DNA Methodologies

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DNA Methodologies

• Sterilization
• Clean the workstation with alcohol and bleach.
• Autoclaving and ultraviolet light (UV radiation).
• Consumables and reagents.
• Equipment
• Pipettes- P20, P200, P1000
• Block heater
• Vortex
• Centrifuge
• PCR machine
• Electrophoresis box and power supply
• DNA sequencing machine
• Computer

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DNA Protocol

• DNA Extraction
• FTA Card
• Chelex
• Spin columns
• Organic- simple and differential.
• DNA Quantitation
• Direct, non-blot
• Direct, slot-blot
• Real Time PCR
• PCR Amplification
• Polymerase Chain Reaction
• DNA Sequencing or Typing
• Analysis and Interpretation

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Human Cell

• How do we get the nuclear and mitochondrial DNA
• out of the cell?

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DNA Extraction Protocols 1

• FTA Card
• Pipette aliquot on card.
• WBCs lyse on paper and DNA is trapped.
• Use hole punch to remove paper for DNA analysis.
• Wash.
• Analysis.

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FTA Protocol
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DNA Extraction Protocols 2

• Chelex
• Incubate blood sample in 5 Chelex at 56C for 30
  min.
• Boil for 8 min.
• Centrifuge to remove inhibitors and cellular
  debris.

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DNA Extraction Protocols 3

• Column Extraction
• Lyse cells and digest proteins.
• Physical, heat, detergent
• Proteinase K
• Bind DNA to silica membrane by centrifugation.
• Wash with 70 ethanol by centrifugation.
• Elute DNA with TAE (Tris-Acetate - EDTA) or water
  by centrifugation.

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DNA Extraction Protocols 4

• Organic Extraction
• Lyse cells and digest proteins.
• Physical, heat, detergent
• Proteinase K
• Organic extraction.
• Phenol-chloroform
• Centrifuge to remove supernatant.
• Precipitate DNA.
• Ethanol or isopropanol
• Centrifugation to pellet DNA.
• Elute DNA with TAE or water.

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Differential Extraction

• Technique used to separate sperm cells from
  non-sperm cells (epithelial cells).
• Vaginal swabs
• 1. Epithelial cells are lysed with a mild
  extraction buffer containing Sodium Dodecyl
  Sulfate (SDS).
• 2. Centrifugation to pellet sperm cells, DNA from
  epithelial cells is in the supernatant.
• 3. Lyse sperm cells using Dithiothreitol (DTT).

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DNA Quantitation

• DNA Quantitation
• Direct, non-blot
• Direct, slot-blot
• Real Time PCR
• Why quantitate?
• U.S. FBI Standards require it!
• 1ng-2.5ng yields consistent typing results.
• 1 cell 6.1pg (164 cells needed for analysis)
• Too much DNA leads to artifacts and too much
  signal.
• Too little DNA leads to allelic dropout.

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DNA Quantitation 1

• Direct, non-blot
• AluQuant (Promega Corp.)
• Human-specific probe that binds to Alu insertions
  (highly repetitious DNA).
• Luciferin-luciferase reaction and a luminometer
  to detect the amount of light.
• Emission is compared against standards.
• Yield Gel
• Standards of known concentration are compared
  against a sample.

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DNA Quantitation 2

• Direct, slot-blot
• QuantiBlot (Applied Biosystems)
• Human-specific probe (D17Z1) binds to DNA.
• Chemiluminescence is used to determine the DNA
  concentration against a set of standards.

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DNA Quantitation 3

• Real Time PCR
• Quantifiler (Applied Biosystems)
• Human genes are amplified.
• Gene number doubles after each cycle.
• Each cycle yields more fluorescence.
• Fluorescence is recorded and compared against
  standards to determine DNA concentration.