a solution will tell you the concentration: OD =  c  for nucleic acids: dsDNA: 6.6 ssDNA, RNA: 7.4 (but these values change with pH and salt concentration!)

41. 0.5 0 Absorbance (1 cm path length) Wavelength (nm) 200 260 400 A typical (good) “scan” (multiple wavelengths) of a DNA sample A 260 = 0.327 A 260 /A 280 : 1.8 is good (lower values indicate significant protein contamination)

42. Example: sample of 250 base pair fragment of DNA has an A 260 =.327 What is its molar concentration? Given: (1.0 A = 50 micrograms/ml DNA) DNA conc. =.327 x 50 = 16.35 micrograms/ml MW of an average bp. = 650 Daltons Therefore 250 bp. Fragment has a MW of 1.6 x 10 5 Daltons Solve for molarity: 1.02 x 10 -7 M, or 102 nanomolar (nM) Important to know how to do this calculation How does A 260 give you the quantity of DNA?

43. 1 ml 16.35 micrograms 1000 ml 1 L 10 6 micrograms 1 gram What is the molarity of a 16.35 microgram/ml solution of a 250 base pair DNA fragment? 1.6 x 10 5 grams 1 mole 1.02 x 10 -7 molar 0.102 x 10 -6 molar [0.1 micromolar (  M)] 102 x 10 -9 molar [102 nanomolar (nM)]

44. Fluorometry: another method for quantitation of DNA Hoechst 33258 (a fluorescent dye) Binds to DNA in the minor groove (without intercalation) Fluorescence increases following binding Good for quantitation of low concentrations of DNA (10-250 ng/ml) rRNA and protein do not interfere But you need a fluorometer

45. Another method for quantitation of DNA: Ethidium bromide (fluorescent dye) binding Compare sample DNA fluorescence to standards of known concentration (dilution series) In solution *or* using gel electrophoresis A commercially available quantitative DNA standard

46. Visualizing DNA: Electrophoresis Allows separation of biomolecules (DNA, RNA, protein) on basis of size A separation matrix, or gel (agarose or polyacrylamide), is saturated with an electrically conductive buffer Samples are loaded, an electric field is applied, and negatively charged biomolecules in the sample travel toward the cathode The larger the molecule, the slower the travel through the gel matrix Dyes allow a visual estimate of the rate of travel through the gel The choice of matrix depends mainly on the size of DNA being analyzed

47. Agarose gels Agarose: a polysaccharide polymer of alternating D- and L- galactose monomers, isolated from seaweed Pore size is defined by the agarose concentration (higher concentration, slower DNA migration overall) The conformation of the DNA (supercoiled, nicked circles, linear) affects the mobility of the DNA in gels Rate of DNA migration is affected by voltage (5 to 8 Volts/cm is close to optimal) Agarose comes in a myriad of types (variable melting temperatures, generated by differential hydroxyethylation of the agarose)

48. Agarose gels Standard gels can separate DNA fragments from 100 bp to about 20,000 bp Pulsed-field gels separate very large DNA fragments (up to 10,000,000 bp, or 10 Mb) This apparatus allows periodic shifts in the direction of DNA migration: 120° refers to the reorientation angle (difference between orientation of electric fields A and B

49. - + time of electrophoresis (progress monitored by marker dyes) Load samples in wells bromophenol blue xylene cyanol Typical agarose gel (the DNA fragments are not visible without some sort of staining) 

51. Polyacrylamide gels Acrylamide monomers (toxic!) polymerized to form gel matrix The gel structure is held together by the cross-linker-- usually N, N'-methylenebisacrylamide ("bis" for short) Pore size defined by concentration of gel (total percentage) and concentration of the crosslinker (bis) relative to acrylamide monomer Very high resolution (better than agarose) Suitable for separation of nucleic acids from 6 to 1000 base pairs in length

52. Polyacrylamide gels Native gels (DNA stays double-stranded) Denaturing gels--run in the presence of high concentrations of denaturant (usually urea) and at high temperature: DNA is single stranded (sequencing gels) (also useful in separation of proteins, when proteins are treated with SDS, which denatures proteins and gives a uniformly negative surface charge)

53. Recipe for a polyacrylamide gel: Acrylamide (anywhere from 4 to 20 %, depending size of nucleic acids or proteins in the gel) Bis-acrylamide (the ratio of Bis to regular acrylamide is important) Water Buffer To initiate polymerization, add APS: Ammonium persulfate -- generates free radicals needed for polymerization TEMED: N,N,N’,N’ - tetramethylethylenediamine -- accelerates free radical generation by APS

54. More about gels There has to be a buffer (for carrying current) TAE (Tris-acetate-EDTA): good resolution of DNA, but buffering capacity is quickly depleted TBE (Tris-borate-EDTA): High buffering capacity, resolution is pretty good Use gel loading “buffers” (relatively simple) Dense material to carry sample to bottom of wells (sucrose, glycerol, or ficoll) Dyes for tracking progress of electrophoresis Bromophenol blue: fast migration Xylene cyanol: slow migration Occasionally denaturant is present (formamide) for denaturing gels (e.g. sequencing gels)

55. Protein electrophoresis oAlmost always polyacrylamide based oThe anionic detergent SDS (sodium dodecyl sulfate) is used to denature the proteins, giving each protein a “uniform” negative charge oProtein separation occurs as a function of size oDiscontinous Tris-Cl/glycine buffer system: oStacking gel: pH 6.8, low polyacrylamide concentration, focuses proteins into thin layer (gives higher resolution upon separation) oSeparating gel: pH 8.8, separates proteins on the basis of size

56. Polyacrylamide gel set up (protein gels) Stacking gel: at low pH, glycine is protonated (no neg. charge), Cl - ions at the leading edge, glycine trailing, steep voltage gradient in between, that’s where the proteins get “focused” into a thin band Separating gel: at higher pH, glycine deprotonates, runs with the Cl - at the leading edge, and the proteins separate based on size

57. ethidium bromide, an anti-trypanosomal drug for cattle Stain works by intercalating in stacked base pairs, elongates DNA helix Fluorescence increases upon DNA binding Stained bands visualized by UV illumination (302 or 260 nm) Staining nucleic acids Ethidium bromide G-C base pair

58. Example of an agarose-DNA gel, Stained with ethidium bromide Direction of electrophoresis Fragments of bacteriophage genomic DNA (48 kb) cut with the restriction enzyme Hind III The fragments are equimolar-- why is the band intensity different?

59. Another ethidium bromide-stained agarose gel The marker lane (M) gives size standards for comparison with the sample lanes M samples

60. Other methods for staining DNA SYBR gold (Molecular Probes, Eugene, OR), more than 10-fold more sensitive than ethidium bromide for detecting DNA, but expensive! methylene blue: not toxic, but the staining protocol is time consuming, and sensitivity somewhat lower than ethidium bromide silver staining: high degree of sensitivity, but the protocols are time consuming, and proteins are also stained by silver

61. Protein detection in gels Coomassie Brilliant Blue R-250: dye from the textile industry that has a high affinity for proteins –Proteins in gels must be “fixed” (rendered insoluble) first with acetic acid/methanol –Dye probably interacts with NH 3 - groups of the proteins, also through van der Waals forces http://www.galab.de/laboratories/services/biopharma/img/sds.jpg

62. Protein detection in gels Silver staining: –100 to 1000-fold more sensitive than Coomassie stain for detecting proteins (need far less sample to see it on a gel) –Process relies on differential reduction of silver ions bound to amino acid side chains (like the photographic process) –There is protein-to-protein variability of staining goodbad (overstained) ugly (keratin)

63. Protein detection in gels Sypro Ruby (Molecular Probes inc, proprietary compound) –As sensitive as silver staining, less variability –Fast protocol –Expensive 1 nanogram of protein Standard gel 2-D gel

64. Visualizing DNA (and RNA, protein): non-specific methods I. Quantitation of DNA (Course Reading 4) II. Electrophoresis (Course Reading 5 ) III. Visualizing DNA (& protein) in gels (Course Reading 6)

65. A. Southern blots (DNA-DNA hybridization)(Methods for labeling “probe” DNA) CR7, MC 6.33 - 6.38 B. Northern blots (DNA-RNA hybridization) CR8, MC 7.21 - 7.26, MC 7.82 - 7.84 C. Western blots (detection of proteins with specific antibodies) CR9, MC A9.28, MC A8.52-A8.55 Methods for detecting specific biomolecules

66. Visualizing DNA, RNA and Protein: detecting specific sequences or proteins Techniques allow one to distinguish specific sequences or proteins in a large, mixed population, e.g. in cell extracts or genomic DNA preparations For DNA and RNA, specific sequence detection is based on DNA and RNA complementarity and base-pairing For proteins, the specific detection is based on antibodies that recognize the protein of interest (or based on a specific assay for activity of the protein)

67. Detecting specific DNA sequences: the Southern blot

68. Agarose or Polyacrylamide gel nitrocellulose or nylon membrane boundary: DNA binds to it A typical capillary blotting apparatus. Electroblotting is also commonly used Immobilization of nucleic acids

69. DNA is fixed to the nylon membrane by: –Baking, 80°C –UV crosslinking (links thymines in DNA to + charged amine groups in membrane), DNA only Probe to detect sequence of interest by base-pairing (hybridization) –Obtain probe DNA: synthetic oligonucleotide or cloned gene (single stranded) –Label probe for later detection Radioactivity Non-radioactive label Southern blotting: Immobilization of target DNA and detection

70. Use T4 polynucleotide kinase --catalyzes the transfer of the gamma phosphate of 32 P ATP to the 5’ end of DNA fragment to be used as a probe 32 P is a high energy beta particle emitter, and provides good sensitivity for detection of hybridization between the probe DNA and the target (blot) DNA Detect radiolabel with --autoradiography (X ray film) --phosphorimager (phosphor coated plates store the energy of the radioactive particle, laser excitation releases photons of light that are collected and represented as a picture, greater dynamic range than film, and faster too Radioactive probes: 32 P labeling

71. …e.g. horseradish peroxidase oxidation Non-radioactive labels

72. …or digoxygenin/antibody-conjugated HRP can also use biotinylated DNA probe oxidation Non-radioactive labels

73. blocking agents (e.g. milk, SDS) prevent non-specific interactions between probes and membrane Volume exclusion agents (eg. dextran sulfate) increase rate and level of hybridization Wash blot with increasing stringency… –Low stringency: high salt, low temperature, probe binds to sequences with mismatches –High stringency: low salt, higher temp., probe binds only to fully complementary sequences Hybridize probes to membranes

74. Southern Blot--one example (RFLPs) (or PCR fragment)

75. Same basic technique as Southern blots, but RNA is run on the initial gel and is transferred to the membrane. Use this method to measure levels of gene transcription in vivo (detecting changes in the levels of RNA transcript under differing conditions) Microarrays for measuring mRNA abundance are based on this principle, but many probes are immobilized in a regular array -- reverse transcribed (and fluorescently labelled) RNA “lights up” the probes on the microarray Northern blots:

76. Proteins are transferred to membranes using the same principle as Southern blots Specific proteins detected by probing blot with antibodies to protein of interest Antibody binding is detected by antibody to the original antibody that has enzyme (horseradish peroxidase, alkaline phosphatase) or radioactivity ( 125 I) conjugated to it Western blots: proteins

77. 1) Separate DNA, RNA, or proteins on the basis of size (gel electrophoresis) 2) Immobilize the separated DNA, RNA, or protein 3) “Probe” the blot with something that will specifically interact with a target a) DNA and RNA: complementary nucleic acid b) Protein: antibody to that protein Methods for detecting specific biomolecules