Forensic DNA Fingerprinting: Using Restriction Enzymes

Learning targets for Restriction Digestion and Analysis of Lambda DNA lab? I will understand the use of restriction enzymes as biotechnology tools I will become familiar with principals and techniques of agarose gel electrophoresis I will be able to generate a standard curve from a series of DNA size fragments I will be able to estimate DNA fragment sizes from agarose gel data I will be able to use DNA profiles to narrow a list of suspects in a hypothetical crime scenario.

Investigation 9 BIOTECHNOLOGY: RESTRICTION ENZYME ANALYSIS OF DNA* How can we use genetic information to identify and profile individuals? Supreme Court Rules DNA Can Be Taken After Arrest The Innocence Project DNA Detectives Track Elephant Poachers Pioneering DNA Forensics

Uses of DNA fingerprinting DNA structure DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping. The Forensic DNA Fingerprinting concepts:

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

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

DNA Fingerprinting Procedure Overview

Laboratory Quick Guide

DNA Fingerprinting Procedures Day One

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

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

How does it work? Enzyme Site Recognition – Each enzyme digests (cuts) DNA at a specific sequence  restriction site – Enzymes recognize 4-, 6- or 8- base pair, palindromic sequences

Common Restriction Enzymes EcoRI –Escherichia coli – 5’ overhang HindIII – Haemophilus influensae – 5’ overhang PstI –Providencia stuartii –3’ overhang 5’ CTGCAG 3’ 3’ CACGTC 5’ 5’ GAATTC 3’ 3’ CTTAAG 5’ 5’ AAGCTT 3’ 3’ TTCGAA 5’

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

Palindromic Sequences 5’ versus 3’ overhang: Sticky Ends Enzyme cuts  5’ GAATTC 3’ 3’ G 5’ 5’ G 3’ 3’ CTTAAG 5’ 5’ G 3’ 3’ CTTAA 5’ 5’ AATTC 3’ 3’ G 5’ Generates 5’ overhang 5’ and 3’ versus Blunt ends

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 humans and other eukaryotic organisms

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 GTTAAC GAATTC GTTAAC CTGCAG GAGCTC CGGCAG GCGCTC 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

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 (standard), 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

Electrophoresis Buffer TAE (Tris-acetate-EDTA) and TBE (Tris-borate-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 migration –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

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

DNA Staining Allows DNA visualization after gel electrophoresis Ethidium Bromide Bio-Safe DNA stains

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

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