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
CCC Food, Housing & Basic Needs Resources 2017 Survey . With information from the 2017 Survey, the Chancellor's Office developed the Basic Needs Survey Report. Provided base line data regarding available resources. Provided best practices and featured best practice models.
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