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Capillary Electrophoresis and the DNA Forensics Renaissance

Capillary Electrophoresis and the DNA Forensics Renaissance

Capillary electrophoresis has driven improvements in throughput and sensitivity for DNA profiling of short tandem repeats

Brandoch Cook, PhD

The first criminal to be caught, tried, and convicted with the aid of DNA evidence was a depraved and elusive recidivist with the apt and grisly surname Pitchfork. The University of Leicester professor Alec Jeffreys, who had developed methods using electrophoresis to analyze minisatellite DNA for paternity and immigration cases, pivoted his technology to address two similar crime scenes that had been canvassed three years apart in the mid-1980s. As it turned out, both crimes were committed by Pitchfork, who tried unsuccessfully to evade an exhaustive regional testing regime. Jeffreys developed DNA profiling techniques in an era that pre-dated the ubiquity of polymerase chain reaction (PCR) technology, and therefore required isolation and resolution of large DNA fragments on agarose or polyacrylamide gels, followed by radiolabeling and development on X-ray film to detect variations in size. In its earliest incarnation, DNA profiling was thus an unwieldy time- and resource-consuming technique.

In principle, electrophoresis can separate and resolve nucleic acids, proteins, or biopolymers by size, by applying a field of voltage across a slab gel or other porous and conductive substance of known composition. The phosphate groups in DNA impart a negative charge, and a corresponding movement away from a negative cathode, with speed proportional to the size of the fragment loaded into the gel. Proteins can be separated by mass or charge, most commonly via SDS-PAGE, in which a strong anionic detergent denatures proteins, coating them with a uniform negative charge. Application of voltage across a polyacrylamide gel works analogously to the electrophoretic separation of DNA in agarose. In both cases, the gel acts as a molecular sieve, with different pore sizes intrinsic to the material.

The incipient potential of DNA profiling took hold at an advantageous time, aligning with breakthroughs in throughput from thermocyclers that automated and streamlined the polymerase chain reaction, and by improvements in electrophoresis apparatuses and techniques, notably through high-performance capillary electrophoresis. Moreover, a switch from minisatellites to analysis of STRs, short tandem repeats of three- to five-base pair stretches of DNA, created conditions in which forensic specialists could realistically identify unique individual DNA fingerprints from unknown samples, quickly resolving size differences in amplified products of less than 500 base pairs. For instance, the five-base pair STR Penta E is present in a range of five to 24 repeats, with two unique alleles being independently inherited from the mother and father. 

In 2017, DNA profiling protocols expanded from examining 13 core STRs to analyzing 20 separate and internationally standardized STR loci, which are evenly distributed across most autosomes. The STR amelogenin can additionally distinguish the Y chromosome and aid in gender distinction, confirm paternity, and decipher sexual assault evidence. The Federal Bureau of Investigation established CODIS in 1998 as a central database for full and partial DNA profiles that satisfy minimum STR thresholds, submitted by users applying approved techniques and reagents. In this way, DNA fingerprints can be compared and potentially matched across different crime scenes to identify patterns and repeat offenders, and to catch or eliminate suspects.

Related Article: A Closer Look: DNA Profiling in Forensic Investigations

Capillary electrophoresis (CE) adapts the principles of gel electrophoresis to make them more versatile and potentially several orders of magnitude more sensitive. A CE apparatus consists of an injection system, a high voltage source, a narrow capillary tube (often treated with a UV-transparent coating), an electrode, and a detector. The readout is an electropherogram with a series of distinct peaks for each allele of each detectable STR that corresponds to its number of repeats. For submission to CODIS, the resulting profile must be at least partially complete, with eight of 13 original core STRs represented. The highest sensitivity detectors are laser-assisted and can resolve several different dyes simultaneously, contributing to throughput by allowing CE apparatuses to run multiplexed reactions. The application of high voltage allows quick resolution of size differences, and capillaries impregnated with substances such as hydroxyethyl cellulose and polyvinyl pyrrolidone can resolve one- to three-base pair discrepancies from STRs as long as 600 base pairs. Changes to capillary type and buffer composition can additionally allow adaptation of CE-based protocols for forensic toxicology.

At the leading edge of CE technology, Promega has developed the Spectrum system to coordinate with its PowerQuant and PowerPlex family of reagents, using 8-dye detection with additional compatibility to reagents supplied by Applied Biosystems and Qiagen. The Spectrum CE system will allow co-amplification of all 20 CODIS STRs, with the ability to directly amplify DNA from FTA cards. Profiling analysis can be completed with integrated and automated GeneMapper ID-X and HID-Spectrum software packages.

As CE-based STR analysis has become the gold standard for forensic DNA profiling, one major caveat is that sensitivity has increased to the point that samples can be amplified and profiled from miniscule starting amounts, such as a couple of skin cells on a door knob. In a realistic crime scene, such a point of contact may be contaminated many times over by confounding evidence, resulting for instance in the resident of a house being over-represented in the ensuing analysis compared to the assailant. A new wave of forensic science is moving toward probabilistic genotyping to analyze data and predict the most likely scenarios based on relative representation within an STR dataset. Both Cybergenetics TrueAllele and STRmix rely on mathematical modeling via Markov chain Monte Carlo algorithms to achieve statistical confidence.

Another emerging development is application of microfluidics technology to DNA profiling. Several STR profiling on-a-chip platforms have the potential to further streamline PCR and analysis by integrating them into microfluidic devices with very small volumes and short run times. Two tangential benefits are to 1) mitigate experimental and replication errors that can be introduced by human hands, and 2) decrease reagent use and therefore waste and expense. The ANDE portable rapid DNA profiling system decreases PCR run times from four hours to 17 minutes via microfluidic integration with high-speed thermocycling, and returns a DNA profile in under two hours to speed evidence gathering at high-pressure areas such as border crossings and police booking stations. The forensic science of DNA profiling has come an incredibly long way in a little over 30 years.

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