Bi430/530Theory 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 techniques2) Emerging science3) Bi530 student presentations
Prerequisite: Molecular Biology (Bi 338)
FRIDAY February 8: Midterm examTHURSDAY March 20: Final exam
Syllabus
RECOMBINANT DNA TECHNIQUES BI 410 and BI 510 MWF 11:30 -12:35
UTS Room 507
INSTRUCTOR Justin Courcelle
725-3866 [email protected]
COURSE DESCRIPTION A study of techni ques and app roac hes
used in DNA mani pulation ge netic engin eering.
Te xtboo k: The “Cour s e Re adings” (CR) packet is availab le a t Sm a rt Copy, 1915 S W 6 th Ave.
Additional re ad ings will be ha nde d out in class an d/or pos ted on the cour s e we bsite. Web si te s : Course home pa ge http://w e b.pdx. e du/~justc/cours e s Office ho urs : M 1:00-3:00 SB2 Rm 432 or by appointme nt
Syllabus--first halfThe basics of DNA manipulation(and the Molecular Cloning Manual)
Week Date Topic Course
Readings 1 Jan 07 Overview, DNA manipulation and safety, intro to Molecular Cloning
manual 1-3
Jan 09 Visualization and detection of DNA, RNA, and protein 4-6 Jan 11 Detection of DNA, RNA, and protein 7-9 2 Jan 14 Isolating DNA, isolating RNA 10-11 Jan 16 PCR and its applications 12-13 Jan 18 DNA sequencing—methodology 14 3 Jan 21 No Class Jan 23 DNA sequencing—bioinformatics (PROB SET 1 HANDED OUT) 15 Jan 25 Genomics and Porteomics: massively parallel measurements 15 4 Jan 28 Manipulating DNA—cutting and pasting 16-18 Jan 30 Manipulating DNA—DNA enzymes and their utility (PROB SET 1 DUE) 19-20 Feb 01 Mobilizing DNA: Plasmids and transformation 21-22 5 Feb 04 Mobilizing DNA: Phages, strain construction, large DNA 23-24 Feb 06 Mobilizing DNA: Specialized vectors 25-27 Feb 08 EXAM I
“Molecular Cloning” (2001), Sambrook and Russel (3rd ed.)
See also Course Reading #2 (detailed table of contents) in course packet
Syllabus--second halfUsing DNA manipulation techniques
6 Feb 11 Gene cloning: Genomic DNA and “library” construction 28-29 Feb 13 Gene cloning: Screening for genes 29 Feb 15 Mutagenesis, Protein engineering and altering the genetic code 30 7 Feb 18 Cloning in bacteria other than E. coli Feb 20 Manipulation of bacterial genomes, genome shuffling Feb 22 Metabolic engineering, development of antimicrobials 8 Feb 25 Cloning in eukaryotes: 2-hybrid system (PROB SET 2 HANDED OUT) 31 Feb 27 Transformation: higher eukaryotes, baculovirus expression 32 Feb 29 Genetic manipulation of plant cells 9 Mar 03 Genetic manipulation of animal cells (PROB SET 2 DUE) Mar 05 Advances in transgenics Mar 07 Embryonic stem cells and organismal cloning
10 Mar 10 Medical diagnostics and development of therapeutics, gene therapy Mar 12 Molecular anthropology/archaeology Mar 14 DNA nanotechnology * Mar 20 FINAL 12:30-2:20
Problem sets: 2 problem sets will be assigned and can be downloaded from the course website on the dates indicate in the syllabus. Homework will be graded, and points will be deducted if they are handed in late.
Exams: Exams will be in the form of short answer and multiple choice questions looking to
determine your understanding of the material as well as your ability to interpret data
Grading: BI410 BI510 Exam I 35% 25% Exam II 35% 25% Problem Sets 30% 25% Presentation 25%
DNA RNA protein
Replication/mutation
Natural selection
Evolution: a dialog between the genome and its environment
environment
DNA RNA protein
Dynamic, immediate, transient modification of DNA program
Organisms respond to their environment via information from sensory input and changes in gene expression
DNA RNA protein
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
environment
DNA RNA protein
Human intervention: genetics (indirect), rDNA (direct)
Human activity: transient modifications of environment, permanent modifications of DNA program
2006: 53 years of DNA structureRosalind 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?
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)
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
The behavior of DNA (genes) is predictable
Gene sequence conservation often indicatesfunctional similarity
Non-protein coding information sequencescan often be detected by homology (promoters for transcription initiation, transcription terminators, ribosome binding sites, DNA binding protein binding sites)
the genetic code
The genetic code and the roots of biotechnology
1961Marshall Nirenberg and Heinrich J. Matthaei:polyU mRNA encodes poly-phenylalanine
1966Nirenberg and colleagues had deciphered the 61 codons (and 3 nonsense codons) for all 20 amino acids
1968Nobel prize for Nirenberg, Holley, and Khorana
1966George 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.”
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
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
"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
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)
http://nobelprize.org/nobel_prizes/chemistry/articles/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”
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/articles/berg/index.html
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
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.
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
(see also CR #3 in course packet)
“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”
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….
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
The biotechnology industry is very newCase in point: Genentech (S. San Francisco)
2006: Genentech to open production facility in Portland (2010)
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
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)
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 A260 (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)
How can concentration be determined by absorbance?
DNA has a characteristic “molar extinction coefficient”
The Beer-Lambert law:
I = Io10- dc
I = intensity of transmitted lightIo = intensity of incident light = molar extinction coefficientd = optical path lengthc = concentration of absorbing material
How much light gets through a solution depends on what’s in it and how much of it there is
The Beer-Lambert law:
I = Io10- dc
Absorbance A measured by a spec is log I/Io
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: 6.6ssDNA, RNA: 7.4
(but these values change with pH and salt concentration!)
0.5
0
Ab
sorb
ance
(1 c
m p
ath
len
gth
)
Wavelength (nm)
200 260 400
A typical (good) “scan” (multiple wavelengths) of a DNA sample
A260 = 0.327
A260/A280: 1.8 is good (lower values indicate significant protein contamination)
Example:sample of 250 base pair fragment of DNA has an A260 = .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 DaltonsTherefore 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 A260 give you the quantity of DNA?
1 ml
16.35 micrograms 1000 ml
1 L 106 micrograms
1 gram
What is the molarity of a 16.35 microgram/ml solution of a 250 base pair DNA fragment?
1.6 x 105 grams
1 mole
1.02 x 10 -7 molar0.102 x 10-6 molar [0.1 micromolar (M)]102 x 10-9 molar [102 nanomolar (nM)]
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
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
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
Agarose gelsAgarose: 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)
Agarose gelsStandard 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
-+
time of electrophoresis(progress monitored by marker dyes)
Load samples in wells
bromophenolblue
xylenecyanol
Typical agarose gel
(the DNA fragments are not visible without some sort of staining)
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
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)
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
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)
Protein electrophoresis
o Almost always polyacrylamide based
o The anionic detergent SDS (sodium dodecyl sulfate) is used to denature the proteins, giving each protein a “uniform” negative charge
o Protein separation occurs as a function of size
o Discontinous Tris-Cl/glycine buffer system:o Stacking gel: pH 6.8, low polyacrylamide
concentration, focuses proteins into thin layer (gives higher resolution upon separation)
o Separating gel: pH 8.8, separates proteins on the basis of size
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
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 bromideG-C base pair
Example of an agarose-DNA gel, Stained with ethidium bromide
Direction ofelectrophoresis
Fragments of bacteriophage genomic DNA (48 kb) cut with the restriction enzyme Hind III
The fragments are equimolar--why is the band intensity different?
Another ethidium bromide-stained agarose gel
The marker lane (M) gives size standards for comparison with the sample lanes
M samples
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
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 NH3- groups of the proteins, also through van der Waals forces
http://www.galab.de/laboratories/services/biopharma/img/sds.jpg
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
good bad (overstained)ugly (keratin)
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
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)
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
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)
Agarose orPolyacrylamide gel
nitrocelluloseor nylon membrane boundary:DNA binds to it
A typical capillary blotting apparatus. Electroblotting is also commonly used
Immobilization of nucleic acids
• 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
• Use T4 polynucleotide kinase
--catalyzes the transfer of the gamma phosphate of 32P ATP to the 5’ end of DNA fragment to be used as a probe
• 32P 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: 32P labeling
…or digoxygenin/antibody-conjugated HRP
can also usebiotinylated DNA probe
oxidation
Non-radioactive labels
• 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
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:
Proteins are transferred tomembranes using the same principle as Southern blots
Specific proteins detectedby probing blot withantibodies to protein of interest
Antibody binding is detected by antibody to the original antibody that has enzyme (horseradish peroxidase, alkaline phosphatase) or radioactivity (125I) conjugated to it
Western blots: proteins
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