forensic dna fingerprinting: using restriction enzymes
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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Forensic DNA Fingerprinting: Using Restriction Enzymes
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DNA Fingerprinting Real WorldApplications • Crime scene
• Human relatedness• Paternity• Animal relatedness• Anthropology studies• Disease-causing organisms • Food identification• Human remains• Monitoring transplants
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Workshop Time Line
• Restriction digest of DNA samples• Introduction to DNA Fingerprinting and RFLP
analysis• Electrophoresis on Agarose gels • Analysis and interpretation of results
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DNA FingerprintingProcedureOverview
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DNA Fingerprinting ProceduresDay One
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DNA Fingerprinting ProceduresDay Two
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DNA Fingerprinting ProceduresDay Three
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DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus
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Model of DNADNA is Comprised of Four Base Pairs
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Deoxyribonucleic Acid (DNA)
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DNA Schematic
OCH2
O
P OO
O Base
CH2
O
PO
O
O
Base
OH
Sugar
Sugar
O
Phosphate
Phosphate
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DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes
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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
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5 vs 3 Prime Overhang
• Generates 5 prime overhang
Enzyme cuts
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Common Restriction Enzymes
EcoRI– Eschericha coli– 5 prime overhang
Pstl– Providencia stuartii– 3 prime overhang
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The DNA DigestionReaction Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor
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DNA DigestionTemperature
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
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Restriction Fragment Length PolymorphismRFLP
Allele 1
Allele 2
GAATTCGTTAAC
GAATTCGTTAAC
CTGCAGGAGCTC
CGGCAGGCGCTC
PstI EcoRI
1 2 3
3Fragment 1+2Different Base PairsNo restriction site
+
M A-1 A-2
Electrophoresis of restriction fragments
M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments
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AgaroseElectrophoresisLoading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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AgaroseElectrophoresisRunning
• Agarose gel sieves DNA fragments according to size– Small fragments move farther than large fragments
Power Supply
Gel running
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Analysis of Stained Gel
Determinerestriction fragmentsizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
Identify the relatedsamples
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Molecular Weight Determination
Size (bp) 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
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Size
, bas
e pa
irsB
A
Fingerprinting Standard Curve: Semi-log