Biological Techniques in Forensic Science: Capillary Electrophoresis and STR Separation

Overview of DNA Processing: STR Separation

  • Module Code: FORE20007 Biological Techniques in Forensic Science.

  • Host Institution: Nottingham Trent University (NTU).

  • Session Focus: Transitioning knowledge from standard gel electrophoresis to capillary electrophoresis (CE) and analyzing how specific errors impact DNA profiles.

  • Learning Objectives (MLO2):

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

    • Apply the principles of gel electrophoresis to the more advanced process of capillary electrophoresis.

    • Critically evaluate how errors within the process can affect the resulting DNA profile.

  • Curriculum Context: This session follows DNA Extraction, Quantification, and PCR/qPCR. It is the first of two parts on STR (Short Tandem Repeat) Separation and is supported by three labs (Online, PCR, and Electrophoresis) and two workshops.

Gel Electrophoresis vs. Capillary Electrophoresis

  • Gel Electrophoresis (GE) Basics:

    • DNA is loaded into wells (entry points/holes) within a gel matrix submerged in a buffer solution.

    • An electrical current is applied, creating a negative $(-)$ charge at the top and a positive $(+)$ charge at the bottom.

    • DNA fragments, which are naturally negatively charged, are forced to migrate through the gel matrix towards the positive $(+)$ electrode.

    • Size Differentiation: Small fragments travel through the gel matrix faster than larger ones.

    • Result Interpretation: Each band visible in the gel represents a group of DNA fragments of the exact same size.

    • Comparison: Standard DNA fragments of known size are run alongside the samples to provide a reference for size comparison.

  • Resolution Differences:

    • Standard gel electrophoresis allows for fragment differentiation of approximately >20 nucleotides.

    • Capillary Electrophoresis (CE) provides significantly higher resolution, allowing for differentiation down to a single base-pair (1 bp1\,bp).

  • Detection Method: While GE often uses staining, CE uses fluorescence detection. Fluorescent tags are bound to the DNA primers during the PCR (Polymerase Chain Reaction) stage.

  • Medium: CE moves through a needle-like capillary tube filled with polyacrylamide gel, as opposed to a flat agarose gel slab.

  • Single-Stranded Requirement: Capillary electrophoresis can only separate single-stranded DNA (ssDNA).

Components of the Capillary Electrophoresis (CE) Machine

  • Major Hardware Components:

    • Mechanical pump: Contains and manages the polymer.

    • Lower gel block.

    • Polymer bottle.

    • Detection window: The specific point where the laser interacts with the moving DNA.

    • Capillary array: A series of needles/tubes through which the DNA travels.

    • Oven: Regulates temperature during the run; associated with the positive $(+)$ electrode.

    • Electrodes: Creates the charge differential needed for migration.

    • Outlet buffer reservoir.

    • Inlet buffer reservoir.

    • Sample tray (Autosampler): Holds the 96-well plate with DNA samples.

    • Fan: Assists in temperature regulation.

  • The Capillary and Polymer:

    • The capillaries are housed within the machine; the well plate is placed underneath for analysis.

    • Capillaries are filled with a polymer (polyacrylamide gel).

    • Inert Walls: The walls of the equipment/capillary are chemically inert.

    • Sieving Mechanism: As DNA fragments move through the capillary, they interact with the polymer and must squeeze through microscopic holes. This is known as "sieving."

    • Polyacrylamide vs. Agarose: Polyacrylamide has much smaller holes than agarose, which is why it provides high resolution (1 bp1\,bp separation).

Setting Up for Capillary Electrophoresis

  • Sample Preparation:

    • Requires DNA samples that have undergone PCR.

    • Samples are fluorescently tagged via the primers used in PCR. This means when new DNA strands are manufactured, they carry a specific color.

    • Four primary colors are used for the DNA samples: Blue, Green, Yellow, and Red.

    • Samples are placed into a 9696-well plate.

  • Detailed Well Contents: Each individual well must contains three specific items:

    1. DNA Sample: Fluorescently tagged (Blue, Green, Yellow, or Red).

    2. Deionised Formamide (DF): Combined with heating to denature the DNA. This is essential because CE requires ssDNA.

    3. Internal Size Standard (ISS): Also known as an internal lane standard. These are DNA fragments of known sizes (e.g., 60 bp60\,bp, 80 bp80\,bp, 100 bp100\,bp, up to 550 bp550\,bp). These are tagged with a specific color (typically Orange) so the machine can distinguish them from the test samples.

The Separation and Migration Process

  • Initiation:

    • The 9696-well plate is placed into the machine.

    • The scientist tells the machine software which wells contain samples.

    • The capillary is filled with fresh polymer.

  • Electrokinetic Injection:

    • The capillaries move into the first set of samples.

    • A positive charge is applied to "pick up" or pull the sample into the capillary.

  • Migration Mechanics:

    • The fragments move from the negative $(-)$ to the positive $(+)$ end.

    • Short fragments: Move quickly due to less physical interaction with the polymer/capillary walls.

    • Long fragments: Move slower due to increased interaction with the polymer matrix.

Detection and Data Capture

  • Mechanism of Detection:

    • An Argon laser is directed toward the detection window.

    • As fragments pass through, the laser excites the fluorescent tags bound to the DNA.

    • The fluorescence emitted by the fragments is detected and documented as a reflection of the laser interaction.

  • Recorded Data Points:

    1. Migration Time: The amount of time taken for the fragment to travel through the length of the capillary.

    2. Color of the Tag: Identified via the Argon laser excitation.

    3. Fluorescence Intensity: Measured in RFU (Relative Fluorescence Units). Higher intensity (higher peaks) indicates more DNA of that specific STR repeat is present in the sample.

  • Data Visualization: The resulting data is displayed on a graph (electropherogram) where the X-axis typically represents base-pair size (increasing from left to right) and the Y-axis represents peak height (intensity).

Sizing and Comparison

  • Internal Lane Standard (ILS) Comparison:

    • The internal standard (e.g., fragments reaching up to 550 bp550\,bp) is detected simultaneously with the DNA sample.

    • Since the base-pair sizes of the standard are known, the machine compares the migration time of the unknown sample fragments against the standard to calculate their exact length.

  • Allelic Ladder Comparison:

    • CE determines the length of DNA fragments.

    • To determine the specific STR repeat number (e.g., Allele 1313, 1414, 1717, etc.), the sample is compared to an "Allelic Ladder."

    • The Allelic Ladder contains various DNA fragments of known STR repeat numbers for specific loci.

    • Example from BALB-JAX (Unknown Sample 1): If a peak aligns with fragment lengths 120.55120.55 and 124.50124.50, and the ladder indicates these correspond to repeat numbers 1616 and 1717, the sample is identified as having a 16,1716, 17 genotype (heterozygous).

Questions & Discussion

  • To Consider Regarding Errors and Protocol:

    • Question: What is the effect on the DNA profile if we do not add formamide into the wells?

    • Question: What happens if we do not add the internal size standard?

    • Question: If the lab temperature is very high, what may happen to the polyacrylamide polymer? How would this affect the migration results?

    • Question: If the Allelic Ladder sample fails to run correctly, will the other samples be successfully sized or genotyped?

    • Question: What results are expected from a positive quality control (QC) sample?

    • Question: What results are expected from a negative quality control (QC) sample?