May 1, 2010 (Vol. 30, No. 9)

Faster, Highly Automated Methods Are Being Developed

A common theme of many of the presentations at the Knowledge Foundation’s “Sample Prep” conference, to be held this month in Baltimore, will be how to purify nucleic acids, including DNA, RNA, and miRNA, from different types of samples using a sensitive and reproducible method that can be performed in as automated a fashion as possible.

Whether these isolation procedures are performed in a research setting, a clinical diagnostic laboratory, or a field or military setting, the goal is to be able to load a sample and the necessary reagents and buffers into an instrument, press a button, and output the pure nucleic acid component for subsequent identification and quantification.

Two main goals drove the development of Norgen Biotek’s nucleic acid sample-preparation technology, which is based on a proprietary resin/matrix for purifying DNA, RNA, and proteins—the ability to isolate total DNA, including miRNA without the use of phenol and the ability to modify this technology for use in resource-limited areas.

To achieve its first goal, Norgen developed a nucleic acid purification strategy that does not require phenol or chloroform. This approach can isolate circulating total RNA from various sample types including plasma, serum, or blood with the sensitivity and reproducibility needed for use in diagnostic applications.

Studies aimed at correlating up- and downregulation of specific miRNAs with the presence of cancer in humans are ongoing, and at the meeting, Yousef Haj-Ahmad, Ph.D., president and CEO, will describe a research project that focuses on prostate cancer.

These types of studies have been hindered by the high degree of sample-to-sample variability and the need to repeat miRNA measurements in each individual sample at least three times. Dr. Haj-Ahmad says that Norgen’s extraction procedure yields a consistent result that can facilitate the development and use of miRNA biomarkers in cancer research.

Norgen has transitioned its nucleic acid purification technology into the diagnostic market with the launch of more than 30 kits—initially available for research use—for applications such as surveillance of drug-resistant pathogens, field surveillance, and epidemiologic studies. The company’s kits target commonly transmitted diseases such as HPV, HIV, herpes simplex virus-1 and -2, chlamydia, and gonorrhea, as well as hepatitis B virus and food-borne pathogens.

Another key feature of Norgen’s technology is its ease of adaptability for use in resource-limited areas due to the properties of the company’s resin, Dr. Haj-Ahmad says. Inhibitor-free nucleic acid isolation from urine samples does not require laboratory equipment such as refrigerators or centrifuges. Extraction involves collection of a urine sample in a tube containing resin to concentrate the analyte of interest. The analyte binds to the resin on mixing, and due to the high density of the resin, the complex falls to the bottom of the tube.

The urine is then decanted, the resin is washed with 1–2 mL of buffer to remove contaminants, and the analyte is then eluted at a 50- to 100-fold increased concentration compared to the original sample. The analyte can then be detected quantitatively or qualitatively depending on the detection method.

Electrophoresis with a Twist

Among the challenges Boreal Genomics sought to overcome in its development of a method for purifying and concentrating nucleic acids are the need to remove contaminants that can interfere with PCR-based amplification, the difficulty in extracting and enriching for low-abundance nucleic acids, the recovery of nucleic acids from dilute samples, and the need to accommodate a variety of sample types, including complex mixtures, viscous samples, and samples containing particulate matter such as soil and sand.

At the heart of Boreal’s approach to nucleic acid sample prep is the synchronous coefficient of drag alteration (SCODA) electrophoretic concentration technology. Traditional electrophoresis separates samples linearly in a gel across which voltage is applied. The distance each molecular species travels through the gel depends on differences in their physical properties such as size, charge, linear charge density, and stiffness/conformational entropy.

SCODA takes advantage of the strong charge of nucleic acids and of their non-linear electrophoretic behavior when exposed to a rotating electric field. The drag on nucleic acids that causes them to move in a spiral pattern rather than a circular orbit and to travel toward the center of the electric field relates to their long length and variable conformation.

With the SCODA technique, injection of up to 5 mL of a cell lysate or other type of sample directly into the gel and application of a rotating electric field, sends the charged molecules in the sample into periodic orbits. After a full cycle of rotation, the nucleic acids will begin to drift toward the center of the gel, whereas contaminants will maintain circular orbits and return to their initial location.

Ultimately, the back-and-forth pattern of field strength will cause the nucleic acid component of the sample to converge and focus to a tightly packed spot from which they can be eluted into a centrally located well in 10–50 µL of buffer.

This method offers “advantages over column- or bead-based methods that rely on chemical affinity to separate mixtures,” says Andre Marziali, Ph.D., president and CSO of Boreal and director of engineering physics at the University of British Columbia.

As SCODA technology relies only on differences in physical properties, it can reject contaminants that have chemical properties similar to those of nucleic acids. Furthermore, “because we are just enriching the nucleic acids that are already there,” compared to PCR amplification-based purification strategies, “we are not introducing any background noise.”

He believes that SCODA will be beneficial in applications ranging from genetic biomarker detection, extraction of pathogen-derived nucleic acids from medical samples, isolation of fetal DNA from maternal blood, forensic studies, and nucleic acid analysis of plant material and soil samples.

In his presentation, Dr. Marziali will describe the company’s second-tier technology that makes the focusing process sequence-specific by attaching short, single-stranded DNA probe sequences to the gel. When electrophoresis is performed at a temperature near the probe-target melting point, the target nucleic acid sequences present in a sample will briefly hybridize with the complementary probes immobilized on the gel, hampering their progress until they are again propelled forward by the transient electric field.

The more time a molecule spends bound to probes, the less circular its orbit becomes. The bottom line, says Dr. Marziali is that by defining the probe sequences, “we can extract focused, matched sequences with single nucleotide resolution.”

Fully Automated Processing

Several design features of Arcxis Biotechnologies’ Xisyl™ sample-preparation system exemplify the company’s emphasis on optimizing the workflow to enable selective batch processing, controlled operations in a contained cartridge, ease of use, and accommodation of large sample volumes and various sample types.

In his talk, Jay West, Ph.D., CTO and founder, will review an automated three-step process that takes samples through to isolation of target nucleic acids without the need for any external processing.

The term “random access sample preparation” describes the system’s ability to run different methods on 12 samples at a time, according to Dr. West. Users can load samples and instruct the instrument, for example, to purify viral RNA from a plasma sample, genomic DNA from blood, and bacterial DNA from a water sample in one run.

Arcxis devised a disposable cartridge that can accommodate a 0.2–1 mL sample volume and a total working volume of up to 6–7 mL. All sample-processing steps are performed in a flow-through system with automated temperature control, mixing operations, reagent additions, and wash/dry steps.

The Xisyl cartridge design ensures that there is no direct contact between the sample and the environment outside the cartridge during processing. The cartridge can be loaded with sample in a biosafety cabinet and then sealed for transport to the Xisyl instrument. Sealing of the cartridge is accomplished through the use of an o-ring system that holds a magnetic disk in the cartridge cap, which prevents leaking during transport.

When the cartridge is placed in the instrument, the magnetic disk is ejected into the sample chamber, allowing reagent dispensing through the newly formed orifice between the cap and chamber. A series of sample-preparation reagents are then dispensed into the cartridge chamber and mixed using the magnet stir disk according to a predefined, automated protocol.

When the sample-preparation steps are completed, including enzyme additions, incubation, and washes, a two-position sliding mechanism built into the cartridge moves the capture material into a sealed conduit, putting it in contact with the top of the cartridge chamber. Air pressure pushes the sample through the capture matrix, which is washed and dried before the nucleic acids are eluted into a microcentrifuge tube.

Dr. West attributed the instrument’s ability to extract even small amounts of nucleic acids and to eliminate PCR inhibitors from the extract to a combination of optimized chemistry, engineering, automation, and cartridge design, as well as the process of washing and drying the capture material to remove contaminants.

The Xisyl consumable cartridge and sample-preparation module from Arcxis Biotechnologies: Consumables are used in conjunction with a module that performs elution tip positioning, noncontact mixing, and thermocontrol of the cartridge. This module was designed to perform stand-alone sample prep by having on-board PID control of the module functions, as well as fluid connectivity to the waste stream. The module is spring loaded to enable reproducible compression of the consumable to the system fluid manifold.

Upping the Throughput

CUBRC has reportedly developed a method that enables rapid and sequential isolation of protein and nucleic acids. The CUBRC approach sequentially purifies PCR-ready nucleic acids and immune-reactive proteins for subsequent analysis from a single sample on an automated system.

Isolation of the nucleic acid fraction utilizes Akonni Biosystems’ TruTip™ nucleic acid isolation procedure, which can extract DNA, RNA, and proteins from samples such as blood, culture, sputum, saliva, nasal swab, or urine, and is applicable for near point-of-care clinical research and forensic applications. The workflow, from lysis and binding to washing, drying, and elution, takes place in a microtube.

A unique aspect of the Akonni TruTip approach is the use of an untreated, solid pored glass matrix formed inside the microtube, in which channels are created as the glass hardens. When a sample flows through these small channels, the DNA or RNA molecules present in the sample bind to the glass matrix; the binding properties will vary depending on the buffers used.

The matrix comes in two forms—one for more viscous and one for less viscous samples. The company has also configured its matrix for insertion into most standard pipette tips, which can be used with both electronic pipettes and automated pipetting stations.

The key, says Kevin Banks, Ph.D., vp of sales and marketing at Akonni, is “to optimize the matrix density and configure the extraction protocol to the sample and material to be extracted.”

The Akonni method offers advantages in speed (the entire workflow takes in four minutes) and portability, as its EDP®3-Plus pipette solutions require no centrifugation or other processing that would necessitate electricity, Dr. Banks says. As a result, it is applicable for in-the-field use.

More recently, the company introduced high-throughput, fully automated capabilities for influenza extraction from nasopharyngeal swab extracts and for genomic DNA from saliva. Under a co-marketing agreement with Eppendorf, a custom-sized glass matrix was developed for the epMotion® automated pipetting system that can isolate nucleic acids from 24 samples in one run in less than 15 minutes, processing eight samples at a time.

In the future, Akonni intends to introduce a PCR product cleanup version compatible with a 96-tip pipettor. Ultimately, Dr. Banks envisions targeting ultrahigh-throughput sample-preparation protocols suitable for use in large-scale genomic or forensic applications or, for example, for population-based infectious disease screening.

TruTip RNA Kits deliver inhibitor-free, PCR-ready RNA in as few as four minutes, according to Akonni Biosystems. Accommodating sample volumes that range from 100 µL to 1 mL or greater, concentration of nucleic acid, on the order of 5- to 10-fold, can reportedly be achieved. TruTips can also process a range of sample sources (blood, culture, sputum, urine), sample viscosities (thick or thin), and types (total RNA including genomic, mitochondrial or viral). Example targets include Adenovirus, Influenza A & B, Venezuelan equine encephilitis virus, and E.coli.

Previous articleCytoscape
Next articlePEPs Gaining FDA Nod but Remain Hard to Take