While the forensic and general communities continue to argue about the merits of the recent Supreme Court ruling on collection of samples from arrestees prior to conviction, I am fascinated by the technology that make this question relevant. The conventional way of generating a DNA profile from a sample by STR (short tandem repeat) analysis is a long process involving a series of steps that require sophisticated expensive equipment, trained personnel and, more importantly, time. The actual process of DNA analysis consists of a) sample collection, b) DNA extraction, c) PCR amplification using 16 or more unique fluorescently labeled primer sets d) capillary electrophoresis to size labeled DNA amplicons, e) software analysis to size DNA fragments and allele calls based on migration of allelic ladder fragments, and f) comparison to known profiles in the database. This entire workflow can typically take days or even weeks, and therefore it is not surprising that we see newspaper reports of backlogs of criminal and other property cases. With these time ranges, the sample collected at a site would be of no practical use to most ongoing investigations.
Current advances in STR technology are focused largely on making the system (reagent kits and the instrument) more sensitive, robust and tolerant to inhibitors such as hematin, typically found in crime scene samples, while the giant leap in this field has been made by miniaturizing the workflow—Lab-on-a-chip is the new mantra. Miniaturization has decreased the space and time factor for many processes such as drug discovery, enzyme assays, as well as multiple cell biology applications. Lab-on-a-chip systems are small, require reduced amounts of reagents and are usually developed for point-of-care use. They are therefore rugged and simple to use with minimal user intervention. While the adoption of this technology in local police stations for arrestee DNA is debatable, there is no doubt that this streamlined automated fast workflow protocol, when adapted by adoption and immigration agencies, would go a long way towards significantly reducing backlogs. The high analytical efficiencies, fast processing times, reduced consumption of expensive biological reagents and portability make it suitable for a range of applications.
How do Miniaturized Systems Make DNA Analysis Faster?
So, what makes the process so efficient and fast in small volume format? Fluids flowing in microsystems have many interesting and useful characteristics such as laminar flow (versus turbulent flow in macro-channels), which accelerate reaction kinetics and reduce assay performance time. Moreover, when an ion-containing fluid (for example, water) is placed in a microchannel that has fixed charges on its surface (such as silicon dioxide or surface-oxidized PDMS) and an electrical potential is applied along the channel, the fluid moves as a plug, rather than with the parabolic-flow profile observed when pumping is accomplished by applying pressure to the fluid. Electro-osmotic flow minimizes the broadening of sample plugs that occurs with many pressure-driven systems and allows very high resolution separations of ionic species. It is a key contributor to electrophoretic separations of DNA in microchannels and helps with resolution of fragments differing in 4 or less basepairs.
There are a few Rapid DNA technologies available commercially. For example, IntegenX and NetBio offer lab-in-a-box formats. Both feature consumable cartridges that accept buccal swab samples inserted into a small instrument that automates the remaining steps in the workflow. It starts by moving the swabs to a lysis solution compartment where the cells are lysed. The lysate is then allowed to interact with silica beads or membranes to allow DNA binding, followed by washes and elution steps. A portion of the eluted DNA is channeled into another chamber, situated on a heating element and containing the reagents for PCR amplification – namely primers, dNTPs and Taq polymerase. Following PCR, a portion of the reaction is mixed with formamide and internal lane standard and separated by electrophoresis. As the fragments resolve on the basis of size they are detected by fluorescence. The data is then analyzed by comparison with migration of allelic ladder fragments, which are run in parallel with the samples. The entire process from sample to profile is completed in about two hours with no user intervention after introducing the buccal swabs into the instrument.
Use of microfluidics devices allows sample analysis in a closed system and, while reducing the number of steps, also minimizes the chances of contamination. The advantages that microfluidic technology brings are quite clear with regard to STR analysis. The only potential drawback is that the user does not have options to optimize the various steps such as template amount, number of PCR cycles in this integrated workflow. However, with a robust system of wide linear dynamic range for signal processing and capable of handling large variations in input sample, Rapid DNA technology is indeed ready for wider deployment.
- Estes, M.D. et al. (2012) Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis. Analyst, 137(23): 5510-9.
- Liu, P. et al. (2007) Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing. Anal Chem 79(5): 1881-9.
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