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Feature Articles : Jan 1, 2011 (Vol. 31, No. 1)

Novel Methods Expedite Nucleic Acid Sample Prep

Technologies Focused at Both the Research and Clinical Markets Stir Excitement in the Field
  • K. John Morrow Jr., Ph.D.

Recent developments in genomic identification using microarray technologies are infiltrating the fields of forensics, diagnostics, and sample identification. They include a combination of new hardware designs and innovative approaches to sample processing. In addition, microfluidics and miniaturization are emerging as key components of powerful new multiplexing platforms. The upcoming “Pittcon” meeting will cover all of the latest advances.

A critical first step in genomic profiling is the preparation of high-quality DNA. Aurora Biomed provides liquid-handling systems, assay services, and reagents for the life sciences, according to Sikander Gill, Ph.D., research scientist.

The company’s robotic workstations automate genomic, proteomic, cell biology, biochemistry, drug discovery, and analytical applications. The Versa series is a group of liquid handlers ranging in throughput and size, providing a customizable solution for any lab-space, Dr. Gill notes.

The Versa Mini Nucleic Acid Preparation Workstation is a small, single-arm system equipped with a single-channel syringe pipettor, while the Versa 1100 workstation has a larger deck capacity and may be equipped with a single channel, 8-channel, or 96-channel pipettor, depending on the user’s requirements.

Various on-the-deck modules such as plate shakers, heater/coolers, etc, enable the systems to fully automate a wide range of liquid-handling applications, allowing researchers the freedom to walk-away during the protocols, Dr. Gill adds.

Dr. Gill says that isolation of DNA from blood samples using Aurora’s high-throughput Magnetic Binding Blood DNA Kit in combination with its Versa automated liquid handlers is straightforward. This magnetic bead-based genomic DNA or RNA system can be used with a range of tissue or fluid samples. This method allows rapid and efficient extraction of genomic DNA without exposing the DNA/ RNA to dry conditions, he adds.

The workstations also provide vacuum-based applications for nucleic acid isolation procedures including centrifugation-based methods. As the Versa platforms are designed for use with 96-well plates for extractions, they can perform multiplexing protocols efficiently, reportedly holding reagent expenditures to a minimum and also for 384 formats.

The magnetic bead based application is also appropriate for purification of amplicons as a post-PCR application, and in next-generation library preps for DNA/RNA fragment size selection and purification of adaptor-ligated fragments, etc.

Dr. Gill says some biological materials like FFPE tissues, plant materials with hard shells, yeast cells with recalcitrant cell walls, are challenging. However, he explains that there are now kits available for processing these tissues that have been accommodated on the Versa workstations. There are other accessories, such as the Bead Beater (a mechanical disruption apparatus using glass or zirconia-silicate beads), that avoid degradation of nucleic acids while effectively preparing the tissues for analysis.

“Our processing technologies avoid the use of centrifugation, as this adds cumbersome and expensive steps to nucleic acid preparation,” Dr. Gill explains. “We also offer column-separation approaches as well as liquid-liquid (aqueous-organic) extraction procedures for preparation of nucleic acids and other cellular components.”

Zygem (www.zygem.com) is developing an integrated microfludics system for forensic analysis, using the enzymes it has long been known for,” according to James Landers, Ph.D., professor of biochemistry at the University of Virginia and also CSO of the company. Dr. Landers is investigating short tandem repeats using Zygem’s microfluidics technology and hardware from Lockheed Martin.

Dr. Landers says that the most important feature of the system is its rapid turnaround time. The company has worked with Lockheed Martin to speed the analytical process to as little as 60 minutes, and Dr. Landers believes that this time frame can be abbreviated further. The partners have concentrated on the three key processes—DNA separation, amplification, and detection—miniaturizing them, reducing the required steps and time frame, and increasing their sensitivity and robustness.

The preparation chemistry takes advantage of an EA1 metalloproteinase from the company’s extremophile collection, which can degrade tissues in buffer conditions compatible with PCR and is inactivated by a simple temperature shift. As a result of the temperature characteristics of EA1, DNA extractions can be carried out at an elevated temperature in a single closed tube or as part of an integrated microfluidics system, improving its efficiency. Because EA1 is inactivated above 95°C, raising the temperature stops the reaction, leaving PCR-ready DNA available for downstream processing.

Just the PCR amplification process alone is ordinarily a three and a half hour process when using conventional technology. The Zygem approach bundles up all three steps in a monolithic format, expediting the time required, according to Dr. Landers. Using special patented polymers for the separation component, Dr. Landers and his colleagues are able to achieve a 30-minute separation in 400 seconds, while still maintaining single-base resolution. In this fashion they are able to progressively drive down the turnaround time.

The microfluidics device, which is the size of a microscope slide, is processed by an instrument about the size of a computer tower. Dr. Landers and his collaborators at Lockheed Martin look forward to a miniaturized system, the size of a laptop, and eventually to a handheld instrument for use in the field. However, at this point the platform is designed for a laboratory environment, so the size of the instrument is not a pressing concern.

It is not clear at this time where the technology will fit into the overall picture of forensic investigation. There is no question that the microfluidics analysis of short-term DNA repeats as a means of identification can speed processing and decrease the backlog of cases in forensic labs around the world. The unknown is whether it would be of value at a crime scene.

“We can envision a time in the future where our instrumentation could be used for onsite identification of a suspect,” Dr. Landers suggests. However, there is no consensus today on whether such technology would be welcome in a chaotic venue where DNA from many individuals would be dispersed. “Only when the technology is available will we know how these approaches will fit into the needs of crime-scene investigators.”

“Portability, speed, and cost of analysis will be welcomed by the genetic analysis community, but there are issues concerning the use of this technology in the field,” Joan Bienvenue, Ph.D., a forensic scientist and program manager at Lockheed Martin, explains. “While you can find people on both sides of the fence, forensic typing for criminal purposes is presently carried out in incredibly well-controlled laboratory situations. The policy changes that would be required for use in the field are not trivial.”

“Even in a very well-organized forensic laboratory, there are three to four instruments that are used, in an analysis that can take ten to twelve hours to process a DNA sample,” explains Paul Kinnon, CEO. “So the first step will be to put our system into use, and as the technology evolves it will migrate into other functions. This will enable investigators to move forward efficiently and get on with their jobs.”

The multiplexibility of the technology opens up a wide range of possibilities for other applications. “With the system’s rapid turnaround, it would be possible to run a molecular diagnostics panel on a patient and discuss the results with him in less than an hour, rather than sending the patient home and doing the consult days later,” says Dr. Landers. “This could have a significant effect on management of patient anxiety while at the same time providing an opportunity for timely clinical intervention.”

Large RNA Molecules

Three-dimensional structural studies of large RNAs (>50 nucleotides) are a particularly difficult task, notes Michael Summers, Ph.D., Howard Hughes professor at the University of Maryland, Baltimore. “Historically, x-ray crystallography has been the approach of choice for investigating the 3-D structure of biomolecules, but RNAs do not yield easily to this technology. Largve RNAs are negatively charged on their exterior and tend to be heterogeneous in terms of their conformation, all of which means they don’t form good crystals.”

RNAs are quite a different target as compared to protein molecules, which are much more easily analyzed through crystallographic techniques. In national data banks there are many more protein structures available than large RNA structures. To expand this slim catalog, one could employ nuclear magnetic resonance for the analysis, but this is also a problematic approach for the large RNAs.

While proteins contain approximately 20 amino acids, there are only four basic nucleotide building blocks in RNA. This means that while the spectral NMR signals are well defined for protein molecules, they crowd together for the RNA molecules, with much overlapping, making the data difficult to interpret.

In nucleic acid NMR investigations, one must rely on aromatic signals for information since the repeated ribose molecule is too homogeneous. Because of the reliance on these ring structures, the use of C13 isotopes is not effective. For this reason, Dr. Summers’ group turned to another strategy.

“We rely on an older technique, that is, using 2-D spectra with duterated nucleotides,” continues Dr. Summers. “We can duterate specific nucleotides to improve our resolution. NMR is very good for obtaining local structural information regarding the location of hydrogen atoms. This works well with proteins, but with nucleic acids it is more difficult, since the hydrogen atoms are sequestered in the middle of the helices and not easily resolvable. So we combine the high-resolution NMR with low-resolution global structural data from cryoelectron tomography.”

Dr. Summers is particularly concerned with how viral genomes assemble in the cell, and for addressing the replication cycle of HIV, a knowledge of the 3-D relationships of the RNA and protein molecules is essential. Dr. Summers argues that the regulation of the assembly process is guided by a specific, highly conserved region within the RNA genome. Antiretroviral drug design depends on a detailed understanding of the 3-D structure of the HIV RNA and protein molecules.

“At present, we don’t know how the assembly process works at the atomic level. However, we are aware that all of the regulation of replication is guided by genes in the 5´ untranslated portion of the genome,” Dr. Summers adds.

It is known that during the process of retrovirus assembly, RNA plays many different roles, as it must be spliced and packaged in a variety of ways in order to produce functional viral particles. Dr. Summers’ investigations are focused on basic science at this point, but it is clear that in the future these finding will contribute to new therapeutics for the control of RNA viral-based diseases.

On-Chip Biomarker Extraction

“While nucleic acid biomarkers are useful for determining the type and stage of cancers, their quantification requires a series of isolation and amplification steps that can take considerable time,” says Adam Woolley, Ph.D., professor of chemistry and biochemistry at Brigham Young University. “To overcome these drawbacks, we have explored the integration of sample-processing steps on microfluidic systems.”

It is generally accepted that cancer screening that takes advantage of the simultaneous quantitation of multiple biomarkers will yield a more sensitive and accurate profiling of malignancies and a concomitant improvement in cancer mortality and morbidity statistics.

For example, while the prostate specific antigen (PSA) is perhaps the most widely adopted test for the early detection of cancer, as a single marker it has a high rate of both false positives and false negatives. Moreover, the PSA test provides no information on the aggressiveness of a tumor, so the result may be that the patient is subjected to costly and unnecessary therapies that offer no benefits, while potentially compromising quality of life.

Most biomarkers are detected using ELISAs, whose performance could be enhanced through extensive validation and quality control. But this would require a multiplexing system, and Dr. Woolley argues that microfluidic format could provide higher speed and lower reagent consumption. “Many process steps, including sample desalting, labeling, and extraction have been successfully performed in microchip systems. “The simultaneous monitoring of multiple biomarkers has the potential to greatly improve the quality of the diagnosis.”

Dr. Woolley believes that although the 96-well immunoassay technology is effective for analyzing large numbers of samples, it is an inefficient approach to smaller volumes, which can be monitored much more economically using the microfluidics approach.

His team has constructed microfluidic devices employing poly(methyl methacrylate) with monolithic columns for immunoaffinity extraction. Using a laser-induced fluorescence detection system, they have quantified α feto protein, a biomarker for liver cancer, down to 1 ng/mL levels in serum samples.

They have now applied the solid-phase extractors for sequence-specific nucleic acid biomarker determination. Monolithic columns were made using glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate. The reactive GMA epoxy groups were employed for the functionalization of the monolith with amine-terminated DNA probes complementary to the target nucleic acid fragment.

Using these columns, they extracted and eluted fluorescently labeled 20-mer target single-stranded DNA fragments in as little as 15 minutes, according to Dr. Woolley. “We tested a number of approaches for eluting the fragments,” he continues, “and settled on dilute (1.5–7.0 molar) urea as the best option. We are extending our investigations to urine as a noninvasive approach to cancer biomarker detection.”

Benefits Accrued

In an era in which cost cutting is all the rage, these new technologies are to be particularly welcomed. While the saying “time is money” lacks originality, it is particularly relevant here, as these technologies are focused at both the clinical and research markets. Pressure from federal healthcare regulation and the demands of the consumer market makes rapid and economic nucleic acid preparation especially alluring.