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Jun 15, 2007 (Vol. 27, No. 12)

Achieving Better Isolation of Nucleic Acids

Researchers Discuss Advances as They Apply to Molecular Diagnostics

  • Broad-based conferences tend to cover everything from soup to nuts. CHI’s “Nucleic Acid-based Technologies” conference in Baltimore promises to be rich with progress in fields ranging from biodefense to personalized medicine.

    Gene-based molecular diagnostics are changing the practice of medicine and will continue to do so for the foreseeable future, says Richard Zhao, Ph.D., associate professor and division head of molecular pathology and director of molecular diagnostics laboratory, University of Maryland Medical Center.

    The major underlying principle of these diagnostic tests is the use of specific nucleic acid sequences as surrogates. Amplification of the surrogate markers enables the detection of pathogens or disease-related gene mutations. Gene targets can be amplified by target-, probe- or signal-based methods, Dr. Zhao notes.

    A variety of choices are available for the detection of amplified amplicons, with the fluorophore-linked nanoparticles as the most sensitive markers. The unique advantages of using covalently linked nanoparticles include the detection of single molecules, the ability to enrich molecules of interest with improved detection sensitivity, and the flexibility of multiple functionalization.

    In the future, a doctors visit will result in a small specimen of blood being drawn, after which, it will be put in the sample reservoir of a “little gadget,” explains Dr. Zhao. Instead of the current exam and series of tests, the gadget will automatically produce an entire genomic profile. Ultimately, this process will define how the individual patient differs from all others and predict drug efficacy, side effects, and drug/drug interactions.

    “Should it be Tylenol or Advil? The computer will provide the answer,” Dr. Zhao says. “Technically, we are there. Not long ago, months were required to sequence a person’s genome; soon it will be only a few days.”

    Dr. Zhao is currently working with the AmpliChip CYP450 test from Roche (www.roche.com) to define the wide patient-to-patient variation in response to drugs based on a genotypic profile of the CYP450 genes. Test results allow physicians to consider unique genetic information to select medications for common conditions such as psychiatric disorders, cardiac diseases, pain, and cancer, Dr. Zhao explains.

  • Biodefense and Environmental Resources

    In the biodefense and environmental areas, two speakers will address new resources that may play important roles. The Biodefense and Emerging Infections Research Resources Repository (BEI Resources) is a source of high-quality materials for research related to biodefense and emerging infections, explains Susan Jones, Ph.D., who refers to the organization as an untapped resource.

    BEI Resources was established by the NIAID and is managed by the ATCC (American Type Culture Collection). Its purpose is to serve the scientific community by ensuring secure access to biodefense and emerging infections-related materials for basic research as well as for the development of vaccines, therapeutics, and diagnostics.

    To do so, it acquires, authenticates, preserves, and distributes—free of charge—biological materials needed to carry out reproducible research on potential bioterrorism agents and emerging infectious disease agents. This includes microorganisms (bacteria, viruses, and protozoa) as well as specialized biological reagents such as antigens/antibodies, genomic DNA, peptides and proteins, peptide arrays, mutant libraries, and toxins. BEI Resources scientists conduct in-house and collaborative research and aid researchers by providing enhanced authentication and validation details for reagents supplied by BEI Resources. The repository is projected to grow in coming years.

    The rapid identification of harmful organisms has also become an important priority in fields such as clinical diagnosis and environmental surveillance. Based on continuing research with Baochuan Lin, Ph.D., at the U.S. Naval Research Laboratory, postdoctoral fellow Marie Archer, Ph.D., will describe “Silicon Microstructures: A New Approach for Molecular Biology Applications.”

    The objective of Dr. Lin’s research team is ultimately to develop a portable and highly automated device that could be used in locations that have no dedicated lab space for making nucleic acid analyses. Units currently on the market are limited in terms of the targets they can detect, Dr. Lin states.

    Since organisms possess a particular genetic signature, they can be identified by their nucleic acid sequence. For this purpose, the nucleic acids have to be extracted from a cell lysate using a solid phase before any further downstream processing. Silicon is an ideal candidate for this purpose given its integration capabilities and the possibility to chemically modify its surface, Dr. Archer says. The surface area of the silicon solid phases can be tailored through micromachining and electrochemical etching to produce structures with large surface areas and feature sizes ranging from microns to nanometers.

    The roughness of the surface is an important factor in solid phases as it increases the effective surface area but it is a complicating factor as well because it makes it more difficult to know the true surface area. The development of these types of solid phases requires an interdisciplinary effort between materials science, chemistry, and molecular biology to address the variables that affect performance.

    Both nonselective and selective solid phases are fabricated with silicon microstructures. Nonselective solid phases are used to separate the nucleic acids from other components that can interfere with downstream processes. This separation is not based on the nucleic-acid sequence, the researchers note, but rather on their intrinsic electric charge, which allows them to interact electrostatically with a charged surface via ionic groups such as hydroxyl and amino groups. After electrostatic bonding, extraneous cell parts are washed away.

    Selective solid phases are used to separate a particular nucleic acid from a population containing various types. In work to date, the team has used adeno and influenza viruses as well as human genomic DNA. Selective separation is based on the recognition of one nucleic acid sequence, used as a probe, by its matching partner or complementary sequence. The goal is to broaden the procedure to identify any target and multiple organisms.

  • DNA Detection and Quantitation

    Both the FDA and the WHO have recognized the safety problems that could be posed by host cell DNA carryover into finished biopharmaceuticals, which include transmitting genetic information to patients receiving the product with the associated risks of insertional gene disruption, malignant transformation, and the production of infectious viruses from viral DNA.

    In his presentation, Scott Kuhns, Ph.D., senior scientist in global cellular and analytical resources at Amgen (www.amgen.com), will present data from the development and qualification of a new qPCR-based method for host cell DNA detection and quantitation that uses target sequences that differ from previous methods. “We’ve employed new targets that afford greater reproducibility and sensitivity,” Dr. Kuhns says.

    Although slot blot and threshold assays are still widely used techniques for quantitating the amount of DNA in biological samples, qPCR is the technology most commonly employed now, Dr. Kuhn says, and has the greatest regulatory acceptance.

    Dr. Kuhn’s presentation will discuss approaches to host cell DNA detection and quantitation in both bulk product and in-process pools. He notes that the actual technique doesn’t differ for bulk versus in-process pools, but that subtle adjustments to the method are often necessary for running the different sample types.

    Well-qualified reagents are critical to the performance of any assay, says Uplaksh Kumar, Ph.D., Digene’s (www.digene.com) senior manager of manufacturing. As assays for analyte detection are becoming more sensitive and instrument-dependent, it is important to have a reagent set that is qualified and stable.

    Reagent formulations such as controls and calibrators that include complex nucleic acids molecules have to be well-qualified to ensure that results and claims made by the assay are authentic and reproducible, Dr. Kumar notes. Such reagents provide a reference to determine if patient samples are above or below normal limits, for example, if there is an infection or not. These are typically special synthetic solutions with solubilization agents for RNA/DNA and other formulation enhancers to expose picogram-range concentrations.

    Components of these complex formulations contain other nonanalyte reagents as part of the matrices, Dr. Kumar adds, which may be from different manufacturers. Some vendors may not have qualified a specific reagent for a particular assay and, as a further complication, some vendors may not be working in a FDA-regulated GMP environment.

    “In our processes, which involve analyzing and detecting the presence of different types of the HPV virus, we have to be certain that the matrices are not contributing to the results,” Dr. Kumar states. “Any analytical noise or interference could be misleading and in our setting negatively impact the company. By putting the right controls in place one can ensure that data from the assay is sample-dependent and not dependent on reagent variability.

  • Isolation from Archived Samples

    Finally, technology reminiscent of Jurassic Park came into play in a presentation by Emily Zeringer, research scientist at Applied Biosystems’ Ambion (www.ambion.com) business unit, titled “Excavating the Archive: Obtaining Useful Data from FFPE Samples.” The capability to isolate nucleic acid suitable for molecular analysis from formalin-fixed paraffin-embedded (FFPE) archived tissue samples, of which 400 million or more are said to exist, enables the retrospective studies of a huge library of tissue representing various diseases, often through their progression, Zeringer notes.

    While FFPE is effective for maintaining tissue structure and preventing putrefaction, it can interfere with molecular analyses on samples due to extensive chemical modification and subsequent fragmentation of the nucleic acids. Zeringer’s presentation will focus on practical elements of optimized FFPE workflows using Ambion’s RecoverAll™ Total Nucleic Acid Isolation Kit.

    The Ambion kit is used to isolate RNA, including miRNA, and DNA from the same sample of either human or mouse tissue. Following deparaffinization and a three-hour protease digestion, the sample can be divided into separate aliquots for isolation of either RNA or DNA using an optimized glass-fiber filter purification protocol, says the company. Digestion time for DNA is longer however—about two days. Zeringer notes the process can be used to look for high-, medium-, or low-expression targets.



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