Scientists from the National Institute of Standards and Technology and Applied Research Associates have published a study demonstrating an improved microfluidic technique to purify, concentrate, and quantify DNA from clean and soil-contaminated buccal swabs prior to STR analysis.
According to the researchers, the technique, called gradient elution isotachophoresis, is able to recover and even quantify DNA from such samples more quickly, completely, and with less effort than existing methods, making it promising for use in human identification and other applied markets.
However, although the method works well for crude samples containing a relative abundance of DNA, it still yields significantly less DNA than existing techniques from highly dilute samples — a shortcoming that the researchers believe they can optimize in future iterations of the technology.
Forensic DNA testing typically requires a skilled technician in a properly equipped laboratory to extract, quantify, and PCR amplify DNA from a sample for STR analysis — a process that can take one to two days, according to a statement from NIST.
However, when the DNA is sourced from crude samples such as soil, the contaminants and particulates mixed in with the genetic material can cofound STR analysis. Thus, additional purification steps are needed, which can further lengthen processing time and reduce the quantity and concentration of DNA delivered — making human identification even more difficult or impossible. Crude samples can also be challenging for more automated and rapid methods of DNA purification. For instance, magnetic bead-based platforms, which are routinely used in laboratories, often do not eliminate all contaminants when purifying DNA from a crude sample.
The new technique from NIST and ARA is based on one the researchers developed four years ago called gradient elution moving boundary electrophoresis. Called GEMBE for short, this technique separates specific components of a sample by using an electric field to push sample in one direction and counterflow of a buffer solution to push it in the other direction. Gradually reducing the buffer flow allows selected components from the sample to pass into a microfluidic channel for analysis while unwanted components are excluded.
The new method, described in a paper published this month in Electrophoresis, is a modified version of GEMBE in which an electric field is applied across a leading electrolyte solution composed of electrophoretically fast ions, and a trailing electrolyte solution composed of electrophoretically slow ions, resulting in isotachophoretic focusing of DNA at the interface between these solutions.
Meanwhile, a pressure-driven counterflow controls the movement of the interface from the sample reservoir into a microfluidic capillary. Further, by choosing appropriate leading and trailing electrolyte solutions, PCR inhibitors may be prevented from focusing along with the DNA, the researchers wrote.
To demonstrate their technique, the researchers extracted, purified, quantified, and concentrated human genomic DNA from both clean buccal swabs and laboratory-prepared "solied" buccal swabs to mimic crude environmental samples
To determine the amount of DNA delivered from each sample, the researchers performed qPCR using a 7500 Real-Time PCR system and Quantifiler Human DNA Quantification Kit, both from Life Technologies. They also performed downstream STR analysis on purified samples using a Promega PowerPlex 16 HS STR Amplification Kit running on a GeneAmp PCR system from Life Tech. In all cases, STR analysis of the delivered DNA consistently yielded full STR profiles from both clean and soiled buccal swab samples.
In addition, the researchers used laser-induced fluorescence to quantify the DNA "on-line;" in other words, during the step in which the purified DNA is delivered into the microfluidic channel. They were able to successfully correlate these measurements with downstream quantitative PCR measurements, thus demonstrating that use of the gradient elution isotachophoresis technique may potentially eliminate the need for quantitative PCR prior to STR analysis.
The NIST and ARA group also tested their method on clean and soiled buccal swabs that they had diluted by either one-third or one-tenth. This dilution step, they noted, resulted in a significant decrease in the amount of delivered DNA and, consequently, a loss of genetic information since STR profiles had an increasing number of alleles lost of loci dropped from the profiles.
In this aspect of the study, they wrote, the gradient elution isotachophoresis method "consistently delivered approximately 1 percent of the DNA in a sample, which is significantly less than the approximately 16 to 33 percent typical of more conventional techniques. Despite this drawback, the results shown here indicate that the [gradient elution isotachophoresis] method is adequate for DNA delivery from crude samples containing a relative abundance of DNA, as demonstrated using the soiled buccal swab samples. Further optimization of the GEITP method is expected to increase the efficiency of DNA purification and concentration for delivery."
The gradient elution isotachophoresis method calls to mind a technology called Synchronous Coefficient of Drag Alteration, or SCODA, which is used in a commercial DNA purification platform called Aurora currently being marketed by Boreal Genomics (PCR Insider, 10/6/2011).
The SCODA technology uses rotating electric fields to selectively concentrate nucleic acids in an agarose gel based on physical characteristics such as charge species, linear charge density, size, and conformational entropy. Boreal has shown that the Aurora system can efficiently extract DNA from samples where there is a low amount of DNA and large amounts of PCR inhibitors — soil, sea sediments, stool, and blood, for example.
Boreal has also incorporated the SCODA technology into a platform to enrich mutant DNA over wildtype DNA, as reported in March in Clinical Sequencing News.
In their paper, however, the researchers from NIST and Applied Research Associates noted that the SCODA method "generally requires additional steps to reduce the ionic strength of the sample solution, and the analysis time is limited by joule heating and DNA reputation."
In general, the researchers noted that their gradient elution isotachophoresis method has several advantages that make it more attractive than existing methods for DNA purification in front of human identification analysis. First, it requires no sample prep aside from suspension in a buffer solution for cell lysis. In addition, no analysis steps are required to further purify or concentrate the DNA after delivery prior to STR analysis, while the "on-line" DNA quantification can potentially eliminate the need for qPCR-based DNA quantification following delivery.
What's more, nanogram amounts of DNA can be delivered into a small volume of less than 10 uL, which is "well-matched to the volume requirements for input into the multiple PCR reaction for STR analysis." And finally, the method delivers DNA rapidly from crude samples in less than five minutes after cell lysis — approximately three to four times faster than typical magnetic bead-based methods and an order of magnitude faster than the SCODA method, the researchers wrote.
A NIST spokesperson told PCR Insider in an email that the group is currently collecting data regarding the speed and efficiency of its technique compared to existing methods and plans to publish these results at a later date.
The researchers also noted that their method is amenable to miniaturization for potential use outside of a laboratory, and is suitable for incorporation as a sample prep step at the front end of a more complete DNA analysis system.
Further, "avenues for improved extraction efficiency are being explored to adapt [gradient elution isotachophoresis] to a broader range of application areas," the researchers wrote. "For example, the ability to extract trace amounts of DNA from other crude samples, such as hair, bone, teeth, and soft tissues, could extend the current work to postmortem identification for forensic or archeological purposes."
So far the researchers have only published peer-reviewed data regarding the use of their technique with clean and soiled buccal swabs. "As we verify the system's success with other crude samples, we will publish those results," the NIST spokesperson said.
The researchers also noted in their paper that their work was supported by a US Army research contract, and that a patent describing the gradient elution isotachophoresis method has been assigned to NIST and licensed by Applied Research Associates.
However, the NIST spokesperson noted that the group is only in the "very early stages of development and testing" for the gradient elution isotachophoresis system, and as such it is too soon to comment on commercialization plans.
Applied Research Associates, according to its website, is an international research and engineering company with a broad range of expertise in defense, civil engineering, computer software and simulation, systems analysis, and environmental technologies.