Download Restriction Enzymes, Gel Electrophoresis and Mapping DNA

Restriction Enzymes, Gel
Electrophoresis and Mapping
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DNA Preparation
The Problem of DNA Analysis Methods
Restriction Endonucleases
DNA Gel Electrophoresis
Restriction Mapping
Functional Mapping
Recombinant DNA
Dry, fibrous DNA
DNA Preparation
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Source of tissue
Dissociate tissue
Remove proteins with phenol extraction
Alcohol precipitation - “salting out”
Remove RNA - RNase treatment
Result - chemically pure, large (~20 kb)
fragments
Physical analysis
• Viscosity—proportional to MW
• Gel electrophoresis—mobility proportional
to MW, random fragments
• Ultracentrifugation—density uniform
(except satellite DNA)
• Hybridization kinetics—complexity of
regions of DNA, no specifics
The Problem
We can make DNA, we can try to characterize it using
biochemistry, we can study crude sequence information with
C0t1/2 and hybridization studies; but we are limited in our
pursuit of specific, single genes. If what we really want is to
study, for example, the gene that is defective in cystic fibrosis
or sickle-cell anemia, how do we do it?
Methods needed
• Fragmenting DNA in a sequence-specific
manner
• Working with small segments of
fragmented DNA that are identified by their
sequence
• Determining the sequence of these small
segments of DNA
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The Pioneers:
Restriction Enzymes
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Bacterial endonucleases that recognize short sequences in DNA before
cutting--producing DNA segments defined by their sequence.
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Physiology or Medicine 1978 awarded jointly to:
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ARBER, WERNER, Switzerland, Biozentrum der Universität, Basel;
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NATHANS, DANIEL, U.S.A., Johns Hopkins University School of
Medicine, Baltimore, MD; and
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SMITH, HAMILTON O., U.S.A., Johns Hopkins University School of
Medicine, Baltimore, MD:
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"for the discovery of restriction enzymes and their application to
Werner
Arbor
Dan
Nathans
Hamilton
Smith
problems of molecular genetics".
The Pioneers:
Recombinant DNA
• Isolating fragments of DNA into replicating populations of
molecules. A clone is a group of genetically identical
individuals.
• The 1980 Chemistry Nobel prize was divided, one half
being awarded to:
Paul Berg
• BERG, PAUL, U.S.A., Stanford University, Stanford, CA,
• "for his fundamental studies of the biochemistry of nucleic
acids, with particular regard to recombinant-DNA"
The Pioneers:
DNA Sequencing
• Determining the sequence of nucleotides within a segment
of DNA
• 1980 Chemistry: “and the other half jointly to:”
• GILBERT, WALTER, U.S.A., Biological Laboratories,
Cambridge, MA; and
• SANGER, FREDERICK, U.S.A., Great Britain, MRC
Laboratory of Molecular Biology, Cambridge:
Walter Gilbert
Fred Sanger
• "for their contributions concerning the determination of
base sequences in nucleic acids".
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Strunk & White Moment
What’s wrong with this sentence?
“Spade, a tanner, was shot to death by an
angry third wife when he accused her,
once too often, of lusting after a polo
player pictured on a popular candy
recipe.” - Sunday Star-Ledger, Sept. 20, 1998, p. 16.
Restriction Endonucleases
• Restricted to cutting specific sequences
• Type II enzymes:
– dimers
– require Mg++
– MW in range of 30-40,000
– Work with Restriction Methylase to confer
species specificity.
Too Complex!
• “Vigorous writing is concise.” p. 23.
• Write simpler sentences!
• In scientific writing, it is tempting to use
complex sentences to sound smart. Don’t
do it! Express your ideas clearly and
concisely.
Host Restriction-Modification
System (hsm)
CH3
5’-GAATTC-3’
3’-CTTAAG-5’
5’-GAATTC-3’
3’-CTTAAG-5’
Methylase
Endonuclease
CH3
5’-GAATTC-3’
3’-CTTAAG-5’
Methylase
5’-GAATTC-3’
3’-CTTAAG-5’
CH3
CH3
Restriction Endonuclease Cut
5’-GAATTC-3’
3’-CTTAAG-5’
Endonuclease
5’-G-3’
3’-CTTAA-5’
+
5’-AATTC-3’
3’-G-5’
Examples
Source
E.coli RY13
Bacillus amyloliquifaciens H
Serratia marcescens Sb
Providentia stuartii
Enzyme
Recognition Site
EcoRI
BamHI
SmaI
PstI
G/A*ATTC
G/GATCC
CC*C/GGG
CTGCA*/G
Fragment
End
5' extended
5' extended
blunt
3' extended
“Sticky Ends”
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Dyad Symmetry
5’-GAATTC-3’
3’-CTTAAG-5’
90°
5’-GAATTC-3’
3’-CTTAAG-5’
270°
5’-GAATTC-3’
3’-CTTAAG-5’
5’-GAATTC-3’
3’-CTTAAG-5’
Recognition Sequences
180°
Recognition site size vs.
fragment size
• Most restriction enzymes recognize 4-6 bp.
• The probability of finding a 6 bp site is:
(1 4 ) ⇒ 1 4096
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Probability of finding one specific base in any one position
is 1 in 4.
Probability of finding 6 specific bases in a row is the product
of the probabilities of finding each base.
Gel Electrophoresis
• A 6 bp-recognizing enzyme produces
randomly-sized fragments with a mean size
of 4,024 bp.
• A 4 bp-recognizing enzyme produces a
mean of 256 bp.
• These are means with normal distributions.
• Base composition affects size distributions
(WHY?).
DNA Gels
DNA Gels
• Mobility is proportional to log (MW)
• Separation is due to sieving action of gel
• DNA is detected by staining with Ethidium
Bromide
• Ethidium bromide bound with dsDNA is
fluorescent (orange) under UV illumination
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Ethidium Bromide
Ethidium-stained gel
Restriction Mapping
pBR322 - a plasmid vector
• Since restriction enzyme recognition
sequences are specific, they should occur at
specific locations on every, identical DNA
molecule.
• Therefore, digestion products are
reproducible.
• Therefore, we can use the recognition
sequence as a “flag” to mark a map.
Single cut of circular DNA
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First commonly-used cloning vector
Circular, dsDNA, 4,361 bp
Contains ApR, TcR, OR genes
Contains only one cut site for EcoRI and
BamHI
Cut pBR322 with both EcoRI
and BamHI
3,986 bp
+
4,361 bp
4,361 bp
4,361 bp
375 bp
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Draw Map - Select Start Site
Draw Map - Select Direction
375 bp
BamHI
EcoRI
EcoRI
Add sites
Two possibilities
• A new restriction enzyme can be mapped by
comparing its product sizes with known
start site and direction indicator
• HincII cuts twice - 3257 bp and 1104 bp
• HincII + EcoRI products:
453
651 EcoRI
453
EcoRI 651
– 3257 bp
– 651
– 453
Direction
• Use second enzyme (BamHI) to determine
orientation
• HincII - 3257 bp and 1104 bp
• HincII + BamHI:
Two possibilities
277
EcoRI
BamHI
829
829
BamHI
277
EcoRI
– 3257 bp
– 829
– 277
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Only one overlapping map!
HincII
829
BamHI
453
EcoRI 651
277
Restriction Map of pBR322
HincII
Map Physical Sites to Genetic
Information
Complementation Analysis
• First done with SV40, a dsDNA, circular,
approximately 5,200 bp.
• SV40 causes transformation of cultured
rodent cells.
• Mutation analysis identified one gene
required for transformation (tsA)
• Relate to restriction map.
Fragment Complementation
Only B complements!
• Combine mutant viral DNA with restriction
fragment of wt virus
• If restriction fragment can complement
mutation, site of mutation must lie within
fragment position on map
• Depends on homologous recombination
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Genetic Map
Recombinant DNA
• First done by Dana & Nathans
• Showed correlation between physical map
of DNA and genetic function encoded
within DNA
• Now that we know where the
transformation gene is, we can isolate and
characterize it!
Ligation of “sticky ends”
5’-G-3’
3’-CTTAA-5’
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• First done by Jackson, Symons & Berg
• Use “sticky ends” produced by restriction
enzymes as sites for recombination
• Use DNA Ligase to attach two DNA
molecules
• Combine plasmid vector with any DNA
fragment
Cut vector, ligate with fragment
DNA Ligase
5’-GAATTC-3’
3’-CTTAAG-5’
5’-AATTC-3’
3’-G-5’
Transfect bacteria, grow clone
Clone
A clone is a group of genetically
identical individuals
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The Major “Breakthroughs” of
Molecular Biology
• Restriction Enzymes
• Recombinant DNA
Techniques
(“cloning”)
• DNA Sequencing
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