Biotechnology: Restriction
Enzyme Analysis of DNA
AP Biology Investigation 9
Gel Electrophoresis Lab
Introduction
• Genetic Engineering: process of
manipulating genes and genomes
• Biotechnology: process of manipulating
organisms or their components for the
purpose of making useful products.
Restriction Enzymes (RE)
• Also called Restriction Endonucleases
• Used to cut DNA strands at specific locations
(restriction sites)
• Discovered in bacteria used to repair DNA
& defend against bacteriophages
• Names: EcoRI (Escherichia coli), HindIII
(Haemophilus influenzae)
• Biologists use RE’s to manipulate & analyze
DNA
• Palindrome = DNA sequence (4-10 base pairs)
that reads same from both directions
– EcoRI: 5’-GAATTC-3’  cuts G | AATTC
– PstI: 5’-CTGCAG-3’  cuts CTGCA | G
• Cuts in 2 places make restriction fragments
that have sticky ends (single-stranded
overhanging ends)
• Cuts exactly in center of restriction site makes
blunt ends
Blunt ends vs.
“Sticky” ends
AP Bio Lab 9 Briefing
• RFLP = Restriction Fragment Length
Polymorphism
• RFLP Analysis: use RE’s to cut DNA so that a
gel electrophoresis can be run to create a
DNA profile
Gel Electrophoresis: used to separate DNA molecules
on basis of size and charge using an electrical current
Gel Electrophoresis
• DNA (- charged) will
migrate towards (+) end
• Shorter DNA  moves
faster
• Longer DNA  move
slower
How RE’s are used to create DNA profiles
How RE’s are used to create DNA profiles
The Disappearance of Ms. Mason
The Crime: Ms. Mason, an AP Biology teacher at
FHS (Fictional High School), is missing. There
are blood drops found at the crime scene (CS).
The Suspects: Five students who are failing Ms.
Mason’s class
– Suspect 1 (S1) – Amy
– Suspect 2 (S2) – Bobby
– Suspect 3 (S3) – Carla
– Suspect 4 (S4) – Drew
– Suspect 5 (S5) – Ellie
What happened to Ms. Mason?
Your Job: Determine whose DNA matches the
blood drops found at the crime scene by
analyzing the suspect DNA profiles created
using gel electrophoresis.
AP Bio Lab 9 Briefing
• Standard Sample: Lambda DNA cut with HindIII
– Sizes of these fragments are known
– Used as a basis for comparison to determine
sizes of unknown DNA fragments
– Create standard curve by graphing known
data on semi-log paper
Standard HindIII Lambda DNA Sample
Standard Curve
(plot of distance migrated vs. DNA size (base pairs)
Data Collection:
Data Analysis
1. Using a ruler, measure the distance from the well to
the center of each DNA band. Record it in the data
table.
2. Using the data from the known HindIII lambda
DNA, plot distance vs. size for Bands 2-6 on the
semilog graph paper. Draw a best-fit line. This will
be your standard graph.
3. Using the standard graph created in #2, determine
the approximate size of each suspect or crime
scene fragment. Fill in the data table.
Lab Write-Up