DNA Quantification

Why Quantify DNA?

• Essential to isolate human DNA from nonhuman sources at crime scenes.
• FBI’s quality assurance standards (Standard 9.4) mandate human-specific DNA quantitation.
• DNA quantification aids in determining optimal DNA template for PCR reactions.
• Optimal input for STR kits: 0.5 to 2.0 ng human DNA.

Quantification Importance

• Helps in avoiding overamplification and allele dropout in PCR.
• Process called normalization adjusts DNA quantity as necessary.
• Effective DNA quantitation conserves samples and streamlines testing.

DNA Quantities Used

• 1 ng human DNA corresponds to ~303 genomic copies.
• Typical range for DNA quantities: 0.1 to 25 ng (~30 to 8330 copies).

DNA Quantitation Methods

• Various techniques include:
  • UV Absorbance: Low sensitivity; can be contaminated.
  • Yield Gels: Requires considerable sample; not DNA specific.
  • Slot Blot: Human-specific, visual comparison; no longer widely used.
  • PicoGreen Assay: Rapid, low sample requirement but not human-specific.
  • AluQuant System: Sensitive, high abundance in human genome; now largely replaced.
  • End-Point PCR: Measures amplifiability; less expensive than qPCR.
  • Real-Time qPCR: Monitors PCR process in real time; preferred method for quantitation.

Real-Time qPCR Details

• Analyzes PCR process via fluorescence; assesses DNA quantity and quality.
• TaqMan probes facilitate quantitation; overall efficiency monitored through C_T values.
• Provides robust data for determining initial DNA template amounts.