June 15, 2006 (Vol. 26, No. 12)
Alternative Methods for Amplifying and Detecting Specific DNA Sequences
Technology development focused on generating tools to advance nucleic acid-based research for functional genomics and gene-expression analysis to drive drug discovery and development and to develop environmental-monitoring and clinical-diagnostic strategies for detecting and identifying low levels of infectious or toxic agents has led to a variety of novel platforms and innovative new products. These include nucleic acid purification, detection, and amplification techniques, tools and materials for bioarrays, and a range of RNA-based products for siRNA and miRNA applications.
Cambridge Healthtech Institute’s (www.healthtech.com) upcoming “Nucleic Acid Based Technologies” conference will feature several presentations on techniques and technologies designed for DNA amplification and detection. These include methods to optimize PCR-based DNA amplification and alternative approaches for amplifying and detecting specific DNA sequences, with a broad range of potential applications in pharmaceutical research, forensics, and biodefense research.
For the detection of small samples of DNA, in the nanogram to picogram range, Argylla Technologies(www.argylla.com) has developed a nanoscale batch chromatography method of DNA purification and concentration for applied genetics. Argylla’s NanoChromatography technology incorporates chemically coated ceramic nanoparticles with a DNA-binding capacity of 10-µg DNA/µL. The submicron size of the particles yields a surface-area-to-volume ratio of 100 cm2/µL. Chemical modification of the nanoparticle surface with biomolecule-specific ligands make them useful for DNA, RNA, or protein-purification applications.
The nanoparticles form a stable colloidal suspension in an aqueous solution. In DNA-purification applications, the particles bind DNA from a sample and can then be centrifuged at low speed to yield a microliter-scale nanoparticlepellet from which DNA can be eluted in volumes as low as 5 µL.
Mike Hogan, Ph.D., managing director of scientific affairs at Argylla, describes the utility of this technology for applications such as crime-scene forensics, population and clinical genetics, and clinical microbiology to detect multidrug-resistant bacteria. The purification process takes place at room temperature in a single microfuge tube and can accommodate any kind of sample, including DNA from thin sections of frozen blood, or DNA soaked off a nasal or cheek swab or off a piece of fabric. The company is exploring a variety of markets including DNA sample prep for microarray-based whole-genome analysis, as well as RNA purification.
Argylla currently offers its PrepParticle MicroKit, which is scalable from 50 µL to 5 mL and can process DNA samples with a DNA content ranging from 100 pg to 10 µg via direct volumetric scale up. Within the next few months, the company plans to launch a kit for isolating both DNA and RNA from the same sample in a two-step process. Argylla will introduce a protein-specific product in 2007.
IQuum (www.iquum.com) developed the Liat Analyzer for rapid, fully automated biological sample testing in a closed system. In March, the company received a Phase II contract from the U.S. Department of Homeland Security Advanced Research Project Agency (HSARPA) for the development of its bioaerosol monitoring systemthe Liat Bioagent Autonomous Networked Detector (BAND)for monitoring outdoor urban areas for bacteria, viruses, and toxins. The company also received a Phase I contract from HSARPA to develop a Food Assay Sensor System for biodefense applications.
A drawback of conventional amplification-based nucleic acid testing is the potential for sample contamination when the sample tubes are opened during processing, Dr. Chen says. The company’s lab-in-a-tube platform is fully self-contained and disposable. The processing tube is only opened at the beginning to load the sample.
According to Dr. Chen, an additional challenge is assay automation. The Liat system utilizes the lab-in-a-tube technology to allow for automation of the entire assay, including sample preparation, target enrichment, inhibitor removal, nucleic acid extraction, PCR amplification, and real-time detection. The system processes the sample through a series of segmented sections in the Liat Tube, with each segment containing the necessary reagent for an assay step.
Peristaltic pumps move the reaction mixture from one segment of the tube to the next, breaking and reclamping the seals that divide the segments. For PCR, one segment is set at the denaturing temperature and another is set at the annealing-extension temperature, and the reaction mixture is alternatively cycled between these two segments.
“Since only the reaction mixture is flow-cycled between the temperatures, the Liat Analyzer can rapidly perform nucleic acid tests,” Dr. Chen adds. The system can complete 30 cycles on a 50-µL reaction volume in seven minutes. This allows an entire genetic test from whole blood to be completed in about 30 minutes, and detection of an infectious agent in plasma takes about 60 minutes.
Dr. Chen’s vision is for nonspecialized laboratory personnel or individuals in a field setting to be able to perform these tests with minimal training. He expects the company to release its first product for research use only by the end of this year and to receive approval for a clinical application in 2007.
IQuum has launched a clinical trial for detection of the infectious agent that causes leishmaniasis and is developing assays for Chlamydia sepsis, cytomegalovirus, and Epstein Barr virus, as well as for biodefense targets, such as anthrax and E. coli. SNP detection for genotyping applications is also in development.
IQuum’s BAND system for environmental monitoring uses the same lab-in-a-tube assay technology as the Liat Analyzer but requires different instrumentation to enable collection of large-volume air samples.
Fractal Systems (www.fractalsystems.com) is developing a PCR-free molecular detection system for biodefense diagnostic applications. The system utilizes an ultrathin conductive polymer film that when exposed to an oxidizing or reducing agent in the environment, changes its conductivity. Measurement of the change in conductivity correlates with the amount of material present in the environment. In this way, the electrochemical response corresponds to the concentration of the analyte, which can be quantified.
The nanocomposites comprised of the conductive polymers are reuseable, as the change in conductivity is reversible. In the presence of the target analyte the redox reaction oxidizes the polymer backbone. Removal of the analyte causes the coated electrode to reverse to its original state.
DNA acts as an oxidizing agent. By exploiting this characteristic to embed a single-stranded oligonucleotide into the polymer backbone, the subsequent recognition and hybridization of a specific, complementary DNA segment will increase the volume of the polymer, displacing the conjugated backbones and altering its conductivity. In addition, hybridization will diminish the interaction between the single-stranded oligo and the polymer backbone, without actually separating the oligo from the polymer. This, too, leads to a change in conductivity.
The company’s first project, funded by an SBIR grant from the Department of Defense’s Threat Reduction Agency, focused on developing highly specific viral sensors. Its second project, funded by the U.S. Army, uses aptamers to detect toxins.
BioHelix (www.biohelix.com) is developing a portable detection system for use in the field to identify biothreat organisms, under a cooperative agreement with the Department of Homeland Security. The company’s isothermal, helicase-dependent amplification (HDA) technology for amplifying and analyzing DNA maintains a constant reaction temperature and eliminates the need for thermocycling equipment. BioHelix plans to commercialize HDA (linked to a downstream detector) as a rapid screening platform for use in the point-of-care molecular diagnostics market. The company’s first assays in development target infectious diseases such as herpes. HDA relies on a DNA helicase that unwinds the two strands of a DNA helix, creating single-stranded templates for primer hybridization and subsequent extension by a DNA polymerase.
Huimin Kong, Ph.D., president and CEO, cites HDA’s low cost and ease of use as two important advantages of the technology compared to traditional PCR approaches. Also, HDA can be used to amplify either DNA or RNA and %is more robust than strand-displacement methods,% says Dr. Kong. In late April, the company entered into an exclusive licensing agreement with Harvard University to commercialize a rapid, primase-based whole-genome amplification technology.
GE Healthcare (www.gehealthcare.com) introduced the illustra Hot Start Master Mix to amplify target DNA sequences that the company says provides an increased level of PCR specificity compared to conventional techniques. The Hot Start Master Mix achieves hot start activation by sequestering primer until the template DNA is denatured. It then releases the PCR primers, and the cycle of amplificationprimer:template annealing, extension, and denaturationthen proceeds using traditional thermal cycling protocols.
Unlike hot start PCR systems that rely on chemically modified or antibody-based methods, HotStart Master Mix does not interfere with the thermostable polymerase, resulting in no loss of enzyme efficiency. By effectively inhibiting primer:dimer formation without compromising enzyme function, Hot Start Master Mix maximizes target amplification specificity and efficiency for better overall PCR results.
A host of companies are targeting the growing market for nucleic acid-based products. These include Invitrogen (www.invitrogen.com), with a broad range of products, such as Dynabeads(r) Oligo (dT)25 for mRNA isolation and Stealth RNAi. Promega’s (www.promega.com) offerings include GoTaq and GoTaq Flexi DNA Polymerase, the PolyATtract mRNA Isolation System, and the siSTRIKE U6 Hairpin Cloning System. Recent additions to Qiagen’s (www.qiagen.com) product line include Mouse Whole Genome siRNA Sets, AllStars RNAi Controls, and the QuantiText Probe or Multiplex PCR kits.
Smaller RNA species are enjoying broad applicability in drug discovery research, as well as in diagnostic and therapeutic drug development. For functional genomics studies, siRNAs are powerful tools for targeted gene knockdown. miRNAs are being exploited for their role in gene regulation, offering another level of control of gene expression, distinct from transcription factors. Traditional siRNAs are chemically synthesized 21-mer RNA duplexes designed to mimic siRNAs found in cells, which are produced by enzymatic cleavage of long double-stranded RNAs via the Dicer endoribonuclease. In contrast, Integrated DNA Technologies’ (www.idtdna.com) Dicer-Substrate RNAi technology utilizes 27-mer RNA duplexes optimized for Dicer processing. “Dicer-Substrate RNAi is a chemical substrate designed for processing by the endogenous cellular machinery,” says Trey Martin, COO. The dicer system processes the 27-mer to form the active species.
By feeding into the Dicer system, the 27-mers yield “more efficient loading into the RNA Induced Silencing Complex, leading to much higher potency,” he adds. With responses achieved at doses of 10 nM and lower”as much as 10 times less than with traditional 21-mers,” Martin points out and good results being reported with doses as low as the 100 picomolar range, Martin contends that the 27-mers are less likely to induce an innate immune response or to produce off-target, adverse effects due to the lower effective concentrations.
IDT’s TriFECTa kits contain three Dicer-Substrate RNAi 27-mer duplexes that target specific genes identified from Genbank human, mouse, and rat gene collections. On the horizon from IDT are Dicer-Substrate RNAi targeting genes in other organisms, stock library sets targeted to protein kinases and other gene families, and 27-mers designed based on splice-variant databases.
Ongoing research efforts to develop siRNA as therapeutic agents to knock out defective genes in vivo must first overcome the daunting challenge of safe, effective, and efficient drug delivery to targeted cells.
Providing an update on recent scientific findings in the field of miRNA and discussing the tools and protocols designed to support miRNA research, speakers from Ambion (www.ambion.com) will present emerging techniques developed for miRNA isolation, enrichment, detection, and expression.
“More than 30% and up to 50% of all human genes may be regulated by miRNA,” says Kathy Latham, Ph.D., marketing manager for miRNA and RNAi at Ambion (Applied Biosystems, www.appliedbiosystems.com, recently acquired the research products division of Ambion). These short, 20-25 nucleotide RNA molecules usually act at the protein level in mammalian cells, blocking translation of messenger RNA into protein.
The main challenge at present in miRNA analysis and isolation is developing techniques to detect these small molecules, according to Dr. Latham. Most glass-fiber filter-based RNA isolation protocols were developed for longer RNA and would not capture miRNA. Similarly, much of the miRNA in a sample would be lost using standard organic extraction and precipitation techniques.
Ambion has optimized protocols that preserve miRNA within a total RNA sample and then isolate and purify the miRNA fraction. The company has developed kits designed for various types of samples, including cell samples, fresh tissue, blood, laser capture microdissected samples, and formalin-fixed, paraffin-embedded tissue samples.
The company’s RecoverAll Total Nucleic Acid Isolation Kit for FFPE, for example, can be used to study archived patient samples linked to detailed patient histories to measure miRNA levels in healthy versus disease tissue samples and understand the role of miRNA in disease processes.
Novel techniques for miRNA detection include qRT-PCR to elongate and amplify miRNA and enable detection from only a few picrograms of total RNA. Applied Biosystem reports that its TaqMan MicroRNA Assays can detect as few as seven copies of a target miRNA. Ambion offers the mirVana qRT-PCR miRNA Detection Kit and Primer Sets.
Ambion’s mirVana miRNA Bioarrays contain a panel of probes to known human, mouse, and rat miRNAs. They enable detection of less than 300 attomoles of miRNA.
Other miRNA products include the mirVana miRNA Detection Kit, the pMIR-REPORT miRNA Expression Reporter Vector System, Anti-miR miRNA Inhibitors, and Pre-miR miRNA Precursor Molecules.
“We are seeing increased demand for specific RNA populations,” says Nezar Rghei, vp of business development at Norgen Biotek(www.norgenbiotek.com). This requires removal of contaminating RNA species. Norgen has expanded its line of RNA-isolation products, adding to its Total RNA Purification Kit three recently launched kits for purifying cytoplasmic and nuclear RNA, leukocyte RNA, and miRNA. The kits isolate RNA without the use of phenol or chloroform. They rely on spin-column chromatography using Norgen’s underivatized resin that is chemically modified to bind preferentially RNA, DNA, or protein based on ionic concentrations.
For cytoplasmic RNA purification, the Norgen kit first fractionates the cytoplasm from the nucleus, thereby eliminating the need for a Dnase step to remove the genomic DNA. The challenge in leukocyte RNA purification is to separate the RNA from the large amounts of red blood cell-derived globin RNA present in blood samples. Norgen’s technique includes a lysis step that involves lysing the red blood cells while keeping the white blood cells intact. The isolated white blood cells can then be lysed and the RNA collected.