Archived | Amplified DNA Product Separation for Forensic Analysts | Principles

Capillary electrophoresis (CE) is an effective tool for the separation of a variety of compounds and materials and is used in the medical and scientific communities.⁰¹ CE is also widely used in the forensic science community in areas such as gunshot residue analysis, explosive analysis, drug analysis, and pen inks analysis.⁰² The focus of this module is routine forensic DNA analysis by CE, which was first introduced in the mid 1990's. It is important to realize that CE is now a well-validated procedure that meets the requirements of Frye, Daubert, and the Quality Assurance Standards for Forensic DNA Analysis.

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A comparison of some features of capillary and slab-gel electrophoresis is presented in the table below.

Comparative Performance of Capillary
Electrophoresis and Slab-Gel Systems
Feature	Capillary Electrophoresis (AB 31x series)	Slab Gel (AB 37x series, FMBIO, MiraBio)
Ease of use	Less time required to: Set up instrument Prepare samples Perform separation	More time required to Set up instrument Prepare samples Perform separation
Automation	Fully automatable (sample preparation, injection, separation and detection)	Requires manual sample loading and some instruments require gel handling for scanning or photographing after electrophoresis⁰³
Reproducibility	Better reproducibility: Bubbles are not problematic Liquid polymer contained in capillary – no thickness issue	Poorer reproducibility: Bubbles are problematic Uneven gel thickness
Resolution	Increased resolution due to more efficient heat dissipation	Decreased resolution due to less efficient heat dissipation⁰⁴
Cross- contamination	Automatic sample loading includes rinsing step and samples are contained within the capillary	Manual sample loading into slab gels can result in leakage into adjacent wells
Sample Consumption	Lower sample consumption: Very small quantities of sample are consumed in the injection step Samples can be easily retested through reinjection from the original sample vial	Higher sample consumption: More sample is required to be loaded in each lane If retesting, the sample must be prepared and loaded into a new gel
Detection and Imaging	Sample data is collected and can be viewed in realtime on the system computer	Viewing in realtime on the computer is not possible with the FMBIO

CE methods can provide quantitative information. For example, forensic analysis of illicit drugs can employ CE methods to determine the quantity of a controlled substance in a sample. The use of CE in forensic DNA analysis provides some quantitative information on the components of an amplified DNA mixture. This does not necessarily represent quantitative differences in the original example because preferential amplification during the PCR process can affect the proportions of detectable DNA.

One major disadvantage of CE is the throughput. Electric fields used with CE instruments are considerably higher (~300V/cm) than those used for slab gel-based instruments (~10V/cm), resulting in faster run times. However, the single-capillary ABI Prism^® 310 Genetic Analyzer can analyze only one sample at a time. In contrast, many slab-gel methods allow numerous samples to electrophorese simultaneously, allowing greater throughput even though the electrophoresis time is approximately twice as long as that in CE.

The development of capillary array electrophoresis (CAE) instruments, which allow for multiple samples to be run in parallel, resulted in throughput capabilities equal to or surpassing that of slab-gel methods.

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Date Created: July 21, 2023