78
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

Bi430/530 Theory of Recombinant DNA Techniques First part of course: Technical aspects of molecular biology work--Molecular Cloning Second part of course:

  • View
    213

  • Download
    0

Embed Size (px)

Citation preview

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%

Recombinant

DNA

Information flow in the cell

DNA RNA protein

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

DNA is very easy to sequence

Etc….

http://www.genomesonline.org/

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

1967“Will Society Be Prepared?” Marshall Nirenberg,

editorial in Science (see WebCT)

"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)

Detecting specific DNA sequences: the Southern blot

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

…e.g. horseradish peroxidase

oxidation

Non-radioactive labels

…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

Southern Blot--one example

(RFLPs)

(or PCR fragment)

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