Procedures for DNA Analysis

1
Procedures for DNA Analysis

2
Procedures for Forensics DNA Analysis

• Sample collection
• DNA isolation and preparation
• DNA quantification
• DNA analysis procedure
• Interpretation

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Procedures for DNA Analysis

• RFLP Analysis
• General Overview of Procedure Steps
• Analysis of and Visualization of DNA
• Advantages/Disadvantages, Power of
  Discrimination, Speed of Analysis

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Procedures for DNA Analysis

• PCR Amplification and Analysis
• Theoretical
• Analysis of PCR Product (Reverse Dot Blot,
  Agarose Gel, Polyacrylamide Gel)
• Applications (DQAa/DQA1, D1S80, STR, Amelogenin)
• Advantages/Disadvantages, Power of
  Discrimination, Speed of Analysis

5
RFLPOverview of Procedure

• Digest the genomic DNA with one or more
  restriction enzymes.
• Transfer the appropriate amount of each digest to
  a sterile microfuge tube.
• Add gel loading buffer.
• Load onto a 0.7 agarose gel and electrophorese
  at a low voltage.

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RFLP Procedure (contd)

• Stain the gel with ethidium bromide or SYBR Gold
  and photograph the gel.
• Denature the gel by soaking in an alkaline
  solution.
• Rinse with deionized water and then neutralize
  with Neutralization Buffer I.
• Transfer to charged Nylon Membrane.
• Wash with Neutralization Buffer II.

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RFLP Procedure (contd)

• Prehybridize the membrane
• Pour off the prehybridization buffer and add new
  prehybridization buffer with DNA probe added.
• Incubate and pour off solution.
• Wash membrane (4X)
• Cover the membrane with sheet of Saran Wrap and
  expose to X-ray film.

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Summarization of RFLP (Textbook Fig. 6.6
pp.72-73)

• Restriction enzyme digestion
• Separate fragments on agarose gel
• Transfer ss DNA to a nylon membrane
• Probing (hybridizing)
• Detection

9

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Autoradiograph FromActual Rape-MurderCase

• Blood samples from victim, suspect and vaginal
  swabs from victim.
• Digested with Pst I
• Hybridized with probes at D2S44, D17S79, D1S13,
  D18S27.

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Detection of DNA

• Radioactivity
• Chemiluminescence

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Power of Discrimination vs- Speed of Analysis

• Very High Power of Discrimination.
• Can differentiate between multiple contributors.
• Can analyze up to 15 different loci.
• Very laborious and time intensive.
• Difficult to automate.

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Polymerase Chain Reaction (PCR) DNA Amplification

• Process to amplify a specific region of DNA.
• The boundaries of the amplified product are
  defined by primers.
• The amplified product is called the amplicon.
• The process is sensitive, rapid not limited by
  the quality or quantity of DNA.

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PCR typically involves three different
temperatures

• Denature (94oC)DNA template strands separate
• Anneal (55-72oC)Primers bind or anneal to the
  DNA template.
• Extend (72oC)DNA polymerase extends the primers
  by copying the target region using the
  deoxynucleotides.

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A Typical PCR Temperature Profile
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DNA Amplification Process
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PCR Reaction

• 5 100 mL reaction volume
• 0.2 mL thin-walled tubes
• 96-well or 384-well plates

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PCR Reagents

• Tris-HCl pH 8.3
• Magnesium chloride
• Potassium chloride
• Deoxynucleotide triphosphates (dNTP)
• DNA polymerase (thermal stable)
• Bovine Serum Albumin (BSA)
• Primers
• Template DNA (1-10 ng genomic DNA)

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PCR Reaction Mix Bead
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Controls Used to Monitor PCR

• Negative Control Entire PCR reaction mix
  without DNA template. Make sure none of the PCR
  reagents are contaminated with DNA
• Extraction Blank Make sure the extraction
  reagents are free of extraneous DNA templates
• Positive Control A standard DNA template with
  known DNA that can be amplified with the same PCR
  primers. Make sure the reaction components and
  reaction conditions are working.

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Stochastic Fluctuation

• Can occur when amplifying very low levels of DNA
  template (lt100 pg DNA)
• Results in false homozygosity. One of the
  alleles fails to amplify.

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Thermal Cyclers

• The instrument circulates liquid at precisely
  controlled temperatures through a heat block.
• A heated lid keeps the PCR reagents from
  condensing at the top of the tube

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GeneAmp PCR System 240
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GeneAmp Thermal Block
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BIO RAD iCycler
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BIO RAD iCycler Thermal Block
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PCR Hot Start

• Optimal temperature for Taq is 72oC, but it
  exhibits some activity at lower temperatures.
• This can result in mis-priming and non-specific
  products.
• Hot start minimizes mispriming products by adding
  the polymerase after the temperature is raised
  above the annealing temperature.

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PCR Hot Start

• Can introduce cross-contamination between samples
  because tubes must be opened at the thermal
  cycler.
• AmpliTaq Gold DNA polymerase is chemically
  modified so that it is inactive until it is
  heated. (usually 95oC for 10-11 minutes)

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Primer Design

• Well designed primers are an important component
  of the PCR reaction.
• Primers must be specific to the target region.
• Must have similar annealing temperatures
• Must not interact with each other (primer dimers)

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Primer Design

• Primer Design software
• Gene Runner, Primer Express, Oligo
• Internet
• http//www.genome.wi.mit.edu/cgi-bin/primer/primer
  3_www.cgi

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Multiplex PCR

• More than one region of the DNA can be copied
  simultaneously by adding more than one primer
  set.
• The multiplex PCR reaction must be optimized to
  obtain a good balance between the amplification
  of the various products.

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Schematic of Multiplex PCR Reaction
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Analysis of PCR Product

• Hybridization
• Agarose Gel Electrophoresis
• Polyacrylamide Gel Electrophoresis
• Manual
• Automated

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Schematic of Hybridization Method for Reverse Dot
Blot
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Agarose Gel Electrophoresis
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Manual Polyacrylamide Gel (Silver Stained)
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Automatic Polyacrylamide Sequencer Detection
Printout
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Applications of the PCR Process in Forensic
Science

• DQAa/DQA1 (Reverse Dot Blot)
• D1S80 (16bp VNTR)
• Amelogenin
• STRs
• Y-STRs

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AmpliType PM DQA1

• A 242 bp region on Chromosome 6
• Reverse dot blot identifies 4 alleles and
  subtypes of alleles 1 and 4.
• Power of discrimination increased by using a
  polymarker (PM) system.
• Perkin-Elmer AmpliType PMDQA1 kit.

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DQA1 Polymarker Reverse Dot Blot
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D1S80

• Locus on Chromosome 1
• Repeat unit that is 16 bp in length
• Unit is repeated 14-40 times in the block
• 29 known different alleles

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D1S80 VNTR
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Amelogenin Gene

• Gene for tooth pulp
• The gene on the X chromosome is 6bp shorter than
  on the Y chromosome
• Can be identified with multiplex STR system

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Amelogenin Typing
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STR

• Short Tandem Repeats
• Thousands of microsatellites have been identified
  in the human DNA
• They occur about every 10,000 bp
• They are scattered on all 23 chromosomes

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Precautions Against Contamination

• Pre Post PCR sample processing areas should be
  physically separated.
• Equipment such as pipettors reagents should be
  kept separate
• Disposable gloves should be changed frequently
• Set up reactions in a laminar flow hood
• Use Aerosol-resistant pipet tips.
• Reagents must be nuclease free.
• UV irradiate workspaces

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Advantages of PCR

• Minute amounts of DNA template are needed
• DNA degraded fragments can be amplified
• Multiplex PCR reactions
• Contaminant DNA such as fungal or bacterial
  sources will not amplify because primers are
  human specific
• Commercial kits are available.

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Disadvantages of PCR

• The target DNA template may not amplify due to
  PCR inhibitors in the extracted DNA
• Amplification may fail due to sequence changes in
  the primer binding region of the genomic DNA
  template
• Contamination from other human DNA sources.

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Steps for Labs (VNTR and Alu)

• Sample collection
• DNA isolation and preparation
• DNA quantification
• DNA analysis procedure
• Interpretation

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VNTR Lab
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Alu Lab
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Alu Lab at UTHCT

• Angies sample
• Jasmines sample
• Jasmines sample
• Raos sample
• Tamekas sample
• -/- Control
• -/ Control
• -/ Control
• / Control
• Molecular Marker

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Alu Lab SFASU

• Molecular Marker
• 1st Sample
• -/- Control
• 2nd Sample
• /- Control
• 3rd Sample
• /- Control
• 4th Sample
• / Control

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Alu Lab SFASU

• Molecular Marker
• 5th Sample
• / Control

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Vocabulary

• Ethidium Bromide
• Southern Blot
• Hybridization
• Chemiluminescence
• Primers
• Amplicon
• DNA Polymerase
• Taq

• dNTPs
• Thermal Cycler
• Denature
• anneal
• Primer dimer
• VNTR
• STR
• Minisatellite
• Microsatellite

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References

• Text Chapters 5 6.
• Forensic DNA Typing John M. Butler Academic
  Press, 2001.
• Molecular Cloning 3rd Edition, Sambrook and
  Russell CSHL Press, 2000.
• DNA Technology in Forensic Science Committee on
  DNA Technology in Forensic Science, 1992.