Forensic DNA Fingerprinting Using Restriction Enzymes PDF

Forensic DNA Fingerprinting: Using Restriction Enzymes Why Teach DNA Fingerprinting? Real-world connections Tangible results Link to careers and industry Laboratory extensions Standards-based T...

Forensic DNA Fingerprinting: Using Restriction Enzymes Why Teach DNA Fingerprinting? Real-world connections Tangible results Link to careers and industry Laboratory extensions Standards-based The Forensic DNA Fingerprinting Kit Can Help You DNA structure Teach: DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping 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 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 Enzyme cuts Overhang Generates 5 prime overhang 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 37°C? 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  PstI EcoRI Restriction CTGCAG GAATTC Fragment Allele 1 GAGCTC GTTAAC Length 1 2 3 Polymorphism RFLP Allele 2 CGGCAG GCGCTC GAATTC GTTAAC Fragment 1+2 3 Different Base Pairs No restriction site M A-1 A-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 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 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 Fingerprinting Standard Curve: Semi-log Weight 100,000 Determination Size (bp) Distance (mm) 10,000 Size, base pairs 23,000 11.0 B 9,400 13.0 6,500 15.0 1,000 4,400 18.0 2,300 23.0 100 2,000 24.0 0 5 10 15 20 A 25 30 Distance, mm