Transcript Restriction Digestion and Analysis of Lambda DNA

Restriction Digestion and
Analysis of Lambda DNA Kit
What can you do with the Restriction
Digestion and Analysis of Lambda DNA
Kit?
Understand the use of restriction enzymes as
biotechnology tools and the mechanics of a
restriction enzyme digest
Become familiar with principals and techniques
of agarose gel electrophoresis
Estimate DNA fragment sizes from agarose gel
data
Understand the importance of restriction
enzymes and their applications
What are restriction enzymes?
Evolved by bacteria to protect
against viral DNA infection
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1950s: discovery of primitive
immune system in bacteria
1962: proof that in bacteria, an
enzyme system recognized and
destroyed foreign DNA while
protecting its own
1960s: E.coli extracts isolated
which cleaved phage DNA, but not
useful
1970s: H. influenzae, HindII,
extract isolated with no
modification activity and cleavage
within the restriction site
Endonucleases = cleave within DNA strands
Exonucleases = digest from the ends of DNA molecules
Types I, II, and III
3,139 known enzymes
How does it work?
Enzyme Site Recognition
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Each enzyme digests (cuts)
DNA at a specific sequence
 restriction site Unambiguous
Enzymes recognize 4-, 6- or
8- base pair, palindromic
sequences
Isoschizomers recognize
identical sequences, but
have different optimum
reaction conditions and
stabilities
Can be unambiguous or
ambiguous
Ambiguous
Palindromic Sequences
5’ versus 3’ overhang: Sticky Ends, subsequent
ligation is very specific
Enzyme cuts 
5’
G
3’ CTTAA
3’
5’
5’
GAATTC 3’
3’ CTTAAG 5’
5’
3’
AATTC 3’
G 5’
Generates 5’ overhang (in the 5’ direction)
Blunt ends: subsequent ligation is
non-specific
5’ GAA TTC 3’
3’ CTT AAG 5’
View the molecular structure of DNA (see the 5’ to 3’ structure):
http://207.207.4.198/pub/flash/24/menu.swf
Common Restriction Enzymes
EcoRI
– Escherichia coli
– 5’ overhang
5’
GAATTC 3’
3’ CTTAAG 5’
HindIII
Haemophilus influenzae
– 5’ overhang
– 1st free-living organism to have its entire
chromosome sequenced
– Type b, Hib, was the leading cause of
bacterial meningitis among children
under 5 years old in the US, before
development of a vaccine.
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5’
AAGCTT 3’
3’ TTCGAA 5’
PstI
– Providencia stuartii
– 3’ overhang
– 5 species of Providencia recognized
today: P stuartii is the most common
species causing human infection.
Extremely common in patients with longterm indwelling urinary catheters due to
Its ability to persist in catheterized urine
because its genetics allows it adhere to
the urinary catheter.
5’
CTGCAG 3’
3’ CACGTC 5’
Examples
Eco RI
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
GGCCTGCG
AATTCCCGATCGAAGGCCCG
CCGGACGCTTAA
GGGCTAGCTTCCGGGCTTAA
Hae III
GG
CC
AATTCTGGCCA
GACCGGT
GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA
CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGT
CCTGCGAATTCCCGATCGAAGG
GGACGCTTAAGGGCTAGCTTCC
CCCGAATTCTGG
GGGCTTAAGACC
CCA
GGT
What is needed for restriction digestion?
Template DNA, uncut DNA, often bacterial
phage DNA
Restriction enzyme(s), to cut template
DNA
Restriction Buffer, to provide optimal
conditions for digestion
Water Bath
Lambda Phage DNA
Genomic DNA of a bacterial virus
Attacks bacteria by inserting its nucleic acid into the
host bacterial cell
Replicates rapidly inside host cells until the cells
burst and release more phages
Harmless to man and other eukaryotic organisms
Bacteriophage lambda (λ)
In 1971 Alan
Campbell showed
that the central third
of the genome was
not required for lytic
growth. People
started to replace it
with E. coli DNA
Lambda genome is
approximately 49
kb in length.
Only 30 kb is
required for lytic
growth.
Thus, one could
clone 19 kb of
“foreign” DNA.
Packaging
efficiency 78%100% of the
lambda genome.
A complete animation of the lytic cycle:
http://www.blackwellpublishing.com/trun/artwork/Animations/Lambda/lambda.html
Bacteriophage lambda
Protein capsule of
lambda has a tight
constraint on the
amount of DNA
that will fit inside it
(~ 55kb)
By the early 1970’s
we knew that a
good portion of
lambda was not
required
“Junk” DNA
COS site: Cohesive
“sticky” ends
Lysis
Replication
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Lysogeny
Head
Tail
Circularized
lambda
Not Quite Bacteriophage
lambda Lysis COS
Eliminate the nonessential parts of
lambda
Can now insert
large pieces of
DNA (~ 20 kb)
Replication
ori
Tail
Head
Restriction Enzyme Digestion
Restriction Buffer provides optimal conditions:
NaCl provides correct ionic strength
Tris-HCl provides the proper pH
Mg2+ is an enzyme co-factor
Different enzymes have different optimal buffers;
Manufacturers package enzymes with buffers for
ease of use
DNA Digestion Temperature
Why incubate at 37C?
Body temperature is optimal for these and most
other enzymes
What happens if temperature is too hot
or cool?
Too hot = enzyme may be denatured, killed
 Too cool= enzyme activity lowered, requiring
longer digestion time
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How do we visualize the DNA?
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
Electrolysis: the splitting of water using
electricity
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current splits water into hydrogen ions (H+)
and hydroxyl ions (OH-)
Electrophoresis: a method of separating
charged molecules in an electrical field;
DNA has an overall negative charge
Used to separate DNA fragments by size
Components of an Electrophoresis System
Power supply and chamber, a source of negatively
charged particles with a cathode and anode
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that DNA migrates
through
Gel casting materials
DNA ladder, mixture of DNA fragments of known
lengths
Loading dye, contains a dense material and allows
visualization of DNA migration
DNA Stain, allows visualizations of DNA fragments
after electrophoresis
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Cathode
Anode
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Buffer
Dyes
Agarose
gel
Power Supply
Bio-Rad’s Electrophoresis Equipment
Power Supplies
Precast Ready
Agarose Gel
Electrophoresis Buffer
TAE (Tris-acetate-EDTA) and TBE (Trisborate-EDTA) are the most common
buffers for duplex DNA
Establish pH and provide ions to support
conductivity
Concentration affects DNA migration
– Use of water will produce no migraton
– High buffer conc. could melt the agarose gel
Agarose Gel
A porous material derived from red
seaweed
Acts as a sieve for separating DNA
fragments; smaller fragments travel
faster than large fragments
1% agarose
– Plinko Model
Concentration affects DNA migration
– Low conc. = larger pores better
resolution of larger DNA
fragments
– High conc. = smaller pores
better resolution of smaller DNA
fragments
2% agarose
Loading Dye
DNA samples are loaded
into a gel AFTER the tank
has been filled with buffer,
covering the gel
Contains a dense
substance, such as
glycerol, to allow the
sample to "fall" into the
sample wells
Contains one or two
tracking dyes, which
migrate in the gel and
allow monitoring of how far
the electrophoresis has
proceeded.
DNA Staining
Allows DNA
visualization after
gel electrophoresis
Ethidium Bromide
Bio-Safe DNA
stains
Agarose
Gel
DNA
Fragments
Complete a Gel Electrophoresis simulation at:
http://gslc.genetics.utah.edu/units/biotech/gel/
Restriction
Enzyme Digest
and Analysis
Procedures
Actual Results of Restriction
Enzyme Digestion
Lane 1, DNA markers
(HindIII lambda digest)
lane 2, uncut lambda
DNA
lane 3, lambda DNA
digested with PstI
lane 4, lambda DNA
digested with EcoRI
lane 5, lambda DNA
digested with HindIII
Analysis of DNA Fragments
Determine restriction fragment sizes
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Create standard curve using DNA marker
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Measure distance traveled by restriction fragments
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Determine size of DNA fragments
DNA Marker Standard Curve
Size (bp) Distance
(mm)
23,000
11.0
9,400
13.0
6,500
15.0
4,400
18.0
2,300
23.0
2,000
24.0
Factors Affecting
Restriction Enzyme Digestion
Temperature, restriction enzymes are
sensitive to prolonged periods of exposure to
heat
Cross contamination of restriction enzymes
Buffer, optimum pH
Incubation temperature, maintain optimum
temperature during restriction enzyme activity
And Finally…Don’t forget to ADD your
restriction enzyme to the reaction!!!
Applications
Recombinant DNA Technology
DNA Cloning
Constructing DNA Libraries
Southern Blot Hybridization
Gene Cloning
Southern Blot Hybridization
DNA is isolated from a sample
such as blood, saliva, semen,
tissue, or hair and purified
The huge genome is cut up
with restriction enzymes to
produce short, manageable
DNA fragments
DNA fragments are then sorted
by size using gel
electrophoresis
DNA is denatured and
transferred and permanently
affixed to a nylon membrane
Attach a radioactive, DNA
probe that is complementary to
a VNTR locus to DNA
sequences on the membrane
Take a picture of it using
special X-ray film
“High-profile” Applications
Forensics (CSI) – “Can you find a match?”
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Crime Scenes
Paternity
Human Remains
Genome Projects
Biological Weapons
“Low-profile” Applications
Can you find similarities?
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Organ Transplants
Anthropology and Human Relatedness
Species Relatedness
Food Identification
Genome Sequencing
Paternity Analysis
No 2 individuals have the
same pattern of
restriction enzyme
recognition sites
Silent mutations on
alleles and RFLP’s
Crime Scene Investigation
VNTR’s: number of
repeats varies from 4 to
40 in different individuals
Variants inherited from
parents; unrelated
individuals not likely to
have same repeats
Length of DNA depends
on the number of repeats
at each locus
Same 3 VNTR loci are
analyzed for 3 individuals
= 6 bands for each
Results can serve as a
DNA fingerprint to
exclude suspects