Forensic DNA Evidence and Collection Techniques

Introduction to Forensic DNA Evidence

  • Quotation by Paul Kirk (1953):

    • "The blood or semen that (the perpetrator of a crime) deposits or collects – all these and more bear mute witness against him. This is evidence that does not forget… Physical evidence cannot be wrong; it cannot perjure itself; it cannot be wholly absent… Only human failure to find, study, and understand it can diminish its value."

    • Emphasizes the reliability of physical evidence in crime investigation.

Overview of DNA Sample Collection

  • Key Topics:

    • DNA Sample Sources

    • Biological Evidence at Crime Scenes

    • Evidence Collection and Preservation

    • Collection of Reference DNA Samples

    • Storage and Transport of DNA Evidence

Crime Scene Evidence Collection

  • Responsibilities of Police Officers and Investigators:

    • Responding to crime scenes to collect biological evidence for forensic DNA testing.

    • Must avoid contaminating evidence with their own DNA.

  • Reference to the National Institute of Standards and Technology.

Sources of Biological Evidence

  • Common Sources:

    • Blood

    • Semen

    • Saliva

    • Urine

    • Hair

    • Teeth

    • Bone

    • Tissue

Blood Sample Collection

  • DNA Extraction:

    • Very small amount of blood needed for DNA profiling.

    • Optimal results from samples with over 100 cells; profiles can be obtained from as little as one cell.

Quantity of DNA in Biological Samples

  • DNA Quantity Breakdown:

    • Diploid cell = 6 picograms

    • Haploid cell = 3 picograms

    • White Blood Cells (WBC): 30 μg/ml

    • Semen: 50,000,000 sperm/ml = 150 μg DNA/ml and 5,000 leukocytes/ml = 30 μg DNA/ml

    • Urine: Not a viable DNA source (requires large sampling area)

    • Saliva: Good source for PCR processes.

Processing of DNA Evidence in Laboratory

  • DNA Evidence Handling:

    • Samples received from the crime scene, victim, or suspect.

    • Sexual assault evidence collection kits include swabs and collection bags.

DNA Collection Methodologies

  • Typical Methods:

    • Use cotton swabs to collect biological materials (e.g., blood, semen).

    • Sensitivity of the PCR process has decreased the DNA amount required for analysis.

    • Samples must be collected efficiently to preserve integrity.

Collection of Reference DNA Samples

  • Blood Samples:

    • Collected by a phlebotomist.

  • Buccal Swabs:

    • Easier, involving scraping cells from the inside of the mouth.

Blood Collection Tubes

  • Color-Coded Cap System:

    • Lavender caps for DNA preservation:

    • Contains EDTA (chelating agent that binds to Mg ions necessary for nucleases to be active).

    • Functions as an anti-coagulant to maintain blood liquid state.

Whole Blood Sample Handling

  • Storage Recommendations:

    • Refrigerate blood promptly after collection.

    • Must be plated within 3-4 days using sterile materials (cotton swatch, FTA paper, filter paper).

    • Never freeze whole blood to avoid cell lysis.

FTA Paper

  • Acronym: Fast Technology for Analysis of DNA.

  • Characteristics:

    • Can be stored at room temperature.

    • Protects nucleic acids from degradation by nucleases.

Buccal Swab DNA Collection Procedure

  • Scrubbing the inner cheek less invasive than blood draw.

  • Dried swabs are essential for storage to prevent mold and bacteria.

Storing DNA Samples

  • Storage Conditions:

    • Air-dried before packaging.

    • Short-term storage at room temperature.

    • Ideally refrigerated at 4°C for weeks.

    • Long-term storage at -20°C or -80°C for extended periods.

Sample Handling Techniques

  • Clean Technique Development:

    • Aims to produce contamination-free samples, adapted from microbiological sterile techniques.

    • Aseptic techniques are not strictly necessary but are beneficial for avoiding contamination.

Avoiding Contamination in PCR Samples

  • Best Practices to Prevent Contamination:

    • Maintain designated lab areas.

    • Clean benches with 10% bleach and utensils with isopropanol.

    • Autoclave tubes and glassware for sterilization.

Personal Protective Equipment (PPE) for Analysts

  • Gloves:

    • Must be powder-free to prevent interference during serology and DNA analysis.

    • Powder may glow under light sources and act as inhibitory in PCR processes.

  • Lab Coats:

    • Designated coats for clean areas, taped sleeves recommended.

    • Each analyst should have distinct coats for DNA processing steps.

  • Protective Eyewear:

    • Use of goggles, safety glasses, and face shields required.

Bloodborne Pathogens Precautions

  • Universal Precautions:

    • Treat all samples as potential sources of pathogens (viruses, bacteria, fungi).

  • Essential Precautionary Measures:

    • Wear PPE, use 10% bleach or isopropanol to sanitize surfaces, discard waste properly, and wash hands after glove removal.

Hazardous Reagents in DNA Analysis

  • Common Hazardous Reagents:

    • Phenol-chloroform-isoamyl alcohol (PCI): used in extraction; phenol is caustic, chloroform is toxic and volatile; isoamyl alcohol is flammable.

    • Ethidium Bromide: used in gels; strong mutagen; handle carefully to avoid contact.

    • Formamide: used in DNA denaturation; teratogen, requiring a chemical fume hood for safety.

Sample Characterization Techniques

  • Tests Employed:

    • Presumptive tests for blood, semen, saliva.

    • Confirmatory tests for assessing blood stains, sperm presence, and saliva identification.

RNA for Sample Characterization

  • Gene Expression Examination:

    • Allows for tissue identification from specimens of blood, semen, vaginal secretions, and saliva.

Factors Affecting DNA Quantity

  • Influencing Factors:

    • Size of the stain, presence of nucleases, exposure to sunlight/heat/moisture, contaminants, and analyst proficiency.

Sample Cutting Guidelines

  • Process:

    • Separate questioned samples and known references to avoid contamination.

    • Only open one sample at a time; label tubes prior to sample insertion.

    • Prevent cross-contamination with clean utensils and frequent glove changes.

Use of Controls in DNA Analysis

  • Types of Controls:

    • Substrate Control: for samples from stained areas to assess DNA prevalence.

    • Manipulation or Reagent Blanks: monitor contamination from reagents and tools.

    • Known Reagent Blanks (KRB) and Question Reagent Blanks (QRB) used to ensure contaminant-free protocols.

Importance of Standard Reference Materials (SRM)

  • Controls Provided by the National Institute of Standards and Technology (NIST):

    • SRM are essential to validate protocols annually or with significant methodological updates.