Forensic DNA Fingerprinting: Using Restriction Enzymes DNA Fingerprinting

Forensic DNA Fingerprinting: Using Restriction Enzymes DNA Fingerprinting

Forensic DNA Fingerprinting: Using Restriction Enzymes DNA Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification

Human remains Monitoring transplants DNA Fingerprinting Procedures Day One DNA Fingerprinting Procedures Day Two DNA Fingerprinting

Procedures Day Three 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 Enzyme Site Recognition Restriction site Palindrome Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair,

palindromic sequences (eg GAATTC) 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 Mg2+ is an enzyme co-factor DNA Digestion Temperature Why incubate at 37C? Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? Too hot = enzyme may be denatured (killed)

Too cool = enzyme activity lowered, requiring longer digestion time Agarose Electrophoresis Loading Electrical current carries negativelycharged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel

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

Power Supply Classroom obstacle course 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 Restriction Fragment Length Polymorphism RFLP Allele 1

PstI EcoRI CTGCAG GAGCTC GAATTC GTTAAC 1 Allele 2 2

3 CGGCAG GCGCTC Different Base Pairs No restriction site GAATTC GTTAAC 3 Fragment 1+2

M Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments + A-1 A-2

Molecular Weight Determination Distance (mm) 23,000 11.0 9,400 13.0 6,500

15.0 4,400 18.0 2,300 23.0 2,000 24.0

100,000 10,000 Size, base pairs Size (bp) Fingerprinting Standard Curve: Semi-log B 1,000 100

0 5 10 15 Distance, mm 20 A 25

30

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