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

Nucleic Acid Sample Prep

Faster, Highly Automated Methods Are Being Developed

  • Fully Automated Processing

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    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.

    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.

  • Upping the Throughput

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    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.

    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.

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