PCR DNA analysis

DNA Typing Protocols Overview

  • Authors: Bruce Budowle, Jenifer Smith, Tamyra Moretti, Joseph DiZinno
  • Affiliation: FBI Laboratory Division and FBI Academy

PCR-Based DNA Analysis

  • Polymerase Chain Reaction (PCR): A core molecular biology technique for amplifying specific DNA sequences.
    • Offers higher sensitivity and specificity compared to older methods (e.g., RFLP).
    • Capable of amplifying small quantities of degraded DNA (picogram/nanogram level).
    • Process timelines: results within a few days, typical amplification in 1-2 hours.

PCR Process Steps

  1. Denaturation: Heating DNA to 95°C to separate strands.
  2. Annealing: Lowering temperature (45°-65°C) allows primers to bind to target sequences.
  3. Extension: Raising temperature to 72°C facilitates DNA polymerase in copying the target DNA.
  • These steps form one cycle; typically repeated 25-36 times for exponential amplification.

Reagents Required for PCR

  • Components: Template DNA, oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), buffer, cations, thermostable DNA polymerase (e.g., Taq).

Considerations for Forensic Samples

  • Potential contamination with inhibitors during DNA extraction.
  • Use of Bovine Serum Albumin (BSA) can mitigate PCR inhibition.

Laboratory Protocol Precautions

  • Implement dedicated equipment for different PCR stages (e.g., extraction, setup, analysis).
  • Use disposable gloves and change them frequently; avoid sample cross-contamination.

Typing Techniques for PCR

  • ASO Probes: Used in reverse dot blot format to identify specific alleles at loci.
  • The HLA-DQA1 locus represents a key application with commercially available kits for multiplex analysis.

Advantages of Multiplex Systems

  • Less DNA is used, yielding more data with reduced labor and risk of contamination compared to single-locus analyses.
  • Efficiently provides high discriminatory power for forensic applications.