ANALYSIS OF PRECUT
LAMBDA DNA
RESTRICTION ENZYMES & GEL ELECTROPHORESIS
VIDEO INTRODUCTION
• AP Bio Lab 6 - Molecular Biology —
bozemanscience 11.57
• Restriction Enzymes 2.19
“Molecular Scissors”
Finds 5 specific sequences…..# cuts? # fragments?
OBJECTIVES
• Understand the use of restriction enzymes as
biotechnology tools
• Become familiar with principles and techniques of
agarose gel electrophoresis
• Generate a standard curve from a series of DNA
size standards
• Estimate DNA fragment sizes from agarose gel data
RESTRICTION ENZYMES
“ENDONUCLEASES”
• Biomolecules that cut DNA at specific sites.
• Recognize specific sequences of DNA base pairs and
cut (chemically separate) DNA at that specific site
• First isolated in bacteria that use them as a natural
defense mechanism to cut up the invading DNA of
bacteriophages. “1st biological immune system”
• Thousands of restriction enzymes. Named for the
bacterium where they were isolated (and #)
• EcoRI (Escherichia coli-1st)
A-A-T-T
HindIII (Haemophilus Infulenzae- 3rd)
A-G-C-T
PstI (Providencia stuartii)
T-G-C-A
STICKY ENDS (UNPAIRED BASES) VS BLUNT
PALINDROMIC
PsiI
“racecar”
“radar”
• Action of enzyme: produces restriction fragments
• cut DNA at specific sequences
• restriction site
• symmetrical “palindrome”
• produces two double stranded DNA
• sticky ends
• will bind to any foreign (complementary) by hydrogen bonding
MANY USES OF RESTRICTION
ENZYMES…
• Now that we can cut DNA with restriction
enzymes…
• we can cut up DNA from different people… or different
organisms…
and compare it
• why?
•
•
•
•
forensics
medical diagnostics
paternity
and more…
Effects of 3 restriction
enzymes on lambda
genomic DNA
LAMBDA PHAGE GENOME
*Smaller DNA fragments
will travel farther than
larger ones
•
•
•
•
Genome = 48,000 base pairs
Cut into several DNA fragments; dye
4 samples: 3 precut; 1 undigested
From (-) to (+) Fragment size : rate ?
GEL ELECTROPHORESIS
• A method of separating DNA in a
gelatin-like material using an
electrical field
• when it’s in an electrical field it moves
toward the positive side
DNA 
–

“swimming through Jello”
+
GEL ELECTROPHORESIS
• DNA moves in an electrical field…
• so how does that help you compare DNA fragments?
• size of DNA fragment affects how far it travels
• small pieces travel farther
• large pieces travel slower & lag behind
DNA 
–

“swimming through Jello”
+
GEL ELECTROPHORESIS
DNA &
restriction enzyme
longer fragments
wells
power
source
gel
shorter fragments
+
completed gel
RUNNING A
fragments of DNA
GELseparate based
on size
cut DNA with restriction enzymes
1
2
Stain DNA
• ethidium
bromide binds to
DNA
• fluoresces under
UV light
3
DNA TECHNOLOGY AND GENOMICS
• http://www.phschool.com/science/biology_place/l
abbench/lab6/praprob2.html
• Recombinant DNA technology 1.13
• Isolates and amplifies DNA sequences; cloned for use with
restriction enzyme and transformation technology
• polymerase chain reaction -1.28
• Denaturing > Anneal primer > DNA polymerase > repeat