Biological Techniques in Forensic Science: Capillary Electrophoresis and DNA Separation

Course Overview and DNA Processing Context

• Module Code: FORE20007 Biological Techniques in Forensic Science.

• Module Topic: DNA Processing: STR Separation.

• Sequence of DNA Processing:

  1. DNA: Recap.

  2. DNA Extraction.

  3. DNA Quantification.

  4. PCR (Polymerase Chain Reaction) & qPCR (Quantitative PCR).

  5. STR Separation I (Current Focus).

  6. STR Separation II.

• Learning Objectives (MLO2):

  • Apply understanding of gel electrophoresis (GE) to the technology of capillary electrophoresis (CE).

  • Explain the range of biological techniques available to forensic scientists.

  • Evaluate how various errors can impact final DNA profiles.

• Associated Labs/Workshops: The module includes three associated labs (Online, PCR, Electrophoresis) and two workshops.

Gel Electrophoresis (GE) vs. Capillary Electrophoresis (CE)

• Fundamentals of Gel Electrophoresis (GE):

  • DNA fragments are loaded into wells within a gel matrix (e.g., agarose) immersed in a buffer.

  • An electrical current is applied; DNA is negatively charged and migrates toward the positive ($+$) electrode.

  • Sieving Mechanism: The gel matrix acts as a sieve. Smaller fragments navigate the matrix faster than larger ones.

  • Bands: Each visible band on a gel represents a specific group of DNA fragments that share the same size.

  • Size Comparison: Sample sizes are determined by running a DNA standard (ladder) of known fragment sizes alongside the unknown samples.

• Advantages and Specifics of Capillary Electrophoresis (CE):

  • Resolution: GE generally allows for the differentiation of fragments with a difference of approximately > 20 nucleotides. CE provides significantly higher resolution, capable of distinguishing differences down to a single base pair ($1 \, \text{bp}$).

  • Medium: Separation occurs within a narrow, needle-like capillary tube filled with polyacrylamide polymer.

  • DNA State: CE is designed to separate single-stranded DNA ($\text{ssDNA}$) specifically.

  • Detection: Fragments are detected via fluorescence as they pass through a detection window, rather than staining the entire gel post-run.

Requirements for Capillary Electrophoresis Setup

• PCR-Amplified DNA Samples:

  • DNA fragments are fluorescently tagged during the PCR process.

  • Fluorescent tags are attached to the primers; therefore, newly synthesized strands carry the color label.

  • Tag Colors: Typically include Red, Blue, Green, and Yellow.

• Sample Plate Preparation:

  • Samples are organized in a standard 96-well plate.

  • Well Contents:

    • Fluorescently tagged DNA sample.

    • Deionised Formamide (DF): Used to denature the DNA. Combined with heat, it ensures the DNA becomes single-stranded ($\text{ssDNA}$) for the CE run.

    • Internal Size Standard (ISS) / Internal Lane Standard: These are known base-pair fragments (e.g., $60 \, \text{bp}$, $80 \, \text{bp}$, $100 \, \text{bp}$) tagged with a unique color, usually orange. The ISS runs simultaneously in the same well as the DNA sample to provide an accurate baseline for size determination.

Components and Mechanics of the CE Machine

• Instrumentation Architecture:

  • Autosampler: Moves the sample tray to position the 96-well plate.

  • Capillary Array: Houses multiple capillaries filled with polyacrylamide polymer.

  • Sieving Matrix: Polyacrylamide polymer has smaller pores than agarose gel, which facilitates the $1 \, \text{bp}$ resolution.

  • Electrodes: Apply the electrical current across the capillary (negative at the inlet, positive at the outlet).

  • Mechanical Pump: Used to fill the capillaries with fresh polymer.

  • Oven: Maintains a stable temperature environment for the capillaries.

  • Detection Window: A specific point in the capillary where the argon laser interacts with passing fragments.

  • Reservoirs: Includes inlet and outlet buffer reservoirs to maintain the electrical circuit and ionic environment.

  • Fan: Assists in thermal regulation of the equipment.

The CE Process: Injection and Migration

1. Preparation: The 96-well plate is loaded, and the user identifies sample locations in the machine software. Capillaries are flushed and filled with fresh polymer.

2. Electrokinetic Injection: The capillaries are lowered into the wells. A positive charge is applied to draw the negatively charged DNA fragments into the end of the capillary tube.

3. Migration:

   • Fragments travel from the negative terminal toward the positive terminal.

   • Short fragments: Move rapidly due to minimal interaction with the polymer matrix.

   • Long fragments: Move slowly due to more frequent interactions and "squeezing" through the polymer pores.

Detection and Data Interpretation

• Detection Mechanism:

  • Argon Laser: Directed at the detection window near the end of the capillary.

  • Excitation: As tagged fragments pass the laser, the fluorescent tags are excited and emit light at specific wavelengths.

  • Documentation: The reflection and emission are documented by the system's sensors.

• Data Parameters Captured:

  1. Migration Time: The time taken for a fragment to reach the detection window.

  2. Color: The specific wavelength/color of the fluorescent tag (Red, Blue, Green, Yellow, Orange).

  3. Fluorescence Intensity: Measured in Relative Fluorescence Units ($\text{RFU}$). Higher peaks on the graph correlate to a higher quantity of that specific DNA fragment (the specific STR repeat) within the sample.

• Determining Fragment Length (Sizing):

  • The machine compares the migration time of the sample fragments against the migration times of the Internal Lane Standard (the orange-tagged fragments of known length).

• Determining STR Repeat Number:

  • Allelic Ladder: A separate sample containing a "ladder" of all common known alleles (various fragments with known STR repeat counts) for specific loci.

  • By comparing the fragment length ($\text{bp}$) of the unknown sample to the Allelic Ladder, the machine assigns a specific STR allele number (e.g., an allele 16 at a specific locus).

Potential Errors and Considerations in CE Analysis

• Missing Formamide: If formamide is not added to the wells, the DNA may not remain single-stranded, potentially preventing it from entering the capillary or resulting in failed/unstable separation.

• Missing Internal Size Standard (ISS): Without the ISS, the machine cannot calibrate the migration time to fragment length ($\text{bp}$), making it impossible to size the DNA fragments.

• Temperature Fluctuations: High laboratory temperatures can affect the viscosity and stability of the polyacrylamide polymer, leading to inconsistent migration times and poor resolution.

• Ladder Failure: If the Allelic Ladder sample fails, the software cannot definitively assign STR repeat numbers (alleles) to the fragments, even if sizing is successful.

• Quality Controls:

  • Positive Quality Control Sample: Expected to yield a known, reproducible profile that matches the manufacturer's or lab's standard specifications.

  • Negative Quality Control Sample: Expected to show no DNA peaks (flat line), indicating the absence of contamination in the reagents or process.