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DNA Typing Methods RFLP- restriction fragment length polymorphism.
AmpliType®PM+DQA1 DNA sequencing Mitochondrial DNA typing. STR- short tandem repeats (PCR based).
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DNA Typing and Criminal Investigations
1980s- Lynda Mann and Dawn Ashworth. Richard Buckland- kitchen porter. Confessed to the second murder. Scotland Yard. Semen samples and blood from Buckland taken. Dr. Alec Jeffreys at Leicester University. DNA Fingerprinting. Buckland was not the rapist/murderer! Voluntary blood submission and DNA testing. Colin Pitchfork and Ian Kelly- bakery workers. Kelly bragged about the blood switch. Colin Pitchfork was arrested and subsequently confessed in 1987. DNA analysis confirmed match.
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Restriction Enzymes An enzyme that cuts DNA at specific internal sites in the nucleotide sequence. They recognize specific double stranded sequences in DNA. Recognition sites are 4-8 base pairs (bp) long. Recognition sites are palindromes. Arrows indicate cut sites.
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Restriction Digestion
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The First Human Genetic Fingerprint
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Gel Electrophoresis Technique used to separate DNA on the basis of size (number of base pairs). DNA samples (mixed with loading dye) are loaded into wells. Samples are pushed through a gel (agarose or acrylamide) while under the influence of an electrical field. DNA is negatively charged due to phosphate groups and thus migrates towards the positive electrode (anode). The larger the DNA, the slower it travels.
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DQA1
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DQA1
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AmpliType®PM+DQA1 DQA1 PM
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PCR- polymerase chain reaction
Kary Mullis- Nobel Prize in chemistry in 1993. PCR is used to target and replicate any segment of DNA. Copy high numbers (1 trillion) of target in 2-3 hours. A technique that involves repeated cycles of 3 steps: Denature Anneal Extension
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DNA Amplification with the Polymerase Chain Reaction (PCR)
Separate strands (denature) 5’ 3’ Starting DNA Template 5’ 3’ Add primers (anneal) 5’ 3’ Forward primer Reverse primer 5’ 3’ Make copies (extend primers)
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Thermal Cycling Temperatures
94 oC 94 oC 94 oC 94 oC Single Cycle 72 oC 72 oC 72 oC Temperature 60 oC 60 oC 60 oC Time Typically cycles performed during PCR
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PCR Components Taq DNA Polymerase- catalyzes the attachment of the nucleotides to the growing strand of DNA. DNTPs- deoxynucleotide triphosphates (A,T,G,C). MgCl2- required to activate Taq Polymerase. Buffer- salt and pH balanced. Primers- a single-stranded short chain of nucleotides (10-30 nucleotides in length). DNA Template Water
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PCR Reaction 25 µL reactions Thermocycler
PCR tubes- thin, for rapid heat transfer. Thermocycler An instrument that is programmed to repeatedly raise and lower the temperature of a heating block. Cycling Parameters 94°C for 3 minutes- initial denaturation. 26-40 cycles of the following: 94 °C for 30 seconds. 55 °C for 30 seconds. 72 °C for 60 seconds. 72°C for 5 minutes- final extension. 4°C until processing.
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PCR Products PCR products are separated on agarose gel.
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PCR products can be sequenced
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ABI 377 DNA Sequencing Machines
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DNA Separation occurs in minutes...
Capillary Electrophoresis (CE) Fill with Polymer Solution Argon Ion Laser m x 27 cm 5-20 kV - + Burn capillary window Inlet (cathode) Outlet (anode) DNA Separation occurs in minutes... Data Acquisition and Analysis
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Capillary Electrophoresis- AB 310
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ABI Prism 310 Genetic Analyzer
capillary Syringe with polymer solution Injection electrode Autosampler tray Outlet buffer Inlet buffer
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Close-up of ABI Prism 310 Sample Loading Area
Electrode Capillary Sample Vials Autosampler Tray
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Capillary System
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DNA Separation Mechanism
+ - DNA- Size based separation due to interaction of DNA molecules with entangled polymer strands. Pumpable polymer can be replaced after each run. Polymer length and concentration determine the separation characteristics.
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ABI 3100 Array Detection
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Fluorescent Dyes Used in 4-Color Detection
JOE (Green) FAM (Blue) FL ROX (Red) CXR NED TAMRA (Yellow)
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Fluorescent Emission Spectra for ABI Dyes
5-FAM JOE NED ROX ABI 310 Filter Set F 100 80 60 Normalized Fluorescent Intensity 40 20 520 540 560 580 600 620 640 WAVELENGTH (nm) Laser excitation (488, nm)
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Principles of Sample Separation and Detection
Labeled DNA fragments (PCR products) Capillary or Gel Lane Size Separation Sample Detection CCD Panel Ar+ LASER (488 nm) Color Separation Detection region ABI Prism spectrograph Fluorescence
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MALDI-TOF Mass Spectrometry
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Short Tandem Repeats (STRs)
AATG 7 repeats 8 repeats The repeat region is variable between samples while the flanking regions where primers anneal are conserved. Homozygote- both alleles are the same length. Heterozygote- alleles differ in length.
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Multiplex PCR Multiple PCR reactions are run simultaneously in 1 tube.
Up to 16 reactions. <1 ng sensitivity. Different fluorescent dyes used to distinguish STR alleles with overlapping size ranges.
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Position of Forensic STR Markers on Human Chromosomes
13 CODIS Core STR Loci CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA AMEL Sex-typing
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STR Primers
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Fluorescent Labeling of STR PCR Products
Dyes are attached to one primer in a pair used to amplify a STR marker. Dye-labeled oligonucleotide is incorporated into PCR product during multiplex PCR amplification giving a specific color “tag” to each PCR product.
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AmpFlSTR® Identifiler™
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 D18S51 TPOX VWA AMEL D5S818 FGA GS500 LIZ size standard 6FAM (blue) VIC (green) NED (yellow) PET (red) LIZ (orange)
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