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
Become familiar with principals and techniques
of agarose gel electrophoresis
Generate a standard curve from a series of DNA
size fragments
Estimate DNA fragment sizes from agarose gel
data
What are restriction enzymes?
Evolved by bacteria to protect against viral
DNA infection
Endonucleases = cleave within DNA strands
3,139 known enzymes
How does it work?
Enzyme Site Recognition
–
Each enzyme digests (cuts)
DNA at a specific sequence
Unambiguous
 restriction site
–
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
Enzyme cuts 
5’
G
3’ CTTAA
3’
5’
5’
3’
3’
G
GAATTC
3’ CTTAAG
G 5’5’
5’
3’
AATTC 3’
G 5’
Generates 5’ overhang
5’ and 3’ versus Blunt ends
Common Restriction Enzymes
EcoRI
5’
GAATTC 3’
3’ CTTAAG 5’
– Escherichia coli
– 5’ overhang
HindIII
–
Haemophilus influensae
–
5’ overhang
PstI
– Providencia stuartii
– 3’ overhang
5’
AAGCTT 3’
3’ TTCGAA 5’
5’
CTGCAG 3’
3’ CACGTC 5’
What is needed for restriction digestion?
Template DNA, uncut DNA, often bacterial
phage DNA
DNA standard or marker, a restriction
enzyme of known fragment sizes
Restriction enzyme(s), to cut template
DNA
Restriction Buffer, to provide optimal
conditions for digestion
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
Restriction Enzyme Digestion
Restriction Buffer provides optimal
conditions
–
NaCl provides the correct ionic strength
–
Tris-HCl provides the proper pH
–
Mg2+ is an enzyme co-factor
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

Agarose Gel Electrophoresis
Electrolysis: the splitting of water using
electricity
–
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
-
Cathode
Anode
+
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
Concentration affects DNA migration
– Low conc. = larger pores better
resolution of larger DNA fragments
– High conc. = smaller pores better
resolution of smaller DNA fragments
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
–
Create standard curve using DNA marker
–
Measure distance traveled by restriction fragments
–
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!!!