Forensic DNA Fingerprinting: Using Restriction Enzymes

Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. Leigh Brown, M.A. Forensic DNA Fingerprinting Kit Instructors

Why Teach DNA Fingerprinting? Real-world connections Tangible results Link to careers and industry Laboratory extensions Standards-based

Forensic DNA Fingerprinting Kit Advantages Standards Based Aligns with AP Biology Lab 6 Use of real restriction enzymes and electrophoresis of real DNA fragments Lab can completed in two 45 minute sessions Sufficient materials for 8 student workstations

DNA structure DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping The Forensic DNA Fingerprinting Kit Can Help You Teach:

DNA Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification Human remains Monitoring transplants

Workshop Time Line Restriction digest of DNA samples Introduction to DNA Fingerprinting and RFLP analysis Electrophoresis on Agarose gels Analysis and interpretation of results

DNA Fingerprinting Procedure Overview

Laboratory Quick Guide

DNA Fingerprinting Procedures Day One

DNA Fingerprinting Procedures Day Two

DNA Fingerprinting Procedures Day Three

DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus

Model of DNA DNA is Comprised of Four Base Pairs

Deoxyribonucleic Acid (DNA)

DNA Schematic O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Phosphate

DNA Restriction Enzymes Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands Over 3,000 known enzymes

Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Palindrome Restriction site Fragment 1 Fragment 2

5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts

Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

The DNA Digestion Reaction Restriction Buffer provides optimal conditions NaCI provides the correct ionic strength Tris-HCI provides the proper pH Mg 2+ 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 the temperature istoo hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time

Restriction Fragment Length Polymorphism RFLP Allele 1 Allele 2 GAATTC CTTAAG GAATTC CTTAAG CTGCAG GACGTC CGGCAG GCCGTC PstIEcoRI Fragment 1+2 Different Base Pairs No restriction site + MA-1A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments

Agarose Electrophoresis Loading Electrical current carries negatively- charged DNA through gel towards positive (red) electrode Power Supply Buffer Dyes Agarose gel

Agarose Electrophoresis Running Agarose gel sieves DNA fragments according to size –Small fragments move farther than large fragments Power Supply Gel running

Chemistry in action…. Or… Ask your friendly chemist… about electro- phoresis and electrolysis.

Chemistry of electrophoresis and electrolysis Electric fields and electric currents

SDS-PAGE, DNA electrophoresis and the need for a gel matrix (sieving medium) Without a viscous medium, all molecules move at ~ the same rate in electric field Gel structure retards larger solute particles DNA molecules, SDS- proteins have ~ equal charge/mass Electrophoresis occurs between the electrodes (field- driven).

Electrolysis always occurs during electro- phoresis. Cathode produces H 2 at twice the rate that anode produces O 2 Current is carried by solute ions. Electrons aren’t soluble in H 2 O. Example: TAE buffer; tris supplies cations (+), acetate supplies anions (-). Electrolysis occurs at the electrodes.

Analysis of Stained Gel Determine restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples

Analysis using Vernier’s Logger Pro software Adaptable to many forms of Gel AnalysisTake or Import Gel ImageSet OriginSet ScaleSet Standard LadderAdd Lanes Leaves more time for students to Explore

Standard Curve and Analysis Easy to View Results include: Gel Image Standard Curve Data Table One convenient page with all the Results

Molecular Weight Determination Size (bp)Distance (mm) 23, , , , , , Fingerprinting Standard Curve: Semi-log

DNA Fingerprinting Lab Extensions Independent studies Plasmid DNA isolation (mini-preps) Plasmid mapping using restriction enzymes Southern blot analysis Introductory labs to electrophoresis: Kool-Aid/FastBlast pH indicator in buffer

Plasmid Map and Restriction Sites Laboratory Extensions BamHI: EcoRI: HindIII: EcoRI+Hind III: 1 linear fragment; 7367bp 2 fragments; 863bp / 6504bp 3 fragments; 721bp/2027bp/3469bp 5 fragments; 721bp/863bp/947bp/1659bp/2027bp BamHI 7367bp EcoRI 863bp 6504bp Hind III 721bp 2027bp 3469bp EcoRI+ HindIII 2027bp 1659bp 947bp 863bp 721bp

Bio-Rad’s Electrophoresis Equipment Electrophoresis Cells Power Supplies Precast Agarose Gels PowerPac ™ MiniPowerPac ™ Basic PowerPac ™ HCPowerPac ™ Universal Mini-Sub ® Cell GTWide Mini-Sub Cell GT

Vernier’s Analysis Equipment White Light Transilluminator BlueView Transilluminator White Digital Bioimaging System Blue Digital Bioimaging System BlueView Transilluminator White Light Transilluminator White Digital Imaging System Blue Digital Bioimaging System

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