Biotechnology Restriction Enzyme Analysis of DNA AP Biology

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: Eco. RI (Escherichia coli), Hind. III (Haemophilus influenzae) • Biologists use RE’s to manipulate & analyze DNA

• Palindrome = DNA sequence (4 -10 base pairs) that reads same from both directions – Eco. RI: 5’-GAATTC-3’ cuts G | AATTC – Pst. I: 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

The Disappearance of Ms. Mason The Crime: 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 (S 1) – Amy – Suspect 2 (S 2) – Bobby – Suspect 3 (S 3) – Carla – Suspect 4 (S 4) – Drew – Suspect 5 (S 5) – Ellie

What happened to Ms. Mason? Your Job: 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 Hind. III – 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 Hind. III 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 Hind. III 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