Poster Session Abstracts

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WITHDRAWN

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Development, Optimization, and Scale-up of a Chromatographic Purification of a Synthetic Oligonucleotide. Fisher, J.; O’Donnell, J.; Rosen, R.; Maikner, J.; Kraus, M.; Juliano, S. Picciotti; TosoHaas, Montgomeryville, PA. Rohm and Haas Company, Spring House, PA, TosoHaas GMBH, Stuttgart, Germany

The objective of this study was to develop and scale-up a chromatographic purification process for a crude, synthetic, DMT-on, phosphodiester oligonucleotide. A separation process was developed on a 1.0cm I.D. x 6.2cm L, lab-scale column, using an experimental 15μm, polymeric reversed phase resin with a triethyl ammonium acetate/acetonitrile mobile phase. The experimental resin was styrene/divinylbenzene with an average pore diameter of 300 angstroms. The process was then scaled to 5cm I.D. x 10cm L, pilot-scale column using a commercially available 35μm polymeric reversed phase resin with similar mobile phase conditions. The process effectively combined removal of failure sequences, detritylation on-column, and elution of a high-purity oligonucleotide. The process was able to deliver purity levels >95% with yields >50% at both laboratory- and pilot-scale. This work was significant because it demonstrated that larger quantities of oligonucleotides can be successfully purified in a one-step chromatographic process for therapeutic purposes.

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Automation of Extracellular Virus Detection Using Microporous Materials for Blood Separation. Alter, J.; Pall Corporation, Port Washington, NY, USA.

Currently, many automated clinical analyzers utilize serum as the starting sample for detection of analytes or viruses. Serum separation from whole blood requires centrifugation, which is not easily automated. In addition, extra steps, resources, personnel and an extended chain of custody are involved.

Future molecular assays should employ methods that allow whole blood to be assayed without the need for centrifugation. One approach is to utilize a solid support that separates plasma from whole blood in a single step. Separated plasma can then be transferred to a secondary solid support for analyte detection. Hemasep V medium is a sophisticated polyester material designed to separate plasma from small quantities of whole blood in under ten seconds. It was originally designed to separate whole blood for immunodiagnostics.

However, it also as utility has a sample preparation material for extracellular viral detection by nucleic acid amplification.

Experiments were performed to demonstrate that Hemasep V could separate small volumes of whole blood containing extracellular virus. Whole blood was spiked with bacteriophage M13 and separated plasma transferred to solid supports was assayed by PCR for the presence of viral DNA. The results indicate that solid phase blood separation can be used in automated equpiment for viral detection via nucleic acid amplification.

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Detection Of PCR Amplicons Using CPT. Bekkaoui, F.; Fong, W.; Parsons, N.; Bryan, R.; ID Biomedical Corp., Burnbay, BC, Canada.

A colorimetric method based on the Cycling Probe Technology (CPT) has been developed for the detection of PCR amplicon. CPT is an isothermal gene detection method based on RNase H activity. When the chimeric probe DNA-RNA-DNA hybridizes to the DNA target, RNase H cleaves the RNA portion of the duplex and the resulting fragments dissociate from the target. A new probe then hybridizes to the target starting the cycle again. The PCR-CPT reaction containing the unpurified PCR amplicons and the CPT components is incubated for 30 min. at 54°C. The uncut fluorescein-biotin labeled probe is then bound to streptavidin coated wells and detected with anti-fluorescein antibody coupled to horse-radish peroxidase. Because of the signal amplification by CPT, it is possible to detect amplicons from significantly fewer PCR cycles, thus reducing the chance of carry over contamination resulting in false positive samples. It is possible to detect colorimetricaly by CPT the equivalent of 100 target copies after only 20 PCR cycles. Using ethidium bromide stained gels, detection of amplicon is possible only after 30 PCR cycles starting from 100 target copies or 20 PCR cycles starting from 10 target copies. CPT is therefore approximately 10-fold more sensitive for detection of amplicons than ethidium bromide stained gel. Furthermore, CPT offers a second level of specificity which prevents detection of non-specific amplicons. A non-specific amplicon of the same size as the specific amplicon would be detected by ethidium bromide stained gel but not by CPT. In addition this method is relatively fast; it is possible to complete the PCR-CPT assay in less than 3 hours starting with a dilution of a crude DNA preparation. PCR-CPT has been applied for the detection of mecA gene in methicillin resistant Staphyloccocus aureus and can be applied to any other targets where a sensitive and accurate detection is required.

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Damage Induced By Chronic UV Exposure Is Closely Associated With P53 Mutations In Skin Cancer. Juhasz, A.; Holmqist, G.; Beckman Research Institute, City of Hope National Medical Center, Duarte, CA.

UV irradiation of DNA induces the formation of cyclobutane dimers (CPD) between neighboring pyrimidines and these CPDs, rather than UV-induced (6–4) photoproducts and photooxidative DNA lesions, are the main cause of the mutations observed in the p53 gene of basal and squamous skin carcinomas. The rate at which CPDs are repaired is sequence specific and codon positions which are hot spots for skin cancer’s p53 mutations tend to show slow repair of cyclobutane dimers. Since non-melanoma carcinomas generally result after repeated exposure to sunlight, we tested the hypothesis, that chronic dose damage is highly correlated with p53 mutations. We irradiated cultures of confluent human fibroblasts with acute doses of UVC, UVB and simulated sunlight, as well as chronic doses of simulated sunlight. CPD frequency was measured at nucleotide resolution along exons 5–9 of the p53 gene by ligation-mediated PCR (LMPCR). Maxam & Gilbert quantitation standards were used for the first time to correct the sequence context dependent amplification signal of LMPCR. The correlation between acute dose CPD frequency and C->T transitions in skin tumors was as follows; UVC≪UVB<simulated sunlight. The differences are presumably due to 5-methyl cytosine positions being selectively damaged by the longer, > 300 nm, wavelengths of UVB than by the 356 nm of UVC. With chronic UV doses- in theory- each nucleotide position reaches a steady state lesion equilibrum frequency which is equal to the arithmetic product of the acute dose damage frequency times the half life for repair. Our experiments support this theory. We show that a chronic dose of simulated sunlight produces a CPD frequency pattern along the p53 gene which is more closely related to the distribution of C->T transitions in skin cancers than is the damage patterns produced by acute doses.

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CPT-Based Gene Testing By A Lateral Flow Device. Bekkaoui, F .; Modrusan, Z.; McNevin, J .; Fong, W.; Zin, B.; ID Biomedical Corp. Burnbay, BC, Canada; Mizuho USA Inc., San Diego, CA, USA.

A rapid and simple method for gene detection based on Cycling Probe Technology (CPT) and a lateral flow device (strip) was developed. CPT uses a specific chimeric DNA-RNA-DNA probe labeled with fluorescein at the 5’ end and biotin at the 3’ end. CPT is carried out at a constant temperature that allows the chimeric probe to anneal to the target DNA. RNase H cuts within the RNA portion of the chimeric probe and the shorter cleaved probe fragments dissociate from the target thereby releasing the target for further cycling. The strip includes four main components: i) the sample pad, ii) the conjugate pad containing anti-fluorescein antibodies conjugated to gold particles (Anti-F-GP), iii) the nitrocellulose membrane imprinted with a streptavidin line and an IgG line, and iv) an absorbent pad. At the end of the cycling, the strip is inserted into the reaction tube and the sample is absorbed by the sample pad. As the sample flows across the conjugate pad, Anti-F-GP binds with fluorescein of the chimeric probe. The complex of uncut probe and Anti-F-GP, if present, will be captured by streptavidin and result in the development of a test line. Such a test line indicates the absence of the target in the sample. The control line results from the binding of the Anti-F-GP to rabbit-antimouse-IgG. We applied this method for the detection of the mecA gene which is present in methicillin resistant Staphylococcus aureus (MRSA) and absent in methicillin sensitive Staphylococcus aureus (MSSA). In a screen of 48 MSSA and 49 MRSA clinical isolates, the sensitivity and specificity were 98% and 100% respectively as compared to PCR. This assay requires minimal hands-on time and no specialized instrumentation. The results are obtained within one hour starting from a bacterial culture. Such an assay will be useful for the detection of a variety of genes.

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Recognition of DNA Base Mismatches by a Sterically Bulky Rhodium Intercalator. Jackson, B.; Barton, J.; Division of Chemistry & Chemical Engineering, California Institute of Technology, CA,USA.

A novel rhodium intercalator, bis(2,2’-bipyridyl) chrysene quinone diimine rhodium(III) trichloride, has been designed which specifically targets DNA mismatches. The sterically bulky chrysene ligand binds by intercalation between DNA base pairs and, upon photoactivation, cleaves the double helix. At micromolar concentrations the metal complex cleaves in the vicinity of mismatch sites but shows little detectable interaction with Watson-Crick B-form intercalation sites. Binding at sets of mismatches in single sequence contexts has been shown to loosely correlate with the helix destabilization caused by the mismatch; it is this destabilization which is thought to allow the binding of the wide chrysene intercalator which should be too large to readily intercalate in unperturbed DNA. Binding of the complex has been examined at all eight mismatches in all possible sequence contexts and detectable cleavage was observed at 80% of the targeted sites. The thermodynamic binding constants of the complex have been determined at several mismatches by photocleavage titration and vary from 1 x 10ˆ7 Mˆ-1 to 3 x 10ˆ5 Mˆ-1. The non-specific binding constant to standard DNA is approximately 4 x 10ˆ4 Mˆ-1. In addition to promoting strand cleavage when irradiated with ultraviolet light, Rh(bpy)2(chrysi)3+ has also been shown to cleave DNA when irradiated in the visible region. This cleavage, which can even be effected by fluorescent room lighting or sunlight, has also been shown to be non-stoichiometric, i.e. the observed cleavage fraction is only explicable by multiple cleavage turnovers by a single complex. In addition to being a prototype for the recognition of DNA mismatches by sterically large DNA intercalating ligands, Rh(bpy)2(chrysi)3+ and its photochemical behavior raise intriguing possibilities for the design of photo-catalytic mismatch detection technologies or potent mismatch directed therapeutic agents. This type of therapeutic would be particularly promising in diseases associated with failure of cellular mismatch repair systems where mismatch recognition could provide a mode of specificity for diseased tissue over properly functioning cells.

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Electrochemical Detection of Single-Base Mismatches by Electrocatalysis at DNA-Modified Electrodes. Boon, E.; Barton, J.; Hill, M.; Division of Chemistry & Chemical Engineering, California Institute of Technology, CA, USA.

DNA-based biosensors sensitive enough to detect single-base mismatches are required for screening of genetic mutation and disease. We have developed a method for electrochemical detection of mismatches based on charge transport through double-stranded DNA monolayers on gold electrodes. DNA-modified electrodes are prepared by the self-assembly of pre-hybridized duplexes in which one of the strands of the duplex is derivatized at the 5’ end with an alkanethiol chain. These modified electrodes are used to monitor the electrochemical signal of methylene blue (MB, a redox active intercalator) bound to fully base-paired and singly mismatched DNA sequences; the presence of a mismatch dramatically decreases the electrochemical response. These absolute electrochemical signals are limited by the surface concentration of MB (∼ 50 pmol/cm), however. To increase the inherent sensitivity of this assay, we have coupled the direct electron transfer to an electrocatalytic process involving freely diffusing ferricyanide (possessing a large negative charge, ferricyanide is electrostatically prohibited from the interior of the monolayer). In this process, electrons flow to intercalated MB, bound primarily at the solvent exposed periphery of the monolayer, and then are accepted by solution-borne ferricyanide. This heterogeneous electron transfer reaction reoxidizes MB, causing more electrons to flow to MB and thus the perpetuation of the catalytic cycle. In essence, the catalytic reaction amplifies the absolute signal corresponding to MB in addition to pronouncing the attenuation of the response in the presence of a mismatch. Furthermore, due to its catalytic nature, the charge measured increases with increased sampling times, and in fact is only limited by the solution concentration of ferricyanide (∼ 2.0 mM). Longer integration times provides even greater absolute signals which increase disproportionately for fully complementary verses mismatched DNA, making it possible to indefinitely increase the differentiation between matched and mismatched sequences. The efficient transport of charge through self-assembled monolayers of thiol-terminated duplexes on gold, coupled to a catalytic reaction in solution, offers a practical means to detect DNA mutations at the single base-pair level.

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Single-tube Quantitation of HCV RNA by Competitive RT-PCR and Fluorescence Detection. Robbins, D.; Pasupuleti, V.; Cuan, J.; Lappin, S.; Chiang, C.S.; Van Nuys, CA, USA.

Hepatitis C Virus (HCV) is the major causative agent for parentally transmitted non-A, non-B hepatitis, affecting approximately 100 million people worldwide. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) permits the direct detection of the virus. The quantitative RT-PCR procedure can be used for monitoring patients undergoing antiviral therapy and is currently used to monitor patient response to alpha-interferon, the newly FDA approved combination therapy, and in clinical trials for HCV therapy. We have developed a one-tube RT-PCR method that quantitates HCV in the range of 2,000 - 10 copies/mL.

A synthetic RNA was generated with HCV sequences on either end, and thus acts as an internal standard (IS) in the assay. A known amount of IS is added to each aliquot of patient plasma or serum and co-extracted with the HCV RNA using commercial viral RNA preparation kits. Isolated RNA is incubated with Reverse Transcriptase (RT), AmpliTaq Gold (Perkin Elmer), and HCV-specific primers. After the RT step, a 95°C incubation simultaneously inactivates the RT and activates the AmpliTaq Gold. During amplification, the IS competes with the HCV RNA (if present). Fluorescent PCR fragment peaks, distinguishable by size via capillary electrophoresis or equivalent, are integrated and the areas compared. The ratio of peak areas for IS and HCV RNAs is linearly related to the HCV RNA copy number on a log-log plot. Thus, the patient sample need not be diluted multiple times as in commercially available quantitative assays. A standard curve of known HCV RNA copies/mL allows translation from peak area ratio to specimen HCV copies/mL. Across the quantitation range, the intra-run CV was 27% or less; the inter-run CV was 31% or less. Assay sensitivity and quantitation is equivalent for HCV subtypes 1a, 1b, 2a, 2b, and 3a. These results show that a competitive RT-PCR with a single patient sample replicate can provide a precise, sensitive method for quantitating HCV RNA amenible to automation.

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Multianalyte Detection of Five Gastrointestinal Bacterial Pathogens Using Fluorescence GeneSTAR^®Technology.Li, Y.; Perkins, T.; Wu, L.; Salt Lake City, Utah, USA.

We present here a method for the rapid detection of multiple pathogens in a single microplate well using GeneSTAR^® DNA detection technology. The DNA from five gastrointestinal pathogens, Salmonella, E. coli O157:H7, Campylobacter, Shigella, and Yersinia, was amplified by multiplex PCR using five primer sets labeled with fluorescein and tetramethylrhodamine. The amplified DNA sample was hybridized to a microplate coated with oligonucleotide probes in a two analyte probes per well format. The plate was washed to remove non-specifically bound DNA and then read on a fluorescence plate reader.

Using two filter sets, one for fluorescein and the other for tetramethylrhodamine, two analytes could be detected in each well with a signal-to-noise ratio of 40. To enhance signal , both PCR primers were labeled. An 80% signal increase was obtained when compared with that of labeling only one primer. To make the test user-friendly, hybridization was done at room temperature. The E. coli primer set, which amplifies the uidA gene, was designed to selectively amplify the single base mutated O157:H7 strain in the presence of wild type E. coli. The kit showed 100% accuracy when evaluated with 24 E. coli isolates that were characterized by the Centers for Disease Control and Prevention and may bear the O157:H7 strain. Preliminary results showed that DNA extracted from fecal samples can also be detected, even though in some samples a lower reading was observed.

This design should be applicable to the large scale screening of enteric pathogens, with the capacity to discriminate point mutations. The test is simple and only requires 3 hours to complete.

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Oligo(Dt)-Immobilized Pipette Tip: Efficient New Methodology For Mrna Preparation And Direct Gene Amplification. Mitsuhashi, M.; Eguchi, K.; Hamaguchi, Y.; Aso, Y.; Shioiri, T.; Ogura, M.; Hitachi Chemical Research Center, Irvine, CA. USA.

Oligo(dT₂₀) was immobilized onto disposable polypropylene pipette tips via the 5’ end to purify poly(A)+mRNA. For quantitation of mRNA hybridization in the tip, captured mRNA was dissociated by aspirating 50 μl of diethylpyrocarbonate boiling water followed by dispensing it into microplates. The resultant mRNA was then stained with Yoyo-1 and the fluorescence was determined by FMBIO II (Hitachi Software). When 250 ng of rabbit globin mRNA was applied to the tips, dissociated mRNA was 91.1±12.4 ng (mean±standard error, n=3). When 5 μg of rat brain total RNA was used, 99.7±19.8 ng of mRNA was collected. Quality of dissociated mRNA was also confirmed by agarose gel electrophoresis. The mRNA was also captured directly from crude cell lysate of human K562 leukemic cells, and resultant captured mRNA was further processed to RT-PCR in the same tip. PCR products of human glyceraldehyde 3 phosphate dehydrogenase was successfully amplified, whereas appropriate PCR products were not amplified when the step of cDNA synthesis was omitted. Because of the variety of advantages, our oligo(dT)-immobilized pipette tips may be acceptable as a new methodology for various mRNA analyses in basic laboratory research, outdoor field research, as well as molecular pathological diagnostics.

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Rapid, Stable And Ambient Storage Of Leukocyte RNA From Whole Blood. Aso, Y.; Shioiri, T.; Hamaguchi, Y.; Kawamura, M.; Mitsuhashi, M.; Hitachi Chemical Research Center, Irvine, CA, USA.

Although many techniques and commercial kits are available for RNA preparation, these require time-consuming multiple steps, and are not suitable when RNA is collected from patients in clinics, hospitals, operating rooms, or emergency room. Here we report a rapid preservation method of RNA from human blood. Mononuclear leukocytes separated by density-gradient centrifugation were filtered through RiboCap syringe filters (AGCT, Irvine, CA), and were immediately dried under vacuum. For whole blood, erythrocytes were first selectively lysed in hypotonic solution followed by filtration through RiboCap syringe filters. Denaturing solution was applied to the filters immediately after cells were trapped, and RNA-containing pass-through fraction was collected. Both dried filters or RNA-containing denaturing solution were stored at room temperature. RNA was then isolated by acid-guanidine/phenol/chloroform extraction. Agarose gel electrophoresis confirmed the presence of 18s and 28s rRNA bands, and glyceraldehyde 3-phosphate dehydrogenase mRNA was amplified by RT-PCR, even from samples stored at room temperature for 2 weeks. The amount of total mRNA extracted from these samples was similar to that processed immediately. We hope this methodology encourages physician scientists to bring more clinical specimens into research fields to bridge between basic science and clinical medicine.

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Direct RT-PCR On Oligo(Dt)-Immobilized Polypropylene Microplates After Capturing Total Mrna From Crude Cell Lysates. Mitsuhashi, M.; Hamaguchi, Y.; Aso, Y.; Ogura, M.; Isreal, C.; Koo, K.; Hitachi Chemical Research Center, Irvine, CA, USA.

In order to simplify the entire process of gene expression analysis, oligo(dT)-immobilized polypropylene microplates (GenePlate-PP, AGCT) were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantitated fluorometrically by using Yoyo-1 or AttoPhos as fluorometric reagents in either FMBIO-II laser scanner (Hitachi Software) or CytoFluor plate reader (Perseptive). GenePlate-PP captured mRNA, but not rRNA, tRNA, and DNA. Moreover, mRNA was prepared directly from crude cell lysates, and approximately 40–50% of applied mRNA was captured by the GenePlate-PP. Hybridized mRNA was then amplified by 1 step RT-PCR with rTth polymerase or 2 step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In 2 step RT-PCR, GenePlate-PP can be re-used for multiple PCRs with same or different primer sets, because synthesized cDNA was covalently attached to the plates at its 5’ end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses including basic research, drug screening, molecular toxicology, as well as molecular pathological diagnostics.

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RNA Chip: Quality Assessment of RNA by Microchannel Linear Gel Electrophoresis in Injection-molded Plastic Chips. Ogura,M.; Agata, Y.; Watanabe, K.; McCormick R.; Hamaguchi, Y.; Aso, Y.; Mitsuhashi, M.; Hitachi Chemical Research Center, Irvine, CA. USA.

Two major components of rRNAs (18S and 28S rRNA) were separated by electrophoresis in injection-molded acrylic chips with a microchannel of 100 μm in width, 40 μm in depth, and 1 cm of separation distance. Microchannels were filled with 0.4% hydroxypropylmethylcellulose as sieving polymer and 5 μg/mL ethidium bromide for RNA stain. The fluorescent signals were detected by fluorescent microscope equipped with photometer and 590 nm emission filter. The assay is rapid (less than 3 min), reproducible, RNase-free, disposable, and requires only 1–2 μL of samples. The sensitivity was approximately 10 ng/μL, 100 folds higher than that of conventional agarose gel electrophoresis. Because only 0.1 nL of loaded samples were used for electrophoresis, the detectable peaks of rRNA were derived from less than single cell. Since the quality of RNA is critical for RNA-related diagnostic tests, our disposable chips will be useful for the quality assessment of RNA.

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Rolling-Circle Amplification Of DNA: Purification Of Circular Molecules Prior To Amplification Increases Specificity. Giffard, P.M.; Hafner, G.J.; Wolter, L. C.; Yang, I.; Stafford, M.R.; CRC for Diagnostic Technologies, Queensland University of Technology, Gardens Point Campus, Queensland, Australia.

In vitro rolling circle (RC) reactions form the basis of a set of emerging methods for the amplification of specific DNA sequences. Target amplification is usually accomplished using a “padlock” probe that is able to hybridise to the target DNA sequence and be circularised by a DNA ligase. This circular molecule is then amplified using an RC reaction. We have been investigating strategies for maximising the sensitivity and specificity of an RC amplification based method that makes use of a padlock probe, a biotinylated “stuffer” oligonucleotide, ampligase ligase, primers designed to anneal to complementary strands of the amplimer, and the moderately thermostable Bst polymerase.

During early phases of this research, it was observed that amplification was not always dependent upon the presence of circular molecules. Incubation of uncircularised padlock probe with the RC amplification primers under RC amplification conditions results in extensive amplification of padlock probe derived sequences. This provides the potential for contamination of the amplimer with unwanted background. It was reasoned that removal of uncircularised padlock probe from the reaction before amplification would minimise this problem. This was accomplished by constructing a padlock probe that when annealed to the target leaves a gap between its ends. Also included in the annealing/ligation reaction is a biotinylated “stuffer” oligonucleotide that is designed to anneal to the target between the ends of the padlock probe. Therefore, the presence of the target allows the ligation of the biotinylated stuffer fragment to the padlock and the consequent formation of a biotinylated circular DNA molecule. This may be purified using streptavidin coated beads and used as a substrate for RC amplification. We were able to detect single copy human genes from 10 ng of genomic DNA, and that no product was produced when a negative control consisting of Escherichia coli DNA was used as the substrate. A modification of this method that makes use of mismatches at the 3’ end of the stuffer fragment is able to distinguish synthetic targets that differ at a single nucleotide.

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Highly Sensitive Bioluminescence Hybridization Assays Based on in Vitro Expression of DNA Reporter Molecules Encoding Apoaequorin.White, S.; Christopoulos, T.; Windsor, Ontario, Canada.

Two expression hybridization assay configurations with high sensitivity for specific nucleic acid sequences have been developed. Both assays use an expressible apoaequorin encoding DNA as a label.

The DNA label (AEQ-PolyA-DNA) was prepared by amplifying the apoaequorin coding sequence, with the T7 promoter, by PCR and then adding a downstream polyadenylation signal. The AEQ-PolyA-DNA was tailed with dATP for use as a reporter molecule in hybridization assays. Expression of this template in vitro by coupled transcription/translation in the presence of coelenterazine indicated that each AEQ-PolyA-DNA can generate approximately 160 aequorin molecules.

The AEQ-PolyA-DNA was tested as a reporter molecule in two hybridization assay configurations. In the “captured target configuration”, biotinylated target DNA was bound to streptavidin coated microtiter wells. After removal of one strand of the target DNA, through NaOH treatment, it was hybridized with dTTP-tailed detection probe. In the “sandwich configuration” a biotinylated capture probe was immobilized on streptavidin coated microtiter wells. This was followed by simultaneous hybridization of heat denatured target DNA to the immobilized capture probe and dTTP-tailed detection probe. In both assay configurations the hybrids were reacted with dATP-tailed AEQ-Poly-DNA followed by in vitro expression of the DNA, in the presence of coelenterazine, to generate aequorin. Aequorin was determined by adding a Ca²⁺-containing light triggering solution and integrating the signal for 3s.

The luminescence was linearly related to the target DNA in the range of 0.5 to 78oo amol and 0.5 to 1500 amol, for the captured target and sandwich configurations, respectively. The average CVs were 10.1% and 8.1% respectively. These assays are about 50 times more sensitive than a hybridization assay which uses aequorin as a label and approximately 40 times more sensitive than an expression hybridization assay which uses luciferase encoding DNA as a label.

The use of apoaequorin encoding DNA as a label provides significant amplification (through transcription/translation) and enhances sensitivity. The assays are readily automatable and allow the detection of sub-attomole levels of target DNA.

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A Neisseria Gonorrhoeae Transcription-Mediated Amplification Assay For The Automated VIDAS Instrument.Burres,E.; Santa Ana, S.; Gilker, M.; Clark-Dickey, K.; Longiaru, M.; Moe, J.; Levasseur, P.; Gen-Probe, San Diego, CA, USA.

Transcription Mediated Amplification (TMA) is an isothermal nucleic acid amplification method utilizing specific primers and enzymes. Targeting ribosomal RNA (rRNA), which is present in bacterial cells in thousands of copies, results in high sensitivity, and ease of use makes it suitable for the clinical laboratory. The VIDAS Amplified N. gonorrhoeae assay (ANG) pairs Gen-Probe’s TMA chemistry with bioMérieux Vitek’s VIDAS automated immunoassay instrument. rRNA from N. gonorrhoeae in a swab or urine specimen is amplified during the TMA reaction followed by solid phase capture of the amplified product, hybridization to a specific alkaline phosphatase-labeled (AKP) probe, and detection of a fluorescent end product in the VIDAS instrument. Reagents are packaged in a unit dose format, and time to results is less than 3 hours.

The ANG assay demonstrates sensitivity of less than one N. gonorrhoeae cell per test, and no false positive results were seen during specificity testing with other Neisseria species. An internal control which monitors success or failure of amplification with no impact on assay methodology or complexity has been incorporated into the VIDAS ANG assay. Internal control monitoring of ANG assay amplification failures in negative clinical specimens has demonstrated an inhibition rate of 0% and 1.6% in swabs and urines respectively.

In a preliminary study, 617 clinical swabs and urines with known PACE results for N. gonorrhoeae were tested in the ANG assay at Gen-Probe Incorporated. Following resolution testing using an alternate in-house amplified method, 520 of 521 negative specimens were negative in the VIDAS ANG assay. Ninety two of 96 positive specimens were positive in the ANG assay.

Preliminary results indicate that the VIDAS ANG assay is sensitive and specific, and addition of an internal control allows the identification of false negative results. The alliance of probe diagnostic technology with an existing automated instrument will provide a rapid method for the direct identification of N. gonorrhoeae.

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Amplified Mycobacterium Tuberculosis Test For The Automated VIDAS Instrument Includes Internal Control. Clark-Dickey, K.; Burns, J.; Darby, P.; Longuaru, M.; Lugo, L.; Quigley, T.; Sitay, A.; McKinley, G.; Gen-Probe, San Diego, CA, USA.

The Centers for Disease Control and Prevention stated, “the main obstacle to effective control of tuberculosis is the lengthy time required for diagnosis.” We are developing a new test that will permit the clinician to diagnose TB infection in patients within 4 hours. Recent enhancements to the M. tuberculosis (M.tb) identification test on the automated VIDAS immunoassay detection instrument include: single dose amplification reagents, a pelletized enzyme reagent, and an internal control to monitor amplification failure. Amplification of a respiratory specimen’s rRNA and the internal control RNA occurs simultaneously during the Transcription Mediated Amplification (TMA) reaction. The amplified products are then hybridized to specific probes and detected on the VIDAS instrument. The entire test takes place within an enclosed disposable strip, and the strip wells are automatically decontaminated with bleach to eliminate any remaining amplicon. The amplified method showed complete specificity for members of the M.tb complex and did not cross react with a panel of 32 species of mycobacteria other than M.tb or 11 related genera. The sensitivity of the system is less than one M.tb cell per test. Four hundred and sixty-three clinical specimens were tested and the results compared to culture. Sixty-one out of 62 M.tb culture positive specimens were also positive by the VIDAS amplified method, including 6 specimens that were smear negative for acid-fast bacilli (98.4% sensitivity). There was no cross reaction with 47 specimens identified by culture to contain mycobacteria other then M.tb. Three hundred seventy-four out of 378 specimens were negative for M.tb by both methods (98.9% specificity). The internal control successfully monitored amplification failure with inhibition rates of 4% in the specimens tested. The union of probe diagnostic technology with the existing VIDAS automated instrument will allow for rapid and convenient identification of M. tuberculosis in respiratory specimens in clinical laboratories worldwide.

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Next Generation Microchip Pcr Instruments. Belgrader, P.; Benett, W.; Hadley, D.; Hanford D.; Long, G.; Mariella, R.; Milanovich, F.; Nasarabadi, S.; Miles, R.; Livermore, CA, USA.

The sporadic outbreak of infectious diseases has created a need for rapid field instruments to detect and identify pathogenic microbes. At LLNL we are developing and testing microchip PCR instruments that perform rapid, real-time DNA testing using the commercial 5’ nuclease fluorogenic assay. These portable instruments are designed to use low power for battery-operation, contain stationary optics for rugged handling, and display a user-friendly interface with an audible alert and red indicator when a positive is identified. Our present 10-chip instrument, the Advanced Nucleic Acid Analyzer (Clin. Chem.1998; in press), was subjected to a blind test on 320 samples consisting of Bacillus subtilis spores, Erwinia herbicola cells, and MS2 virions which are surrogates for pathogenic agents. Successful performance on this test has initiated the production of a third generation of instruments comprising a 4-chip hand-held unit and a 24-chip suitcase unit. These devices will incorporate new chip and optical technology for faster heating and cooling, less power consumption, and multiplex analysis capability. In addition, real-time detection has been demonstrated using sample preparation devices that incorporate automated microfluidics in place of manual pipetting. This work was supported by the Central Measurement and Signatures Intelligence Organization and was performed under the auspices of the U.S. DOE by Lawrence Livermore National Laboratory, Livermore California under Contract No. W-7405-Eng-48.

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Detection Of Nucleic Acid Hybridization By Thin Film Formation On Optical Surfaces. Hopkins, D.; Laumark, A.; Ryan, E.; Ostroff, R.; Polisky, B.; BioStar, Inc., Boulder, CO, USA.

We have developed a nucleic acid hybridization assay utilizing Optical ImmunoAssay (OIA^® ) thin film technology. This technology allows direct visual or instrumented detection of the physical change in optical thickness of molecular thin films resulting from the binding of the appropriate analyte to a capture molecule on the surface. The direct visual signal is generated by the change in reflection of light through the molecular thin films formed on an optically coated solid phase. In this assay, the thin film is generated by hybridization of a surface-bound capture oligonucleotide to the appropriate target, followed by addition of a biotinylated detector oligonucleotide, an anti-biotin antibody-HRP conjugate and enzyme substrate. Results are obtained in as little as 25 minutes. Data from three experimental systems will be presented. Initial experiments involved a simple 2-oligonucleotide “capture-detector” model system. This assay was extended to a more clinically relevant 3-oligonucleotide “capture-target-detector” system based on sequences from the S. aureus mecA gene encoding methicillin resistance. Finally, we have developed a high throughput screening format for multianalyte diagnostic assays. This detection technology is adaptable to a wide variety of applications including medical diagnostics, environmental and food testing, drug screening, expression analysis and genomics.

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Genetic Heterogeneity In Human Acatalasemia. Goth, L.; Sherimani, A.; Kalmar, T.; Department of Clinical Chemistry, Medical University, Debrecen, Hungary.

The enzyme catalase is the main regulator of hydrogen peroxide being involved in physiological phenomena and in the pathogenesis of various diseases.

Acatalasemia, a genetic deficiency of erythrocyte catalase is inherited as a rare, autosomal trait. The peculiar and clinical signs of this syndrome are poorly understood and its two types (Swiss, Japanese) are characterized by clinical, biochemical and molecular biological methods.

The asymptotic Swiss-type is caused by a point mutation resulting in a truncated protein with a less stability. Of 46 families with the Japanese-type, four patients revealed a splicing mutation (G to A at position 5 of intron 4) and five patients with a T(358) deletion at position 10 of exon 4.

Firstly, in Hungary we have reported one acatalasemic and nine hypocatalasemic families with two acatalasemic and 42 hypocatalasemic members. The characterization of their catalase protein excluded the similarity to the Swiss-type of this syndrome.

The causal mutation(s) for these patients were examined using the PCR products of all exons and exon/intron junctions for single strand conformational polymorphism, heteroduplex formation, and nucleotide sequence analyses.

The result showed neither the 358 T deletions in exon 4 nor the splicing mutation in intron 4 demonstrating that the Hungarian acatalasemia/hypocatalasemia differs from the Japanese-type.

The search for the desease causing mutation yielded 8 mutations at the promoter region and exon 1 with 4 novel mutations. They were in no correlation to hypocatalasemia. Heteroduplexes of exon 2 could be detected for all the hypocatalasemic members (22) and none of the normocatalasemic members (24) of the acatalasemic and three hypocatalasemic families. This pattern was not found in 22 hypocatalasemic and 15 normocatalasemic members belonging to the other 8 families. Sequencing of these samples are in process to detect the exact change causing the heteroduplex formation.

From these results we may conclude that the Hungarian acatalasemia/hypocatalasemia is a new type of this syndrome and it is heterogen at DNA level.

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Rapid Amplification (RAMP): An Isothermal Method For Amplification Of Nucleic Acid. Wick, J.; Rifelj, M.; Jolly, J.; Milwaukee, WI, USA.

RAMP is a rapid (10–30 min), isothermal method for amplification of nucleic acid. For RNA targets this method relies on AMV reverse transcriptase and RNase H to synthesize double-stranded cDNA and a thermophilic DNA polymerase and restriction endonuclease to subsequently amplify the cDNA. Two primers, each containing a 3’-hybridization sequence and a 5’-tail containing a site for a restriction endonuclease, delineate the amplification region within the RNA molecule. The reaction mechanism involves first strand cDNA synthesis by AMV reverse transcriptase initiated by the hybridization of a primer to the RNA. RNase H degrades the RNA and a second primer hybridizes to the first strand cDNA. DNA polymerase initiates second strand cDNA synthesis at the second primer. A modified restriction site is generated by the DNA polymerase which incorporates a modified dNTPαS into the strand opposite the first primer. The restriction site is rendered nickable but non-cleavable since the primer strand contains unmodified nucleotides while the filled-in sequence on the opposite strand contains a modified nucleotide located precisely at the restriction endonuclease cut site. The restriction enzyme nicks the primer strand at the restriction site and the polymerase displaces the downstream DNA strand. The displaced strand hybridizes to the first primer, leading to subsequent rounds of DNA synthesis, nicking of the regenerated restriction site, and displacement of the single strand downstream from the nick site. The reaction amplifies the target RNA molecule yielding DNA amplicon. RAMP primers have been designed for the amplification of Cryptosporidium RNA. Amplicon was detected either colorimetrically in a microplate sandwich assay using a sequence-specific capture probe and a HRP-oligonucleotide conjugate complementary to the amplicon or using a labeled sequence specific probe analyzed by PAGE. Using these systems we show that RAMP produced detectable amounts of DNA in 10 min starting from a single Cryptosporidium oocyst. This assay method may be suitable for the rapid detection of a variety of other pathogenic microorganisms.

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Intercalation Activating Fluorescence DNA Probe And Its Application To Homogeneous Quantification Of A Target Sequence By The Isothermal Sequence Amplification In A Closed Vessel. Saitoh, J.; Yokoyama, A.; Ishizuka, T.; Taya, T.; Ishiguro,T.; Tosoh Corporation, Ayase-shi, Kanagawa, Japan.

It is no doubt that polymerase chain reaction, PCR, is a powerful tool to detect pathogen-related sequences in high sensitivity. On the other side, however, the PCR-based methods have several obstacles in engineering to attempt the high-throughput automation for upcoming clinical settings. A homogeneous nucleic acids assay with isothermal sequence amplification could be a strategy in order to overcome these drawbacks.

We developed a new type of fluorescent DNA probe, which named INAF DNA probe, or Intercalation Activating Fluorescence DNA probe, which can emit enhanced fluorescence by binding to a complementary oligomer. INAF DNA probe is an oligonucleotide equipped with oxazole yellow (YO) through a linker, -(CH₂)₂NHCO(CH₂)₂S-, which would readily deliver the dye moiety into adjacent base pairs of formed double stranded complex with a complementary target nucleic acids.

We thus attempted to apply the YO-linked DNA probe to real-time monitoring of the production of RNA in isothermal sequence amplification on the basis of cooperative two-enzyme feed-back reaction of in vitro transcription by SP6 RNA polymerase and the conversion of the product, RNA, by AMV RTase into the promoter bearing double stranded DNA.

The optimized reaction condition brought to 10-fold amplification of starting RNA, 10 copies, in 3-hour incubation at 50°C ddTT was attached at 3’ terminus of the probe by terminaltransferase to avoid unfavorable elongation as a primer in the concerted reaction.

After one-hour incubation, the fluorescence intensity in the reaction tube observed dramatically increased with the progress of the concerted reaction, which finally yielded comparable product bands on agarose without any interference caused from the presence of the YO-linked DNA probe. It was found that the rate of the increase of the fluorescence depended on starting copies of RNA ranged from 10 to 10 copies and the target RNA in a sample could be quantified on the basis of the fluorescence profile obtained.

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Homogenous Detection Of Nucleic Acid Targets Using Acridinium Ester-Labeled 3-Way Junction. Escobedo, M.; Nelson, N.; Gen-Probe, San Diego, CA, USA.

The Hybridization Protection Assay (HPA) is a homogeneous nucleic acid assay system based on the differential hydrolysis properties of the highly chemiluminescent acridinium ester (AE). Typically, short oligonucleotide probes are covalently labeled with AE. AE associated with unhybridized probe is rapidly hydrolyzed and rendered non-chemiluminescent, while AE associated with probe hybridized to its complementary sequence is protected from hydrolysis and, thus, yields a chemiluminescent signal proportional to the amount of target nucleic acid present. A mismatch in the target adjacent to the site of AE attachment in the probe disrupts protection, rendering the AE once again subject to rapid hydrolysis. In this way, matched targets can be discriminated from mismatched targets.

However, there are applications where discrimination between mismatches is not desired. For example, HIV is prone to random mutation, yet each strain resulting from such mutation still must be detected as HIV. It was the goal of this work to develop an HPA-based system that would tolerate, rather than be sensitive to, mismatches.

This goal was accomplished using an AE-labeled 3-way nucleic acid junction probe which consists of two separate oligonucleotide strands, each of which has a target-binding region and an arm region. The arm regions of the two strands are complementary, but designed not to form a stable duplex in the absence of target. One of the strands is labeled with AE in the arm region. In the absence of target, AE rapidly hydrolyzes and yields a background chemiluminescent signal. In the presence of target, the two target-binding regions bind to target and position the arms regions in close proximity, allowing formation of a stable arm duplex. The AE is thus protected from hydrolysis and yields chemiluminescent signal. Since the AE is not in the target-binding region of the probe strands, mismatches in the target do not affect AE protection and are thereby tolerated. A 3-way junction probe specific for HIV-1 was constructed and shown to detect a number of strains which contained genetic mutations.

In conclusion, AE-labeled 3-way junctioned probes have been developed which function in HPA to sensitively and quantitatively detect nucleic acid targets and tolerate genetic mutations.

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Direct Electrochemical Detection Of Nucleic Acids. Eckhardt,A.; Thomas, R.; Mikulecky, J.; Napier, M.; Research Triangle Park, NC, USA.

The detection of nucleic acid hybridized to a complementary capture probe is of central importance in genomic research and pathogen diagnostics. Current methodologies require the covalent attachment of an enzymatic, fluorescent, chemiluminescent or radioactive label to the hybridized target for detection. We have developed a direct electrochemical detection method for DNA and RNA that does not require covalent attachment of reporter molecules. The method is based on the electrochemical oxidation of guanine by the soluble metal mediator ruthenium (2,2’-bipyridine) (Ru(bpy)₃²⁺). Ru(bpy)₃²⁺ abstracts electrons from guanine via a catalytic cycle when the appropriate potential is applied and shuttles the electrons to an indium tin oxide (ITO) electrode thereby generating an electrical current. The oxidation of guanine by Ru(bpy)₃²⁺ can be utilized for the detection of single and double stranded DNA as well as RNA without the need for reverse transcription to form cDNA. This method, unlike other electrochemical techniques, does not require the use of duplex specific redox-active indicators or that the target nucleic acid be in close proximity or directly wired to the electrode.

The detector format currently in use is a 6 mm diameter ITO electrode upon which a monolayer is self assembled and an oligonucleotide probe attached to the monolayer. A nucleic acid target is allowed to hybridize to the probe and after washing, Ru(bpy)₃²⁺ added and the signal acquired. Substitution of hypoxanthine for guanine in the probe reduces the current to background levels because hypoxanthine is 1000 fold less reactive than guanine. Currently, using the 6 mm electrode, seven fmoles of a 34-mer oligonucleotide containing 23 guanines generated a current of 0.16 mA over background.

The specificity and sensitivity of the system is being optimized using synthetic oligonucleotide probes and targets, PCR products, mRNA from cultured cells, various signal acquisition methods and miniaturization of the electrodes. The detector system can be readily multiplexed and we are designing a 96 well microtiter plate with multiple individually addressable electrodes in each well. The technology is also useful for the simultaneous monitoring of protein expression using detector antibodies labeled with guanine.

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Chemiluminescent Microplate-Based RNA Detection Assay For Sensitive Quantitation Of Target RNA. Boldt-Houle, D.; Martin, C.; Liu, B.; Costa, S.; Aristarkhov, A.; Palmer, M.; Voyta, J.; Bronstein, I.; Bedford, MA, USA.

A powerful technique for ribonucleic acid detection in a microplate assay has been developed by combining sequence specific labeled capture and detection probes with highly sensitive chemiluminescent detection. Typical quantitation of RNA targets in assays such as Northern hybridization or RNAse protection assay require large quantities of RNA and several days to complete. We have developed a highly sensitive chemiluminescent solid phase capture assay for quantitation of target RNA molecules that requires much less RNA, and can be performed in one day. In our basic assay, RNA samples are captured with biotin-labeled sequence specific capture oligonucleotides bound to an avidin coated plate and detected with alkaline phosphatase labeled sequence specific oligonucleotides. The RNA-oligonucleotide hybrids are quantitated through the signal generated from chemiluminescence with CSPD® 1,2-dioxetane, an alkaline phosphatase substrate, and Sapphire II™ polymeric enhancer. A c-fos specific RNA target, 2.145 Kb, was detected at a sensitivity of 20 attomoles. Variations on the basic assay include using longer (300 bases to 1.1 Kb) biotinylated capture probes, or peptide nucleic acid (PNA) probes for capture and detection. Preliminary results indicate an increase in target sensitivity using the PNAs for quantitation. The enhanced sensitivity of chemiluminescence coupled with the simpler and faster protocol for RNA quantitation in a microplate format creates an excellent alternative to approaches such as quantitative reverse transcriptase-PCR, and may be adapted easily to a higher throughput format.

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Quantitative Polymerase Chain Reaction Based On A Dual-Analyte Chemiluminescence Hybridization Assay For Target DNA And Internal Standard. Verhaegen, M.; Christopoulos, T.; University of Windsor, Ontario, Canada.

We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was co-amplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24 bp sequence centrally located. Biotinylated PCR products from both target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe and the hybrids were determined using an antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a specific fluorescein-labeled probe and the hybrids determined using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was determined by measuring luminescence following addition of a Ca²⁺ containing light-triggering solution. Alkaline phosphatase was subsequently determined by measuring luminescence generated after addition of a dioxetane chemiluminogenic substrate. The luminescence was linearly related to the concentration of target DNA and IS amplification products over the range of 0.97–500 pmol/L and 5.5–700 pmol/L, respectively. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430–315 000 target DNA molecules. Day-to-day coefficients of variation (including the PCR step and dual-analyte hybridization assay) for the ratio of luminescence values obtained for 1300, 13 000, and 130 000 target DNA molecules co-amplified with a constant amount of IS were 7, 9 and 17%, respectively (n=4). Average within-run coefficients of variation for target DNA and IS were 5.9 and 5.5%, respectively (n=5). The designed dual-analyte system is expected to facilitate the automation and routine use of quantitative PCR.

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Enzyme Amplification In Aequorin-Based Bioluminescence Hybridization Assays. Laios, E.; Ioannou, P.; Christopoulos, T.; University of Windsor, Ontario, Canada.

We have introduced enzymatic amplification in aequorin-based bioluminescence hybridization assays for increased sensitivity. In a model sandwich hybridization assay, 8 nmol/L of a capture probe, tailed with biotin-dATP, is bound to streptavidin coated microtiter wells. Heat-denatured target DNA (495 bp) is then hybridized (60 min at 42°C) simultaneously with the capture probe and 4 nmol/L detection probe tailed with digoxigenin-dUTP. The hybrids are reacted (20 min, RT) with antidigoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzes (20 min, RT) the oxidation of a digoxigenin-tyramine conjugate by hydrogen peroxide, resulting in the covalent attachment of multiple digoxigenin moieties to the solid phase. Anti-digoxigenin antibody conjugated to aequorin was then allowed to bind to the immobilized digoxigenins. The luminescence of aequorin was measured by adding a Ca²⁺-containing (0.1 mol/L) light triggering solution. The signal was integrated for 3 s. The digoxigenin-tyramine conjugate was prepared by reacting equimolar amounts of N-hydroxysuccinimide derivative of digoxigenin to tyramine in dimethylsulfoxide. The antidigoxigenin-peroxidase concentration was studied in the range of 6.2–200 U/L with an optimum signal/background (S/B) ratio at 100 U/L. The digoxigenin-tyramine concentration was studied in the range of 0.9–59 umol/L at the optimum S/B ratio was found to be at 15 umol/L. The hydrogen peroxide concentration was studied in the range of 0.2–15,625 umol/L with an optimum S/B ratio at 25 umol/L. We observed that the optimized assay gave a 12-fold improvement of the signal and a 7-times enhancement of the signal/background ratio compared to the assay that used only anti-digoxigenin aequorin without the peroxidase amplification step. The CV was in the range of 5.5–7.3%. In conclusion, the combination of enzymatic amplification with the aequorin bioluminescence, leads to a significant enhancement of the sensitivity of hybridization assays. The assays are performed in microtiter wells and are easily automatable.

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Universal Molecular Connectors For DNA Hybridization Assays. Tannous, B.; Chiu, N.; Christopoulos, T.; University of Windsor, Ontario, Canada.

We constructed two universal molecular connectors for attaching enzyme-coding DNA reporter molecules to molecules that provide molecular recognition (probes or antibodies). The molecular connectors were prepared by conjugating an oligodeoxynucleotide with either streptavidin or antidigoxigenin antibody. A protected sulfhydryl group was introduced to a 5’ NH₂-modified oligo (5 nmol) by reacting with a 10 fold molar excess of S-acetylthioacetate and purifying by gel filtration. Streptavidin and antidigoxigenin antibody (Fab fragments) were derivatized with a 40 and 25 molar excess of sulfo-SMCC, respectively and purified by ultrafiltration. Subsequently, the oligo was mixed with the derivatized protein in molar ratio of 10:1. The reaction was initiated by deprotecting the SH-group with hydroxylamine. The conjugate was purified from the free oligo by ultrafiltration. The oligo of the conjugate was then tailed with dATP. To demonstrate the applicability of molecular connectors, a sandwich-type hybridization assay based on an expressible enzyme-coding DNA label was developed. Microtiter wells were coated with 25 uL of 5ug/mL anti-fluorescein antibody. Then, 62.5 fmol/well of a capture probe, tailed with fluorescein-dUTP was bound to the well. Denatured target DNA (233 bp) was simultaneously hybridized (60 min) with the capture probe and 200 fmol/well of detection probe tailed either with biotin-dATP or digoxigenin-dUTP. The appropriate molecular connector (200 fmol) was then bound to the immobilized detection probe. Subsequently, 40 fmol of a DNA label comprising a T7 RNA polymerase promoter and a firefly luciferase encoding sequence was tailed with dTTP and bound to the molecular connector. The luciferase-coding sequence was expressed by in vitro coupled transcription-translation and the activity of synthesized luciferase was measured by using its characteristic bioluminescent reaction with luciferin and ATP and O₂. As low as 0.4 fmol of target DNA could be detected. The CVs were in the range of 7–9%. Therefore, the proposed molecular connectors could link any dT-tailed DNA reporter molecule to any recognition molecule (probe or antibody) that has been labeled with biotin or digoxigenin.

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Signal Amplification System Using An Expressible Firefly Luciferase-Coding DNA Fragment As A Reporter Molecule. Chiu, N.; Christopoulos, T.; University of Windsor, Ontario, Canada.

A DNA fragment that consists of an upstream T7 RNA polymerase promoter and a firefly luciferase gene is used as reporter molecule. The detection of the DNA fragment (LucDNA) is accomplished by a two-step process. In the first step, the luciferase gene is subjected to in vitro coupled transcription and translation. Transcription generates several mRNA copies from each DNA template. Translation, in turn, results in the synthesis of multiple molecules of active luciferase in solution. At the second step, the activity of generated luciferase is measured, without further purification, by using its characteristic bioluminescent reaction with luciferin, ATP and O₂. The proposed signal amplification system is used to develop a two-site (sandwich type) immunoassay for serum prostate specific antigen (PSA). The luciferase encoding DNA is biotinylated and complexed to streptavidin. PSA is captured on microtiter wells coated with 25 uL of 5 mg/L monoclonal anti-PSA antibody and the sandwich is completed by the addition of 0.23 mg/L biotinylated polyclonal anti-PSA antibody. The immunocomplex is reacted with 0.86 ug/L streptavidin-LucDNA complex. The solid phase bound luciferase-coding DNA fragments are subsequently expressed into luciferase by a one-step in vitro transcription-translation reaction. The luminescence signal is found to be linearly related to the concentration of PSA. With a sample volume of 12.5 uL, as low as 30ng/L of PSA can be detected with a signal to background ratio of 2.3. The linear range is extended to 3000 ng/L of PSA. Serum specimens are diluted 30 fold prior to analysis. The proposed immunoassay correlates well with the IMx PSA immunoassay (R=0.97, n=23). The CVs for 2.1, 6.8, and 16.9 ug/L PSA are 9.4% (n=5), 3.4% (n=4) and 5.6% (n=5), respectively. The proposed system provides significant amplification and, therefore, high sensitivity. Furthermore the DNA itself (instead of the protein) is used as a label, thus avoiding the problem of inactivation of luciferase upon chemical conjugation to antibodies or oligonucleotide probes.

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Immobilized Sample Amplification (Isa) Analyses Of RNA And DNA In Tumors. Stapleton, M.; Wei, K.; Durham, NC, USA.

Immobilized Sample Amplification, ISA, is a novel molecular diagnostic tool for assessing the gene expression status in human tumors. Briefly, specimens are collected by surgery or biopsy of both tumor and benign tissues from the same patient. This sampling procedure provides a dynamic range of representative samples so that tumor progression is examined systematically. Samples are immobilized onto individual matrices of the ISA collection devices to render the samples noninfectious. ISA does not require extraction, isolation, or purification of nucleic acids. It incorporates analysis of both DNA and RNA simultaneously so that gene amplification and gene expression are measured together. ISA on tumor samples has revealed changes in the gene expression pattern between tumor and benign tissues from tumor specimens. Further development of ISA methodology is needed to identify these RNA species that are differentially expressed in the benign and tumor tissues. The rationale is that a correlative study of gene expression in a clinical population and treatment outcomes will discover expression patterns which can be used as prognosticators. Correlating gene expression patterns and different modalities of treatment with patient outcome may provide information to assist physicians with effective therapy regimens after, or in place, of surgery. The same principles are easily adapted for high through-put screening system for anti-tumor drugs, methods for tumor biology studies, and risk assessment determination for environmental toxicants.

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Direct Immobilized Sample Amplification (ISA) And Quantitation Of Retroviral RNA. Stapleton, M.; Wei, K.; Durham, NC, USA.

Immobilized Sample Amplification (ISA) is a novel molecular means for amplification, detection, monitoring, and quantitative determination of pathogens from a minute amount of samples. A novel quantitative ISA assay for retroviruses is described here. A replication-defective recombinant retrovirus (vGT-1) is utilized to establish and validate a quantitative ISA assay for retroviruses. This method detects retroviruses at single digits of infectious retroviral particles as determined by both colony forming assay and electron microscopy studies. Sample volumes, as large as 1 ml or as small as 5 to 10 μl, are readily immobilized on a matrix by heating at 50°C to 80°C for a few minutes. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. This is especially important when only a small amount of viral sample is available or a diluted viral sample needs to be concentrated. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.

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Frequency Of Hepatitis C Virus Genotypes As Determined By Direct Sequencing Of The NS5B Region In A Clinical Reference Laboratory. Hamdan, H.; Duong, D.; Kagan, R.; Fortich, M.; Khuu, R., Zhu, S.; Fenwick, R.; Lewinski, M.; Quest diagnostics, San Juan Capistrano, CA, USA.

Hepatitis C Virus (HCV) is the main causative agent of non-A, non-B hepatitis. HCV infection may be treated with alpha interferon alone or in combination with the antiviral drug Ribavirin. Isolates of HCV exhibit up to 33% nucleotide sequence variability over the entire viral genome resulting in differential antigenic and biological attributes of each isolate. Six major types (designated 1–6) and 12 major subtypes (designated 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a, 5a, 6a) have been characterized based on nucleotide sequence divergence. Evidence suggests that HCV genotype may be an independent predictor of a patient’s response to interferon therapy. Patients infected with genotypes other than type 1 appear to respond better to interferon therapy. A genotyping assay based on direct sequencing of a 222 bp region of the NS5B gene was developed. Viral RNA extracted from serum is reverse-transcribed and the NS5B region is amplified by PCR. The PCR product is sequenced by solid phase or cycle sequencing methods. Sequences are input into a BLAST database and BLASTN searches utilizing established cutoffs are run as a quality control measure. Sequences are then aligned (CLUSTALW) to 13 consensus sequences representing subtypes 1a to 6a and the HCV type and subtype are determined from the phylogenetic distances. Analysis of approximately 1000 patient samples showed Type 1 to be the most common genotype (76%: 1a=54.5%, 1b= 22.0%) followed by Type 2 (13.1%: 2a=2%, 2b=11%), Type 3 (8.8%: 3a=8.7%, 3c=1%), Type 4 (4a=1.5%) and Type 6 (6a=0.1%). Subtypes 1c, 3b and 5a have not yet been observed in our laboratory. Using this technique we were also able to identify cases of mixed subtype HCV infections. These results indicate that sequencing of the HCV NS5B region is discriminating and reliable for genotyping of HCV in a clinical laboratory.

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Typability Of HIV-1 Specimens Received For HIV-1 Genotyping For Drug Resistance In A Clinical Reference Laboratory. Hamdan, H.; Cohen, M.; Green, B.; LeBault, J.; Duong, D.; Kagan, R.; Fenwick, R.; Lewinski, M.; Quest Diagnostics, San Juan Capistrano, CA, USA.

Current treatment of HIV-1 infections involves a combination of protease and reverse transcriptase inhibitors. However, drug therapy favors the replication of viruses carrying mutations that confer drug resistance. A genotyping assay based on direct sequencing of the protease and reverse transcriptase genes was developed in order to predict HIV-1 resistance to antiretroviral drugs. HIV-1 is concentrated by centrifugation from plasma and viral RNA is extracted. A one-step reverse transcription and PCR amplification of a 1.4 kb product followed by nested amplification with gene-specific primers is carried out and a 1.3 kb PCR product is cycle sequenced. Sequences are input into a BLAST database and BLASTN searches utilizing established cutoffs are run as a quality control measure. The sequences are analyzed for mutations at 37 codons associated with resistance to antiretroviral drugs. Approximately 1400 specimens were analyzed to determine the percentage of specimens that could be typed in this assay. 8% of the specimens could not be amplified by PCR. A majority (82%) of the unamplifiable specimens had viral loads of <600 copies/mL. There were, however, 5 cases (0.35%) with viral loads greater than 3000 copies/mL that gave insufficient PCR amplification for sequencing. Four additional cases (0.29%) were untypable due to repeatedly ambiguous sequence data. Dilution experiments showed that mixtures of viral species were observed at viral loads of 10,000 copies/mL or more, but only single species were detected at lower dilutions (<1000 copies/mL). When plasma from different specimens with equivalent viral loads were mixed, mutations were reliably identified if at least 40% of the variant was present in the specimen. In conclusion, the typability of specimens in the HIV-1 genotyping assay largely depends on sufficient viral load. However, a small percentage of specimens are not typable in spite of high viral loads and successful amplification, possibly due to viral sequence variability at the primer binding sites.

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Evaluation Of A Novel PCR-RFLP Technique For Apolipoprotein E Genotyping. Ossendorf, M.; Hafner, G.; Lohse, P.; Prellwitz, W.; Institut für Klinische Chemie und Laboratoriumsmedizin der Universitätsklinik Mainz, Mainz, Germany.

Apolipoprotein E (apoE) is a 299-amino acid protein. The three main apoE isoforms are related to the polymorphic gene located in chromosom 19 with three common codominant alleles (?2, ?3 and ?4). ApoE-phenotype is usually determinded by isoelectric focusing (IEF). For genotyping, PCR-amplification of genomic DNA followed by HhaI-digestion is widely used. A high rate of misclassification has been reported for both techniques (1). Recently we presented a new rapid and easy PCR-RFLP technique for ApoE-genotyping using the restriction enzymes AflIII and HaeII and a mutant PCR-primer (2). Here we present first results of the ongoing evaluation of this new technique. On specimen of 28 subjects, sent to the laboratory for ApoE-typing, we could perform IEF for phenotyping and the new genotyping technique. On another 16 specimen we performed the new and the established genotyping technique. Both groups included all six genotypes. If discrepancies in apoE allele assignment occured, both tests were repeated. IEF for phenotyping and genotyping using HhaI-digestion was performed as described before. For the new technique a 318-bp fragment was amplified in a standard PCR protocol using Taq DNA polymerase and Q-Solution of QIAGEN, Hilden, Germany and the primer E2mut, 5’-ACTGACCCCGGTGGCGGAGGAGACGCGtGC and E3, 5’-TGTTCCACCAGGGGCCCCAGGCGCTCGCGG. The PCR products were digested separatly with AflIII and HaeII. The fragments were seperated on a 4% agarose gel. There was 100% concordance between the two genotyping techniques. Comparing the new genotyping with IEF technique four specimen had to be reassessed, whereby the genotyping result could be confirmed in all cases. Our results indicate that the new genotyping technique is superior to IEF phenotyping, were there is no difference between the two DNA techniques so far. However, all results should be confirmed on a greater number of specimen.

Reference:

Lahoz C et al. Clin Chem 1996; 42:1817–1823

Ossendorf M, Prellwitz W. J Lab Med 1998; (2):104–105

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PROBING ENZYME-INDUCED DNA DISTORTIONS VIA LONG-RANGE ELECTRON TRANSFER.Rajski, S.; Kumar, S.; Roberts, R.; Barton, J.; California Institute of Technology, Pasadena, CA, USA.

Oxidative DNA damage promoted from a remote site readily occurs resulting from electron migration through the π-stacked array of heterocyclic DNA bases. This process relies heavily upon the integrity of the DNA π-stack. We demonstrate herein the utility of DNA-mediated electron transfer in probing DNA-protein binding (and persuant base flipping) by the methyltransferase M. Hha I. Utilizing a rhodium(III) metallointercalator complex to photoinduce a radical cation “hole” into the DNA π-stack, we show that hole (or electron) equilibration over the DNA duplex is significantly hindered in an enzyme-dependent fashion. Incorporation of the M. Hha I binding site 5’-GCGC-3’ between two 5’-GG-3’ doublets (one proximal to the metal and one distal from the metal) allowed protein-induced π-stack distortions (ie.- base flipping) to manifest themselves as alterations between the two 5’GG-3’ damage events. Significant decreases in distal:proximal 5’-GG-3’ damage ratios occurred over a series of DNA and enzyme concentrations with concommitant retention of proximal 5’-GG-3’ damage efficiency. M. Hha I-dependent changes in distal oxidation were typically on the order of 2–3 fold lower than in the corresponding non-enzymatic case. Slight variations were found to depend on the specific DNA duplex substrate used and on conditions used for protein-binding. Notably, these alterations in damage ratios were insensitive to differing concentrations of the co-factor S-adenosyl-homocysteine (SAH) and were retained in the presence of significant molar excesses of non-specific competitor DNAs.

These results show the amenability of DNA-mediated electron transfer towards the investigation of protein-induced DNA structure distortions. Specifically, we have shown that DNA base-flipping is readily visualized by monitoring the oxidative DNA damage events resulting from intraduplex electron transfer. We feel that this general strategy offers a new and significantly different method from those currently available for investigating DNA distortion events mediated by biologically important macromolecules.

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Use Of Transcription-Mediated Amplification For The Direct Detection Of HIV-1. Stringfellow, L.; Lee, S.; Nunomura, K.; Galarpe, G.; Goto, S.; Egan, R.; Gen-Probe, San Diego, CA, USA.

In order to reduce the time to detection of HIV-1 for the Blood Banks in Japan, an ultrasensitive assay employing Transcription-Mediated Amplification (TMA) has been developed. The resultant assay is highly sensitive and specific and provides for the detection of all known subtypes of HIV-1.

TMA is an isothermal, amplification methodology that utilizes reverse transcriptase and RNA polymerase to achieve exponential amplification of RNA targets. This technology was coupled with (1) a sample processing method that specifically captures the target RNA and eliminates inhibitors; and (2) a sensitive AE-label based detection method called Dual Kinetic Assay (DKA). This system provides for simultaneous amplification and detection of an internal control and multiple HIV-1 targets.

Sensitivity was determined using an individual donor reproducibility panel constructed from patients known to be HIV-1 positive; >98% sensitivity was achieved at ∼100 copies/ml. Using the BBI Worldwide HIV-1 Performance Panel, detection of subtypes A-F and O was 100% (40/40). Furthermore, full-length RNA transcripts of subtypes F-H (single viral specimens) were detected as well. In testing of more than 3000 normal donor specimens, the assay was 100% specific. No interference was observed in normal plasma containing potentially interfering substances. No crossreactivity was observed in patient specimens infected with other blood borne pathogens.

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PNA: Synthesis And Use In Hetero - And Homogeneous Assays. Koch, T.; Kofoed, T.; Borre M.; Ørum, H.; Copenhagen, Denmark.

The full potential of PNA in diagnostics and monitoring will be reached when fast, flexible and efficient synthesis strategies on a routine basis are available. We have developed a novel synthesis strategy to complement the already known strategies. The protecting group strategy is based on tert-butyloxycarbonyl (Boc) and on acyl protection of the exocyclic amines on the bases. The strategy combines easy monomer preparation and high oligomer quality with a very broad modification potential of the PNA during the synthesis. Such flexibility is necessary to produce the variety of PNA-conjugates needed for assay development.

In a heterogeneous assay the probe is typically bound to a solid surface and its association with the targeted nucleic acid will elicits a measurable response. We have developed a general method to immobilize PNA on solid surfaces. PNA is conjugated to anthraquinone and after photolysis the quinone mediates a covalent bond between PNA and the surface. The technique is generally applicable when introducing PNA in heterogeneous assays and may be used in many different formats and on a variety of solid surfaces.

Homogeneous nucleic acid assays in solution generally rely on the spatial dependence of the interaction between groups on the probe and on the target strands to signal hybridization. We have developed a detection system based on electron transfer between a donor (quencher, Q) to an acceptor (fluorescer, F). Due to the low conductivity in the PNA/DNA duplex fluorescence quenching is obtained by placing the Q and F closely together in a PNA hairpin. Separation of Q and F and subsequent signal generation is obtained when the hairpin is hybridized to a piece of complementary DNA.

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A Solid Phase Plate Assay (Sppa) For Hiv-1 Genotyping. Gonzalez-Villaseñor, L.; Wu, K.; BBI-Biotach Research Laboratories, Gaithersburg, MD, USA.

The extensive HIV-1 genetic diversity, large number of worldwide circulating HIV-1 subtypes, and AIDS pandemic pose a formidable challenge for virus diagnosis, transmission, infectivity and vaccine development, and underscore the need of monitoring the geographic distribution and identification of HIV-1 genetic subtypes. Since antibody binding to gp120 and virus neutralization assays have failed to identify neutralization serotypes, and peptide based immunoassays do not distinguish all HIV-1 subtypes, we developed a nucleic acid based Solid Phase Plate Assay (SPPA) for the detection and differentiation of the major HIV-1 subtypes of groups M and O. Well characterized HIV-1 reference controls of known subtype and short length (25–28 bp) subtype specific capture oligonucleotide probes targeting the V1 to V5 loops of the envelope gene of HIV-1 were used to develop the assay. The oligonucleotide probes were bound to microtubes by covalent binding and biotin-labeled primers were used to amplify a 0.7 kbp fragment. The PCR products were hybridized to the capture probes in the microtubes and colorimetrically detected by a chromogenic reaction. The reference subtypes specifically hybridized to the corresponding capture probes and no cross-hybridization between subtypes was observed. The SPPA provides a convenient method for (1) HIV-1 subtyping of clinical samples, (2) the production of molecular controls and kits useful for developers of candidate vaccines and antivirals, (3) for physicians in the assessment of anti-retroviral treatment and drug resistance of HIV-1 strains, and (4) for scientists and clinical laboratories to evaluate the performance of HIV-1 detection assays. Manufacturers of microdevices, microchips and automated systems could adapt this assay into a biochip format to create a HIV-1 genotyping system.

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Use of the FMBIO® II Fluorescence Imaging System in Diagnostic Assays. Colby, S.; Chapman, W.; Lou, Y.; Mitsuhashi, M.; Moorthy, T.V.; Zoha, S.; Allnut, T.; Mizeracki, A.; North, P.; Musk, P.; Rogers, Yu-Hui.; Smead, D.; Hitachi Genetic Systems, South San Francisco, CA; Hitachi Chemical Research Center, Irvine, CA; Bio-ID Diagnostics, Inc., Saskatoon, Saskatchewan, Canada; Martek Biosciences, Columbia, MD; Arkansas Childrens Hospital, Little Rock, AR; Palmetto Richland Memorial Hospital, Columbia, SC; Molecular Tool, Inc.; Baltimore, MD.

Fluorescence is gaining in popularity for nucleic acid detection because it offers multiple label detection, permits sample quantitation, facilitates high sample throughput, and is convenient and safe relative to using radioisotopes. The FMBIO II Fluorescence Imaging System is a laser-based scanning system designed for versatility in sample processing. We demonstrated that the FMBIO^RII can be used to generate and analyze image data in several fluorescence-based diagnostic assays, including virus identification and paternity testing. The Analysis Software’s 1-D, 2-D, and sequence tools were used to view and analyze scanned images. Because the scanner is independent of an electrophoresis apparatus, we obtained high sample throughput in a variety of assay formats. Successful formats were low- to intermediate-density arrays, microtiter plates, and electrophoretic gels. The scanner’s two photomultiplier tubes were used for simultaneous multicolor imaging of fluorescein-and rhodamine-labeled moleular beacon sample quantitation. Finally, the instrument provided the sensitivity needed for viral detection and other diagnostic assays.

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Detection Of Nucleic Acids Using A Novel Enhanced Lumiol System. Strachan, B.; Jackson, J.; Sylvers, L.; Pierce Chemical Company, Rockford, IL, USA.

Many nonisotopic methods have been developed for nucleic acid detection, and recently the sensitivity of these methods have been improved to the point where many equal the sensitivity of their isotopic counterparts. However, these sensitive methods, usually based on 1,2-dioxetane substrates, tend to suffer from a high degree of signal :noise variability. Aside from the lack of robustness of most of the current methods, a further drawback comes from the inordinate amount of processing time involved in the procedure and length of exposure time needed to obtain the desired sensitivity. These drawbacks undoubtedly contribute to the lack of wide acceptance of nonisotopic nucleic acid detection methods. We have recently developed a complete system for the chemiluminescent detection of nucleic acids in Northern and Southern blot applications that combines a novel enhanced luminol substrate with optimized hybridization and blocking steps that ensure consistent results with sensitivity equivalent to P. This robust system also has the advantages of greatly reduced processing and film exposure time. Post-hybridization processing time has been reduced from the standard 2.5 hours to 1 hour. Film exposure times range from 0.5–10 minutes with the substrate emitting light with relatively constant intensity over a six hour period, thus allowing for multiple exposures.

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Development Of An Internal Control For Monitoring Assay Performance In Individual Specimens. Hotaling, S.; Kolk, D.; Burrell, T.; Linnen, J.; Riggs, M.; Giachetti, C.; Gen-Probe Inc., San Diego, CA, USA.

Traditionally, diagnostic assay controls have been external positive and negative controls which assess reagent performance within each run. Implementation of nucleic acid testing for blood donor screening presents new challenges. For example, the potential severity of a false negative result is potentially very serious. A false negative result can lead to a transfusion-associated transmission event. To provide an extra level of assurance that assay performance is satisfactory for each specimen tested, an internal control (IC) was incorporated in the Gen-Probe HIV-1/HCV assay.

The IC consists of an RNA transcript comprising HIV capture, amplification and detection sequences. The transcript was added to each sample before sample processing to be captured, amplified and detected in the same reaction as the viral targets. The amplified transcript does not react with HIV or HCV probes and it is detected with an unique IC probe. Dual Kinetics Analysis, using differential chemiluminescent probes, was used to distinguish IC from target. A reduced or negative IC signal indicates specimen interference and/or problems with assay execution or reagents.

IC performance was verified using normal plasma populations run by different operators over several days. Running of ∼1000 normal samples indicated an average IC signal of 139,000 relative light units [RLU] with a standard deviation of 9,500 RLU and a run to run, operator to operator CV of 7.4%, showing excellent reproducibility. None of the 1000 normal samples were inhibitory to the IC amplification. To assess the utility of the IC in identifying false negative reactions due to reagent/instrument/technician failure, several protocol steps were altered and the effects on IC signal and target signal were evaluated. These experiments showed excellent correlation between IC signal and HIV and HCV signals. Failures in the HIV or HCV signal always corresponded with failures in the IC signal, indicating that the IC provides an useful indicator for assay steps and it is very effective at confirmation of amplification performance in individual specimens.

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Development Of Multiplex ARMS For The Newly Identified –186 G/T And –491 A/T Apolipoprotein E Promoter Polymorphisms. Donohoe, G.; Turku, Finland.

Apolipoprotein E is a polymorphic protein constituent of lipoproteins and plays a major role in lipid metabolism. The Apo ε4 allele is an established risk factor in both atherosclerosis and Alzheimer’s disease (AD), whereas the ε2 allele confers protection against AD and lowers lipid levels. Recently, two new mutations in the Apo E gene have been identified. The –186 G/T promoter mutation alters the expression of the Apo E alleles leading to an increased deleterious effect for ε4 carriers and increased protective effect for ε2 carriers. Whereas the AA genotype of the –491 A to T polymorphism is independently associated with an increased risk for the development of AD. We have developed a new, rapid and inexpensive screening method for both these mutations based on the Amplification Refractory Mutation System (ARMS). Using published sequence data we designed a set of primers to amplify both loci. The primers were constructed so that the 3’-most nucleotide of the specific primers recognized either the mutant or normal allele, and the 3’-penultimate nucleotide was mismatched to enhance specificity of the reaction. In addition, a common primer was designed to pair with both the mutant and wild-type-specific primers. Using these primer sets the six different Apo E promoter genotypes [GG, GT, TT (-186 G/T)/AA, AT, TT (–491 A/T)] were unambiguously determined and the validity of the method was verified by sequencing. This assay is reliable, simple and gives clear results, thus we believe that it will be a valuable tool for future epidemiological studies.

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Two Novel Schemes For Integrated Detection Of Cascade Rolling Circle Amplification Products. Thomas, D.; Lizardi, P.; Marmaro, J.; Gerdes, J.; Kozwich, D.L.; Winn-Deen, E.; Oncor, Inc., Gaithersburg, MD, USA.

A novel isothermal amplification system has been developed called cascade rolling circle amplification (CRCA) that is simple, rapid, and highly sensitive. Generic primers are used to amplify circularized DNA probes instead of the target, achieving over a billion-fold amplification within one hour without using a thermocycler. The initial step is formation of a circularized padlock probe resulting from ligation of the ends of a linear probe annealed to adjacent target regions. Using two primers and strand-displacing Bst DNA polymerase (large fragment), rolling circle amplification ensues which triggers a near-exponential cascade reaction of continual primer binding, extension and strand displacement, forming a ladder of products from one to many probe unit lengths. To minimize carry-over contamination, the amplification and detection steps are performed in a closed system. Homogeneous fluorescence detection is achieved when one of the primers is an energy transfer-labeled primer which generates a signal only when incorporated into the amplicon. Using model systems for HIV and Chlamydia trachomatis, as few as 100 target copies were detected in 60 minutes. CRCA is also being developed to integrate into a platform for point-of-care detection of environmental pathogens called a Self Contained Isothermal Partical Device (SCIP). The device contains separate but interconnectable compartments for DNA extraction/amplification, liquid waste collection, and detection. To assess the feasibility of CRCA for this system, each of the two primers were uniquely tagged at the 5’-end with haptens (fluorescein and biotin) to enable detection by the SCIP lateral flow system. Using ∼1000 target copies of the HIV gag gene, cascade products were easily detected within 40 minutes. Model systems for various environmental pathogens such as Cryptosporidium parvum are currently being tested to further assess the utility of CRCA for this platform.

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Evaluation Of A Semi-Automated Quantitative HIV-1 BDNA Assay With Reporting Threshold Of 50 Copies/Ml. Irvine, B.; Barr, S.; Sawyer, L.; Booth, J.; Ocampo, A.; Leung, K.; Duey, D.; Hendricks, D.; Kolberg, J.; Chiron Diagnostics, Emeryville, CA.

Performance characteristics of a Quantiples™ HIV-1 RNA 3.0 Assay (bDNA) with increased sensitivity were evaluated. The limit of detection was 50 copies/mL with a specificity of 99.5%. The assey has a dynamic range of 4 logs (50 to500,000 copies/mL) and quantities HIV-1 subtypes A-F. Precision between 50 and 500,000 copies/mL was <0.21 logSD. The data genorated in this evaluation was analyzed both as single wells and as duplicate wells and the variation was found to be similar over the range of the assay. Comparison of this assay with the previous Quantiplex™ HIV-1 RNA 2.0 Assay showed that approximately 80% of specimens with results of <500copies/mL in the earlier version also had < 500 copies/mL in the new version.

The sensitivity improvements relative to Quantiplex™ HIV-1 RNA 2.0 Assay were achieved by a combination of developments which lowered the assay background and increased the level of amplification. Background reduction was achieved, in part, by incorporating non-natural nucleotides (i.e. Iso-G and 5’methyl Iso-C) into some of the amplification sequences, reducing non-specific hybridization of these sequences to the other assay components. In addition, background reduction was achieved in the redesign of the “target probes” which mediate binding of the first layer of amplification (pre-amplifier) to the captured target. Increased amplification was achieved by designing a new target probe set which increased the number of potential binding sites for the pre-amplifier.

The ease-of-use and throughput of the assay is greatly facilitated by use of a semi-automated instrument, the Quantiplex™ System 340. This instrument system provides a platform wherein the multiple incubations, washes, final luminescence readout and data reduction are all automated. Using the instrument a single technician can process two 96 microwell plates (i.e. 164 patient results) per run. HIV-1 RNA can be quantified with precision, accuracy, and at very low concentrations with this assay.

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DNA Technologies In Human Disease Detection-1998. Bean, P.; Millennium Strategies, Madison, WI.; Specialty Laboratories, Santa Monica, CA, USA.

The emergence of HIV mutations associated with resistance to reverse transcriptase (RTIs) and protease (Pis) inhibitors are recognized consequences of the use of these drugs as treatment of HIV infection. In order to treat HIV-infected patients more effectively, the use of DNA sequencing for monitoring the appearance of mutations associated with decreased drug susceptibility was investigated. From an initial cohort of 47 HIV-infected individuals undergoing antiviral treatment for HIV, four patients were identified who, although they had been treated for at least 6 months with triple therapy, had shown no decrease in their viral load (mean value =270,000 copies/ml). Viral isolates taken from retrospective blood specimens of each of the four patients were examined for mutations associated with decreased drug susceptibility by sequencing relevant regions of the HIV pol and protease genes. The DNA sequences of the earliest viral isolated indicated that each of the four patients was initially resistant to Zidovudine (AZT) upon starting triple-drug therapy as each viralisolate contained the primary characteristic zidovudine-resestance mutation at codon 215 as well as one or more secondary mutations at condons 67,70,210 and/or 219 of the HIV pol gene. Over the six months course of treatment, additional mutations developed at condons 10,64 and 82 of the protease gene which have been characterized as decreasing susceptibility to protease inhibitors. Thus, none of these four patients received the true benefit of triple drug therapy for their HIV infections. It is possible that, had they been screened and monitored by DNA sequencing, their HIV infections could have been managed more effectively since the start of triple therapy.

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Preparation Of Specimens In Preservcyt ® Solution For RCR Amplification Of HPV. O’Connell, M.; Cohenford, M.; Lentrichia, B.; Boxborough, MA, USA.

INTRODUCTION: PreservCyt^® Solution (Cytyc Corp.) is a methanol-based collection and transport medium, used in conjunction with the ThinPrep^® Pap Test™. A variety of molecular applications have been performed on samples in PreservCyt, however detailed methods of DNA extraction for polymerase chain reaction (PCR) amplification are not well documented. Standard DNA purification methods fail to consistently remove inhibitors of PCR, and often produce insufficient yields of DNA. We describe a simple and rapid method of DNA purification, which efficiently removes inhibitors without compromising DNA yield.

OBJECTIVE: The focus of this study was to amplify Human Papillomavirus (HPV) DNA using gynecological (gyn) specimens in PreservCyt with the Epicentre MasterAmp™ Buccal Swab DNA Extraction Kit (Epicentre, Madison, WI).

METHODS: HPV status of gyn samples was established using Digene’s Hybrid Capture HPV Assay (HC)(Digene, Silver Spring, MD). Primers (MY09/MY11) targeting the HPV L1 conserved region were used for amplification purposes. Control cells in this study included; cultured CaSki cells (600 viral copies of HPV DNA/cell), HeLa (20–60 copies/cell), SiHa (1–2 copies/cell) and C-33A (negative for HPV DNA). β-globin primers (gh20bg1/gh20bg2) served as an internal control. PCR reactions were done using optimal concentrations of MgCl (3mM-7mM). Amplified products were analyzed using standard electrophoresis techniques.

RESULTS: β-globin amplified for all samples. All cultured cells positive for HPV DNA were amplified by PCR. No HPV DNA was observed for C-33A cells. Twelve of seventeen gyn samples, which were positive by HC, were positive for HPV DNA by PCR. Nine of ten samples were negative by HC and PCR. Five of seventeen gyn samples, which were positive by HC, yielded negative PCR results. However, the β-Globin fragment of each of these HPV negative samples was amplified, indicating effective removal of PCR inhibitors. There was one sample that was negative by HC, yet positive by PCR. The Epicentre kit yielded sufficient DNA for all specimens in PreservCyt with no evidence of inhibitor carry-over for PCR.

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Development Of A Chemiluminescent Hybridization Assay For Quantitative Determination Of Residual DNA In Recombinant Protein Drugs Expressed In E. Coli. Ji, X.; Dipaolo, B.; Venkat, K.; King of Prussia, PA, USA.

Impurity assays for recombinant protein products are essential in order to meet the FDA’s criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, radioisotopic assays have traditionally been used for products derived from bacterial expression systems in order to obtain the required sensitivity. In the present study, we describe the development of a sensitive non-radioactive chemiluminescent hybridization assay to quantitate residual E. coli DNA levels in purified protein drugs.

A slot blot hybridization method was used for this analysis. E. coli DNA (both genomic and plasmid) was isolated and spiked into 1 milligram protein at 10 and 50 picogram levels.. Protein was extracted prior to blotting using the WAKO DNA extraction kit. Serial dilutions of E. coli DNA (5, 10, 25, 50, 100 and 200 picogram) were used to generate a standard curve for each assay. The probe was labeled with digoxigenin (DIG) using a random primer labeling procedure. A horseradish peroxidase labeled anti-DIG antibody and an enhanced chemiluminescent substrate was used to detect the DNA signal. The results were recorded on a x-ray film and quantitated using densitometric image analysis. The DNA concentration in protein products and spike-recoveries were calculated using the standard curve. Parameters such as probe concentration, hybridization buffer, antibody concentration, and blocking solution were optimized to achieve high sensitivity, low background blots. Our data revealed that a high probe concentration (1 ug/mL) is required to obtain low (< 10) picogram detection. A high background was experienced which was not affected by probe or antibody conjugate concentration. This background, however, could be reduced by adding appropriate blocking reagent to the hybridization buffer.

Taken together, our data showed that this non-radioactive chemiluminescent hybridization analysis allowed us to specifically detect 5 picogram of E. coli DNA using a slot blot format. This newly developed, sensitive, and non-radioactive method is feasible for detection and quantitation of DNA contamination in recombinant protein products expressed in E. coli.

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Thermal Gradient Affinity Electrophoresis (TGAE) for SNP Typing and Haplotyping. Weir, L.; Hammond, P.; Kenney, M.; Mamaeva, N.; Dhanda, R.; Boles, C.; Boston, MA.

We have developed a new electrophoretic technique for typing single nucleotide polymorphisms (SNPs) and for haplotyping closely linked markers. Oligonucleotide probes modified with 5’-acrylamide groups are copolymerized within polyacrylamide electrophoresis gels. Previously, we have shown that single-stranded nucleic acid targets hybridize with great efficiency and specificity to complementary gel-immobilized probes when electrophoresed through such gels. During electrophoresis, complementary targets are themselves immobilized whereas non-complementary molecules migrate through the gel unimpeded. When a temperature gradient decreasing from top (origin) to bottom is imposed on a gel containing immobilized probe, complementary targets electrophorese normally through the upper, hotter regions of the gel, but stop moving and form a sharp band at the point in the gel where the temperature is low enough for hybridization to occur. The distance migrated within the gel is a sensitive measure of the melting temperature of the target-probe complex. Targets that are fully complementary to the probe hybridize at higher temperatures and form bands near to the top of the gel. Targets that are mispaired with the probe form bands lower in the gel where the temperature is lower. We tested this method for SNP typing using model oligonucleotides and single-stranded PCR products from patient samples as targets.

For haplotyping closely linked SNPs, we used composite capture probes in which two short probe sequences, complementary to two polymorphic loci within the target, are covalently joined by short linkers of poly-T or PEG. For two human genes that have a closely linked pair of biallelic markers, the ß2-adrenergic receptor and ApoE, the TGAE system could unambiguously type all four alleles in a single minigel run. These data demonstrate that TGAE is a high resolution SNP typing method that is also capable of analyzing linkage between closely spaced markers. The use of temperature gradients significantly reduces both the labor involved in optimization during development of new tests and the need for precise, expensive temperature control. TGAE also has excellent potential for automation.