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1999 San Diego Conference |
1
Direct Electrochemical Detection of Nucleic Acids. Jill C. Mikulecky, Robert S. Thomas, Allen E. Eckhardt, Mary E. Napier, Natasha D. Popovich, Jonathan L. Baron, Mark D. Gelardi, XANTHON, Inc., Research Triangle Park, NC 27709.
The detection of target nucleic acid hybridized to complementary capture probe is of central importance in genomic research and pathogen diagnostics. Present detection methods require attachment of enzymatic, fluorescent, chemiluminescent, or radioactive labels to the hybridized target. Furthermore, these methods require amplification of rare targets to detect low copies of a specific nucleic acid. These procedures are costly and time consuming, require complex instrumentation, and may introduce sample contamination. We have developed a novel detection method for DNA and RNA that does not require reporter molecule attachment or target amplification. The target nucleic acid is electrochemically detected using a soluble, metal mediator, tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)32+). When the appropriate potential is applied, Ru(bpy)32+ oxidizes guanine via a catalytic cycle and current is produced. This method, unlike other electrochemical techniques, does not require the target to be directly wired to the electrode or the use of duplex specific redox-active indicators.
The present detector format utilizes a 6 mm diameter electrode, upon which a monolayer pre-coupled with oligonucleotide probe is self assembled. Complementary nucleic acid target is allowed to hybridize to the probe, unhybridized target washed away, Ru(bpy)32+ added, and the signal acquired. Using this format, 100 femtomoles of a synthetic 124-mer oligonucleotide target containing 41 guanines generated a current of 0.18 mA over background. To improve sensitivity, the detector format is transitioning to a 200 micron diameter electrode, where approximately 100 attomoles of a synthetic 21-mer oligonucleotide containing 5 guanines generated a current of 600 nA over background.
The specificity and efficiency of the system is being optimized using synthetic oligonucleotides, PCR products, mRNA from cultured cells, and various signal acquisition methods. For example, successful hybridization has been achieved at the electrode using peptide nucleic acid probes, with 30% of the RNA target captured in 2 hours. The detector system can be readily multiplexed, and we are designing a 96 well microtiter plate with five individually addressable test electrodes in each well. This design will allow the detection of five different sequence specific nucleic acid targets per well or up to 480 analyses per plate.
2
A Novel Molecular Screening Tool for High-Throughput, Multiplex Analysis of Mutations Involved in Thrombotic Disease. Daniel D. Kephart, Richard B. Rhodes, Ken Lewis, and John Shultz, (Promega Corporation); Suzanne Huber, Karl V. Voelkerding, (UW-Madison WI); Debra G.B. Leonard, (Univ. of Penn., Phil., PA); Gregory J. Tsongalis, (Hartford Hospital, Hartford, CT).
Mutations in the genes encoding Factor V and Prothrombin are the two most common defects associated with inherited thrombophilia. This study was designed to develop a high-throughput, multiplex Factor V/Prothrombin screening assay to demonstrate the capabilities of Promega's READITTM molecular analysis tool. READITTM Technology utilizes a coupled enzyme format that interrogates a specific DNA sequence of interest to generate light using Luciferase. The READITTM System offers a high resolution, microtiter plate format capable of automated mutation detection. Because target-directed primers provide specificity, the READITTM System represents a modular platform that has been used to detect a variety of sequence variations including SNPs, deletions, insertions, and chromosomal translocations. We used READITTM to analyze over 400 residual clinical samples from three independent diagnostic testing laboratories for two specific SNPs correlated with thrombotic disease. Analysis software allowed rapid statistical analysis of the READITTM data, with automated sample assignments. READITTM analysis gave rise to greater than 6 standard deviation separation between wild type and mutant assignments. Repetitive sample measurements indicated less than 2% r.s.d. between READITTM analysis. The simple and robust nature of the READITTM assay enables extreme confidence in a variety of genotype assignment applications.
3
Direct Hybridization Detection Using Optical Chip Technology. Bernard H. Schneider, M. Danna Vach and Beth Dickinson, Photonic Sensor, Atlanta, GA 30308.
There is a growing demand for nucleic acid diagnostics that are able to achieve real-time, quantitative measurement of multiple gene sequences in a simplified assay format. The optical chip technology under development in our laboratories has the inherent characteristics to meet these demands. This device functions by real-time measurement of the increase in refractive index caused by hybridization of nucleic acid target sequences to oligonucleotide probes immobilized on the chip surface. Using integrated interferometric detection, background non-specific binding can be minimized without compromising specificity. Furthermore, the proprietary configuration of the optical chip is compatible with low density arrays (<100) in a small chip size (3 cm by 1 cm).
An assay for the detection and differentiation of mycobacterium species is being used as a model system to demonstrate feasibility. This assay detects mycobacterial ribosomal RNA without the necessity of nucleic acid amplification techniques. Preliminary studies, using synthetic oligonucleotide targets, have shown that nucleic acid sequences of M. tuberculosis (MTB) could be differentiated from M. avium (MAV) target sequences using an MTB-specific probe on the chip. For oligonucleotide levels at or above 4 ng/ml (0.2 picomoles in a 1 ml sample volume) no target amplification or signal labelling was required, for <1 hour assays. At lower concentrations a signal amplification step is used to improve detection sensitivity. Colloidal gold amplification has produced at least a 250-fold improvement in the sensitivity of the optical chip for immunoassay measurements (detection levels of 20 pg/ml), and this approach is currently being applied to nucleic acid detection.
4
Compound Heterozygotes for Familial Hypercholesterolaemia and Familial Defective Apolipoprotein B Mutations Detected in a Malay Family. Seng Tzer Jing1, Tai E. Shyoug2, and Evelyn SC Koay1, 1Department of Pathology, National University of Singapore, 2Department of Endocrinology, Singapore General Hospital, Singapore.
Classical familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the low density lipoprotein receptor (LDLR) gene. Clinical characteristics are premature atherosclerosis, elevated LDL-cholesterol levels, and familial occurrence of tendon xanthomas. However, genetic defects in apoliprotein B-100 (ApoB-100) gene in familial defective apolipoprotein B-100 (FDB) result in identical phenotypes. Since clinical distinction between FDB and FH is not possible, we have developed a mutation screening assay based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) method. This method allows screening for point mutations and minor insertions/deletions in the promoter region and the 18 exons with flanking intron sequences of the LDLR gene, and the codon 3453-3553 region of the ApoB-100 gene. Mutations were detected in the exon 9 (codon 407) of LDLR gene and codon 3500 in ApoB-100 gene in a Malay patients. Seven of her 1st-degree relatives were recruited for the screening of these known mutations. The LDLR gene mutation was detected in one of them, while the ApoB-100 gene mutation was detected in the other four of them. The present study has provided additional information on the mutational spectrum in Malay FH-families and has shown the general applicability of our DGGE-based mutation screening assay for genetic diagnosis of FH and FDB. Our studies also indicate that the search for genetic defects in families with hypercholecterolaemia should begin by analysis of the ApoB-100 gene mutations, as almost all reported mutations are found in codon 3500. In contrast, there are more than 190 different mutations in LDLR gene and hot-spots for alterations are not known.
5
Highly Efficient Detection of Gene Expression in Patients' Tumor Samples. Agnes Juhasz1, S. Lin2, J.H. Doroshow1. 1City of Hope Natl. Med. Ctr., Medical Oncology Department, Duarte, CA, 2Clinical Microsensor Inc., Pasadena, CA.
In the last several years laboratory investigators have suggested that the relative amounts of various enzymes in tumors are associated with sensitivity to different treatments. Danenberg et al. developed a RT-PCR based assay which is an effective and sensitive method to measure a gene expression using ß-actin as a reference gene within a given tumor sample. Their data show that the actual gene (TS)/ß-actin ratio might help to predict the outcome of a 5-FU treatment; patients with lower ratios have longer survival.
In our laboratory we improved the efficiency and through-put of the traditional 32P-labeled RT-PCR-PAGE assay by using primers labeled with ABI fluorescence dyes. To increase the accuracy of the PCR step, reactions were carried out with ABI Prizm 877 Integrated Thermal Cycler. The fluorescence-labeled PCR products were loaded on an ABI 377 DNA Sequencer using GeneScan 3.0 software for data collection; for further quantitative analyses we launched to MS Excel.
This method allows us to use 3 dyes to detect the genes of interest in a single lane on the denaturing 6% polyacrylamide gel: ß-actin reference gene was labeled with HEX (yellow); Dihydropyrimidine Dehydrogenase (DPD), Thymidylate Synthetase (TS), and Multidrug Resistance (MDR1) were labeled with 6-FAM (blue); and Gluthatione Peroxidase (Gpx1), Ribonucleotide Reductase M2 (RR/M2) and Excision Repair Cross-Complementing (ERCC1) were labeled with TET (green). The genes labeled with the same dye were distinguished by their size (position on the gel). We obtained data from 25 samples of 17 patients who participated in an ongoing clinical trial. From the results it is obvious that the fluorescence method has a lot of advantages over the isotope technique: the sensitivity is ~20 fold higher, it offers multiple labeling, the sample throughput is high and the accuracy is increased. This allows quantitative, and safe and convenient analysis of gene expression; the labeled PCR primers and products are stable for years.
6
Analytical Sensitivity of TMA HIV-1/HCV Assay. K. Greenbaum, R. Wang, M. Klemm, J. Dockter, T. Burrell, G. Yapit, D. Madriaga, C. Giachetti, and J. Linnen, Gen-Probe Incorporated, San Diego, CA.
Transcription-mediated amplification (TMA) provides a sensitive method for detection of HIV-1 and HCV viral RNA in plasma or serum samples. The TMA HIV-1/HCV Assay uses a system of redundant capture oligonucleotides, amplification primers, and detection probes that allows detection of diverse genetic variants of HIV-1 or HCV. To determine the analytical sensitivity of the TMA Assay, we tested dilutional sensitivity panels comprised of HIV-1 (type B and group O) and HCV subtypes (1a and 2b) at concentrations of 300, 100, 30, 10, 3, 1, and 0 viral copies/mL. Six kit lots of the TMA HIV-1/HCV Assay were used to test the panels. For each kit lot, up to eighty replicates of each copy level were tested. The TMA Assay achieved 99 to 100% detection at 100 copies/mL and 88 to 100% sensitivity at 30 copies/mL. The study also showed greater than 50% positivity at 10 copies/mL for both viruses. To confirm the analytical sensitivity results obtained with our in-house panels, we tested several Nucleic Acid Amplification Testing (NAT) standard panels obtained from several institutions, including CBER and WHO. The results obtained with currently available HIV-1 and HCV NAT standards confirmed the analytical sensitivity of the assay determined with in-house panels. Furthermore, to provide additional support for the analytical sensitivity of the TMA HIV-1/HCV Assay, we performed sensitivity studies with co-infected samples (samples containing both viruses in combinations of high and low titers). The results indicated that the presence of a high titer of one virus does not impair the detection sensitivity of the target virus. The TMA HIV-1 and HCV Discriminatory Assays obtained 100% detection at 100 and 30 copies/mL of one virus in the presence of a high titer, 107 copies/mL, of the other virus. Our analytical studies demonstrate that the TMA HIV-1/HCV Assay provides sensitive detection of HIV-1 and HCV viral RNA in both single- and co-infected samples.
7
Direct Detection of HBV Using the Gen-Probe Transcription-mediated Amplification/Dual-Kinetic Assay System. S. Lee1, T. Yoshimura2, M. Hirose2, M. Yagasaki2, M. Blankenbiller1, O. Montano1, L. Stringfellow1. 1Gen-Probe Incorporated, San Diego, CA, and 2Chugai Diagnostics Science, Co., LTD., Tokyo, Japan.
The blood supply in America and in Japan is screened for HIV-1, HBV, and HCV using assays that rely upon detection of viral antigens or antibodies. While these assays have improved the safety of the blood supply, it is well documented that a window period exists between time of infection and time of seroconversion during which these blood-screening methodologies cannot detect infection. We have developed a blood-screening assay for hepatitis B using Transcription-Mediated Amplification (TMA) technology that can detect infection prior to seroconversion.
TMA is an isothermal amplification methodology that utilizes reverse transcriptase and RNA polymerase to achieve exponential amplification of HBV targets. This technology has been coupled to a sample processing method that includes target capture; HBV DNA and an internal control target are specifically captured and co-amplified. The resultant products are detected simultaneously using a modified form of Gen-Probe's Hybridization Protection Assay (HPA) known as the Dual-Kinetic Assay (DKA). The assay has also been developed to be compatible with the fully automated Gen-Probe TIGRIS system, which has a throughput capability of 500 tests in eight hours.
Studies performed at Gen-Probe have demonstrated that the HBV assay is highly sensitive and can detect HBV ayw and adw targets with 100% sensitivity at 50 EU/mL. In addition, the assay showed 100% specificity with normal donor specimens (N = 720). Furthermore, the assay has been demonstrated to reduce the seroconversion window (N = 18 panels) by an average of 14 days compared to a sensitive HbsAg assay.
8
ACE Gene Polymorphism in Association with Essential Hypertension in Indian Population. T.F. Ashavaid, K.K. Shalia, K.G. Nair, J.J. Dalal, P.D. Hinduja National Hospital & Medical Research Centre, Mumbai, India.
The deletion polymorphism situated in intron 16 of angiotensin I converting enzyme (ACE) gene (17q23) has been observed to be associated with an increased risk for myocardial infarction and left ventricular hypertrophy in Caucasian populations. The homozygous genotype for the deletion allele (D/D) has additionally been observed at greater frequencies in hypertensive individuals of African-American and Japanese origin. To determine whether ACE gene polymorphism may be responsible for essential hypertension in Indian population, we have studied this polymorphism so far in 53 patients and 100 controls. Serum ACE levels are observed to be highest in subjects carrying D/D polymorphism followed by I/D and I/I polymorphisms. The genotype frequencies of I/I, D/D and I/D in hypertensive subjects are 38%, 25% and 38% respectively, while in controls the frequencies are 32%, 23% and 45% respectively. The I and D allele frequencies of the patients are 0.57 and 0.43 while in controls, it is 0.54 and 0.46 respectively. There is no significant difference in the allele frequencies between patients and controls. Thus in our test population, so far the ACE gene polymorphism is not found feasible to be used as a risk for essential hypertension.
9
Single-Base Mismatch Detection Based on Charge Transport through Double-stranded DNA. Elizabeth M. Boon1, Shana O. Kelley1, Thomas G. Drummond1, Michael G. Hill2, and Jacqueline K. Barton1, 1California Institute of Technology, 2 Occidental College.
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. 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 through the DNA duplex to intercalated MB, bound primarily at the solvent exposed periphery of the monolayer, and then are accepted by solution-borne ferricyanide. The latter electron transfer event reoxidizes MB, causing more electrons to flow to MB and maintaining the catalytic cycle. In essence, the catalytic reaction amplifies the absolute signal from the reduction of MB mediated by the DNA stack, and thus also dramatically attenuates 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 provide larger absolute signals which increase disproportionately for fully complementary versus mismatched DNA, thus substantially increasing the differentiation between matched and mismatched sequences. All possible single-base mismatches in various sequence contexts have now been studied using electrocatalysis. The efficient transport of charge through self-assembled monolayers of thiol-terminated DNA duplexes on gold, coupled to a catalytic reaction in solution, may offer a novel and practical means to detect DNA mutations at the single base-pair level.
10
Performance of the GeneMachines® OmniGridTM Microarrayer for Printing DNA Chips for Gene Expression Analysis. Scott Hunicke-Smith, Jaques Fayet-Faber and Poonam S. Medberry, GeneMachines, San Carlos, CA.
Functional genomics and gene expression profiling have recently evolved from the synthesis of oligonucleotides on a chip to a wide range of DNA chips produced by microarraying spotters. As more and more researchers encounter the need to create their own arrays, the demand increases for flexibility, low production costs and high throughput in a microarraying system.
The OmniGrid offers a robust, high-speed and fully integrated solution for making DNA arrays. Data on the instrument's precision, accuracy, flexibility and throughput are discussed. As an example of these parameters, the OmniGrid is capable of printing more than 20,000 spots/slide in 10 hours onto 100 slides using 32 pins. Additionally tip technology, cross-contamination and alignment issues are addressed. Results demonstrate that the OmniGrid achieves high-throughput while maintaining a high quality of DNA chips with spot sizes averaging 150µm and contamination levels below 0.5%.
11
A High-Throughput, Plasmid Template Preparation Machine at $0.05 per Prep. Carl U. Buice and Nancy Bergsteinsson, GeneMachines®, San Carlos, CA.
Based on the array microcentrifuge technology developed at Stanford University, we have developed a machine capable of fully-automated isolation of DNA from plasmids for less than $0.05 per sample at a rate of two 96-well plates an hour. The key to the cost savings is the replacement of expensive filtration steps common to most automated DNA isolation machines with inexpensive centrifugation steps.
The array microcentrifuge consists of 96 individuals rotors arrayed in the standard 96 well format and spacing, allowing completely automated handling of samples by our 96 channel pipetter. To achieve reduced centrifugation times, each rotor spins at 60,000 rpm, subjecting each sample to forces of nearly 14,000 gs.
The Revolution PrepMachineTM (RevPrepTM) integrates the array microcentrifuge into a robotic workstation including a 96 channel pipetter, a bulk reagent dispenser, an 8 position rotating deck and robotic server arm with access to a total of 40 plates. The combination of reduced centrifugation times, low reagent and disposables cost, and long unattended run times makes the RevPrepTM a superior technology for high-throughput, low cost template prep.
12
Detection of HIV-1/HCV/HBV in a Single Assay System: New Method for Blood Screening. Qi Meng, Christine Wong, Lawrence Cheng, Jim Araujo, Darren Clarke, Helen J. Lee, Anu Rangachari Thara Ramankutty, Ellen Fiss, Masato Sasaki, Toru Hirose and Chaka Impraim, Roche Molecular Systems, Inc., Pleasanton, CA, and Roche Diagnostics K.K., Tokyo, Japan.
Nucleic Acid Amplification Tests (NAT) have shown their power for detection of blood-borne viruses in blood and plasma screening. Compared with the current serological methods, NAT has the potential to reduce the "window period" of blood-borne infections. We are developing a high throughput assay for detection of HIV-1, HCV and HBV in donated plasma. In this assay, multiple targets (both RNA and DNA viruses) are captured with specific capture probes by an automated nucleic acid isolation system GT-12 (Roche Diagnostics, Japan); and amplified and detected simultaneously with TaqMan® technology on the ABI Prism 7700. Both internal and external controls are utilized in this assay. The total assay time is about 4 hr for 96 samples.
Preliminary data of this multiplex assay have shown that the sensitivities for all three targets are in the range of 50–150 copies/mL. Preliminary studies with seroconversion panels of HIV-1, HCV and HBV show significant reduction of window periods. Specificity was obtained at 100% (0/191 individual patient samples). No target or amplicon carry-over was observed with samples containing up to 106 HIV RNA copies/mL.
We have demonstrated the feasibility of detection of HIV-1, HCV and HBV in a single assay format with high sensitivity and high throughput. Further optimization studies and evaluation of this multiplex assay are in process.
13
Polymorphism Scanning Utilizing Whole Blood. Krajnik, K. and O'Kane D.J., Dept. Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905.
Scanning for genetic variation is generally performed utilizing DNA isolated from blood containing anti-coagulants. A minimum purification is required to remove inhibitors of PCR including hematin and heme generated during storage and purification. An alternative approach is to lyse RBCs permitting removal of hemoglobin, and then releasing DNA into a crude lysate. A commercial kit (GeneReleaserTM, BioVentures, Murfreesborough, TN) is used to generate crude DNA lysates for polymorphism analysis of the gene encoding cftr (exon 10) and Factor V (exon 10). Scanning for polymorphsims was accomplished through branch migration inhibition (BMITM, Dade-Behring, Inc)1 in which base-pair mismatches in the presence of high concentrations of Mg++stabilize Holliday junctions. These cruciform DNA structures are captured to microwell plates and detected with an anti-digoxygenin-alkaline-phosphatase conjugate binding to digoxygenin-modified bases incorporated into the amplified DNA. Signal generated in this format indicates the presence of a polymorphism (heterozygous). Samples that are negative are re-incubated following addition of homozygous wild-type DNA, and re-assayed. Generation of a signal at this stage indicates a homozygous polymorphism; low signal indicates the homozygous normal allele. Frozen whole blood samples (1 to 6 months at–20C) were prepared for PCR using GeneReleaserTM. Genotyping was performed by RFLP analysis (Factor V) or SSCP (cftr). In a blinded study, of 137 samples scanned for polymorphisms in exon 10 of the gene encoding Factor V, 78/78 heterozygotes were correctly identified. Samples homozygous for the normal allele (N=78) were correctly identified with one apparent false-positive that was found to have an unsuspected polymorphism upon DNA sequencing. For cftr exon 10, 42/42 heterozygotes were identified correctly; 55/55 normal individuals were correctly identified; and 2 novel polymorphisms were detected that were missed by SSCP.
1. Lishanski A, Kurn N, Ullman EF. A homogenous mutation detection method based on the inhibition of DNA branch migration. 28th Annual Oak Ridge Conference, 1996.
14
Homogeneous Amplification and Detection of Nucleic Acid Sequences Using DzyNATM-PCR. Alison V. Todd, Helen L. Impey, Tanya L. Applegate, Margaret A. Haughton and Caroline J. Fuery, Johnson and Johnson Research Pty Ltd, Sydney, NSW, Australia.
DzyNATM-PCR is a general strategy for the detection of specific genetic sequences associated with disease or the presence of foreign agents. The method provides a system that allows homogeneous gene amplification coupled with signal detection in a single closed vessel. The strategy involves in vitro amplification of genetic sequences using a DzyNATM primer which harbors the complementary (antisense) sequence of a 10:23 DNAzyme (PNAS 1997; 94: 4262-4266). During amplification, amplicons are produced which contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mix. The accumulation of amplicons during PCR can be monitored by changes in fluorescence produced by separation of fluoro/quencher dye molecules (eg FAM/TAMRA) incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts.
Real time fluorometric measurements were performed on the ABI Prism 7700 Sequence Detection System. Experiments where a K-ras plasmid was amplified by DzyNATM-PCR indicated the technology allows quantification of DNA over at least 7 orders of magnitude. When the log of the copy number was plotted against the Ct value (ie the cycle number at which a threshold value is reached), a standard curve (r = 0.990) was generated. This allowed estimation of the amount of DNA in reactions containing unknown numbers of copies of the K-ras gene. These experiments also indicated that as little as 10 copies of the gene could be detected. In summary, DzyNATM-PCR is a flexible, simple, rapid and sensitive technique for homogeneous amplification and detection.
15
Localised Mutation Detection with Padlock Probes and Rolling Circle Replication. Mendel-Hartvig M., Banér J., Nilsson M., and Landegren U., Deparments of Genetics and Pathology, Uppsala University, Sweden.
We have demonstrated that padlock probes (Nilsson et al 1994, Science, 265, 2085) can be applied in situ to distinguish single nucleotide sequence variants (Nilsson et al 1997, Nature Genet, 16, 252). Padlock probes are synthetic oligonucletides with target recognition sequences situated at both the 5'- and 3'-ends. When hybridised to a target molecule the ends are brought adjacent to each other, and with the help of a ligase they can be covalently joined. The circular molecules so formed can be used to template rolling circle replication (RCR), resulting in a specific target-dependent signal amplification (Banér et al 1998, Nucl Acid Res, 26, 5073). However, the RCR reaction is effectively inhibited for topological reasons when a padlock probe is catenated to a target DNA molecule, unless the target strand has a free 3'-end close to where the padlock probe hybridises.
In the present work we have addressed the topological problem that prevents RCR. Ligated padlock probes could template RCR if the target strand was first cleaved with a restriction enzyme, allowing the free 3'-end to serve as a primer for the RCR reaction. Strand-specific cleavage can be effected, either using a resident restriction site in the target strand or more generally by hybridising an oligonucleotide including recognition sequence for a type IIS restriction enzyme (Podhajska et al, 1985, Gene, 40, 175). This approach should allow direct visualisation of haplotypes of sets of single nucleotide polymorphisms (SNPs) in chromatin fibres. Moreover, the method could be applied to detect multiple DNA or RNA sequence variants in arrays of either samples or probes.
16
Apo E Genotyping by Restriction Fragment Length Polymorphism in Myocardial Infarct Survivors and Healthy Controls. Hergenc G1, Ilcöl Y2, Budak Y2, Taga Y3, 1Kocaeli Med Fac Dept Biochemistry, Kocaeli, 2 Uludag Univ Dept Biochemistry Bursa, 3Hipokrat Laboratories, Istanbul, and 3Marmara University Medical Fac, Dept. Biochemistry, Istanbul, Turkey.
Apo E gene is known to have effects on the susceptibility to atherosclerosis, coronary heart disease and primary dyslipoproteinemia. Apo E gene is known to be polymorphic. Three common allleles determine six phenotypes which can easily be detected by restriction length polymorphism. Role of Apo E in late onset Alzheimer's Disease and cholesterol efffux have drawn much attention and interest on phenotyping and genotyping of apo E.
Amplification of the 237 nucleotide long apo E gene was followed by treatment with Hha1 and Taq1 enzymes in order to specify the genotype of the patients according to restriction fragment length polymorphism. This method can detect the isoforms that have been detected before.
The relative frequency of apo E genoforms in our patient group was found as : 0.82 E3/3, 0.14 E3/2, 0.04 E3/4, 0.00 E2/4; The relative frequency in the control group: 0.80 E3/3, 0.10 E3/2, 0.075 E3/4, 0.025 E2/4. Relative alllele frequency for the patient group was found to be 0.91 for e3, 0.07 for e2, 0.02 for e4 and for the control group 0.875 for e3, 0.067 for e2, 0.058 for e4.
Both e4 and e2 allele frequencies of the control and the patient groups are lower that that of Caucasians in Europe and the USA.
17
Detection of Mycoplasma Species in Blood of Patients with Rheumatoid Arthritis. Nasralla M, Haier J, Nicolson GL, Institute for Molecular Medicine and International Molecular Diagnostics, Inc., Huntington Beach, CA.
Mycoplasmas can enter a variety of tissues and cells and cause various signs and symptoms. These infections are associated with several acute and chronic illnesses. Using a forensic Polymerase Chain Reaction (PCR) procedure 28 patients (14 female, 14 male) diagnosed with Rheumatoid Arthritis (RA) were investigated for the presence of different mycoplasma species in their blood leukocytes. Amplification was performed with genus- or species-specific primers, and specific radio-labeled internal probes were used for Southern hybridization with the PCR product. Patients were investigated for presence of Mycoplasma spp., and the following species: M. fermentans, M. hominis, M. pneumoniae and M. penetrans. The Mycoplasma spp. sequence, which is not entirely specific for mycoplasmas, was amplified from the peripheral blood of 15/28 patients (53.6%), and specific PCR products could not be detected in 13 patients (46.4%). Significant differences (p < 0.001) were found between patients and positive healthy controls in the genus-test (3/32) and in the species-specific tests (0/32). Moreover, the incidence of mycoplasmal infections was similar in female and male patients. Using species-specific primers, we were able to detect infections of M. fermentans (8/28), M. pneumoniae (5/28), M. hominis (6/28) and M. penetrans (1/28) in RA patients. In 36% of the patients we observed more than one mycoplasma species in the blood specimen. This detection of multiple mycoplasmal species occurred only as combinations of M. fermentans with other species. The results suggest that a high percentage of RA patients have systemic mycoplasmal infections which may be an important cofactor in the pathogenesis of RA, and their role needs to be further explored.
18
Enhanced Fluorescence Signal from Molecular Beacon-based Asymmetric PCR compared to Symmetric PCR: Amplification of Adenovirus Hexon Gene. Poddar, S. K., Espina, R., University of California, La Jolla, CA, USA.
A symmetric PCR results in exponentially grown double stranded DNA. An asymmetric (where concentration of one of the primers is higher than the other) PCR generates one of the strands by linear amplification and a fraction of its total product as double stranded DNA limited by the concentration ratio of the primers used. In liquid phase assay, the fluorescence signal from a molecular beacon based symmetric PCR arises only from the probe fraction that hybridizes with the target successfully in competition with the strand complementary to target strand of the amplified DNA. So far symmetric PCR has been used in a molecular beacon based liquid phase assay. This communication reports a comparative study on the level of fluorescence signal detectable from a symmetric versus an asymmetric PCR conducted in presence of a molecular beacon. A DNA fragment (307 bp) from an adenovirus specific gene (hexon) was amplified by symmetric and by asymmetric PCR in presence of a molecular beacon. In asymmetric PCR, the appropriate primer was added in higher concentration so its single stranded product contained the target sequence. The concentration ratio of the two primers was also optimized for a maximally efficient amplification. The beacon probe was complementary to an internal sequence of the amplified fragment. 5'-6 Fluorescein (FAM) and DABCYL were used as the fluorophore and quencher, respectively, for the beacon. The fluorescence signal from the post-PCR sample was measured at 515 nm, the emission maxima of FAM with excitation at 491 nm. The fluorescence analysis demonstrated that an appreciably higher level of fluorescence signal is detectable from the asymmetric PCR compared to symmetric PCR conducted in presence of the beacon probe.
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Simultaneous Detection of HBV, HCV, and HIV in Plasma Samples Using a Multiplex Microplate Capture (MMC) Assay. Jay Ji, Kezuo Wu, Lijuan Yang, and Mark Manak, BBI Biotech Research Laboratories, 217 Perry Parkway, Gaithersburg, MD 20877.
The primary risk to the safety of the blood supply worldwide is the potential transmission by transfusion of viral diseases such as hepatitis and HIV. Currently the FDA requires all blood and blood products to be screened against HBV, HCV and HIV. The conventional screening tests are serological based assays, which indirectly measure exposure to viral infection. The FDA has recently required the screening for HIV RNA by nucleic acid amplification assays be also included to reduce the size of the "window" period between time of actual infection and the appearance of antibodies. Blood samples for the European market must also be screened for HCV RNA. We have developed a PCR based multiplex microplate capture (MMC) assay to simultaneously screen for HBV, HCV and HIV nucleic acids in plasma. The nucleic acids including both viral DNA and RNA are purified from the plasma samples in a single extraction procedure. A mixture of degenerate biotin-labeled PCR primers specific for the HBV, HCV, HIV-1 M and HIV-1 O were used to amplify any of these viruses which may be present in the plasma. PCR amplified products were captured on microtiter wells by hybridization to immobilized capture sequence, and colorimetrically detected by a chromogenic reaction using plate reader. An internal control vector containing a synthetic fragment flanked by sequences corresponding to the HBV primers was designed to monitor sample recovery during extraction, amplification and detection. Three copies per assay, equivalent of 100 copies per ml, were detected consistently without the requirement for a virus pre-centrifugation step. All major subtypes of HBV, HCV and HIV-1 including HIV-1 type O can be detected by this assay. Our newly developed MMC assay for the detection of HBV, HCV and HIV can be used as a highly sensitive and robust screening method for multiple analytes in blood or blood products.
20
A Quantitative, Signal-amplified Assay for mRNA Expression in an Inducible Cellular System. Zakel J., Williams I., Seyfried D.M., and Lazar J.G., Digene Corporation, Beltsville, MD.
RNA expression analysis is emerging as a major tool for use in the fields of pharmaceutical screening, lead discovery and disease monitoring. RNA expression analysis enables investigators to rapidly obtain high quality information on changes in gene expression in biological systems due to exposure to experimental compounds or infectious agents. Digene Corporation has developed the Expression Analysis System (EAS) based on patented Hybrid Capture® technology that provides quantitative, signal-amplified detection of RNA from crude cell lysates, in a 96-well format. EAS does not require the purification of RNA from samples prior to analysis. Target RNAs are detected directly from cell lysates by hybridization to a biotinylated ssDNA probe. RNA:DNA hybrids are captured onto a streptavidin coated microplate and detected with an alkaline-phosphatase conjugated antibody specific for RNA:DNA hybrids. The bound antibody is detected by the addition of a chemiluminescent substrate. The EAS assay has an analytical sensitivity of 1000 RNA copies, with a dynamic, linear range of 103–107 RNA copies per well. The assay typically has coefficients of variation of 15% or less. Analysis of drug dose response has been demonstrated in several cellular models using cultured A-10 and Jurkat cells. From A-10 cells stimulated with 30 ng/ml of platelet derived growth factor (PDGF), a 25 fold induction in c-fos mRNA expression was observed relative to unstimulated cells. From Jurkat cells stimulated with 1 ug/ml PMA and 1 ng/ml Ionomycin, a 12 fold induction of IL-2 mRNA expression was observed relative to unstimulated cells. These observed levels of induction are similar to those determined by other methods as reported in the literature. EAS technology can provide a rapid, accurate, and universal method for measuring gene expression in high throughput systems.
EAS is marketed and distributed for high throughput pharmaceutical screening as Xpress ScreenTM by Tropix, a subsidiary of PE Biosystems.
21
A Novel Thermal Cycling Apparatus for Strip- or Solution-based Nucleic Acid Amplification or Detection. Ross Barnard1, Deema Al-Sheikhly1, Peter Devine1,2 and Irina Elarova1. 1Co-operative Research Centre for Diagnostic Technologies, Queensland University of Technology, Brisbane, QLD 4001 and 2PanBio Pty Ltd, Windsor, QLD 4030, Australia.
We have developed prototype thermal cyclers, which utilize oscillating interconnected cells with Peltier driven, synchronized heating and cooling of opposed, interconnected cells.1. We have used these prototypes to carry out strip based hybridization reactions using the factor V sequence as a target, strip based primer extension reactions, and solution phase polymerase chain reaction in three model systems, one from genomic DNA (factor V gene). We are currently diversifying the applications to other model systems. The machines are light weight (approx. 1.2 kg) and would be amenable to point of care or field application. A variety of physical embodiments of this type of machine is possible.
1. Barnard R, Elarova I. Improved thermal cycling apparatus and method. PCT WO 99/15622
22
Enhancements to the VIDAS PROBE Neisseria gonorrhoeaeTest. E. Burres1, M. Gilker1, P. Hodge1, M. Longiaru1, T. Wang2, B. Rice2, 1Gen-Probe Incorporated, San Diego, CA, 2bioMérieux, Inc., Rockland, MA.
Gonorrhea remains a significant and under reported disease in the United States, which if undiagnosed can result in complications costing millions of dollars annually. The VIDAS PROBE Neisseria gonorrhoeae Test is a rapid, automated assay in which Transcription-Mediated Amplification (TMA) of Neisseria gonorrhoeae (NG) ribosomal RNA (rRNA) and detection of the amplified product occur in a VIDAS Probe strip. Processed samples are added to disposable VIDAS PROBE strips containing all reagents necessary for amplification and detection. The strip is placed in a new instrument (AMPstation) which automates TMA inside the enclosed strip. The VIDAS PROBE NG Test also contains an internal control which co-amplifies and monitors inhibition of amplification. Detection in the VIDAS instrument utilizes solid phase capture of amplified product and specific fluorescence-generating reporter probes. Time to results is less than four hours including sample processing. Seventy five endocervical and vulval swab pairs with known endocervical NG culture results, and 318 male and female urines with known NG culture or PACE NG results were tested at Gen-Probe in the VIDAS PROBE NG Test. Sixty of 60 (100%) NG culture negative endocervical swabs were also negative and 15 of 15 (100%) NG culture positive endocervical swabs were also positive in the VIDAS assay. The vulval swabs showed 100% correlation with the paired VIDAS PROBE NG Test endocervical swab results which suggests that the less invasive vulval swabs are a viable sample alternative. No internal control amplification failures were seen during testing of the swabs. Following resolution testing, sensitivity and specificity in male urines was 100%; female urines demonstrated a sensitivity of 90% and a specificity of 100%. Male and female urines had inhibition rates of 0% and 5.9% respectively. The VIDAS PROBE NG Test, co-developed by Gen-Probe and bioMérieux, is a rapid and sensitive method for the direct identification of N. gonorrhoeae. The automated format, unit dose reagents, closed amplification system, and internal control provide additional convenience for the clinical lab.
23
An Epidemic of Hepatitis E Virus in South India: Its Detection by Polymerase Chain Reaction. Arshia. N., Jameel, S., Panda, S.K., Habibullah, C.M., Dept. of Virology, Central Laboratory & Blood Bank, Riyadh, Saudi Arabia.
Viral hepatitis is endemic in India. Periodically it assumes epidemic form. During the past two decades major outbreaks have been reported from India. This investigation describes an epidemic of Non-N, Non-B hepatitis involving 250 patients in Southern India.
The study was aimed to evaluate the role of hepatitis E Virus in Non-A, Non-B related epidemic. We utilized PCR amplification for detection of HEV genome sequence in clinical isolates. Stools from acutely infected individuals were tested for HEV sequence.
Using guanidinium isothiocyanate acid phenol method carried out extraction of RNA from stools. Nested PCR was performed for detection of HEV RNA. The selection of primers for PCR amplification was based on the nucleotide sequence available in the literature. Nested RT-PCR was carried out using external & internal primers for 25 cycle in a thermocycler. The amplification primer pair 3043/3044 and HEV-1/HEV-3 was selected to amplify DNA fragments of 591 & 343 bp respectively. PCR amplification specificity was confirmed by Southern hybridization. Stools from 5 patients were tested by PCR amplification for HEV (serologically) excluded for HAV, HBV and HCV. Two of five samples (40%) were found to be positive for HEV RNA.
Thus by the use of PCR amplification strategy we established the epidemic contained an HEV related disease component.
24
Gene Expression Analysis Using RNA/DNA Extracted from Minute Formalin-fixed, Paraffin-embedded Tissue Samples. Jean-Philippe Stephan, Stanley Chao, Siao Ping Tsai, (Genentech, Inc., CA).
Archives of formalin-fixed, paraffin-embedded tissues are a major source of material for gene expression analysis. However, even though numerous methods have been described to extract DNA or RNA from formalin-fixed tissues, only a few are routinely usable for reverse transcription and PCR amplification.
In this study, using a novel procedure in which we modified and combined a DNA extraction protocol [EX-WAXTM DNA Extraction Kit (for paraffin-embedded tissue); Intergen] with a total RNA isolation kit [RNA STAT-60TM; Tel-Test, Inc.], followed by a DNA isolation kit [DNeasyTM Tissue Kit, Qiagen], we purified RNA and DNA from formalin-fixed, paraffin-embedded human breast carcinoma cell lines SKBr3 and MCF-7. Subsequent to overnight incubation, the entire procedure is completed in less than three hours. From 1 mg of material, we can recover up to 9 µg of total RNA and 5 µg of DNA from the same sample.
As a first approach, we used quantitative RT-PCR/PCR to compare HER-2/neu gene expression in fresh and fixed SKBr3 cells using total RNA/ DNA. Next, the expression of GAPDH and ß-actin, along with eight genes [HER2, Progesterone Receptor, PCNA, CEA, OA, Estrogen Receptor, PS2, and p53], previously described as potential breast cancer prognosis factors, was analyzed using RNA from fresh and fixed SKBr3 & MCF-7 cells and rat prostate. Finally, utilizing microarray technology (GeneFiltersTM; Research Genetics), we compared the gene expression profiles of total RNA from fresh and fixed rat prostate.
Our data show that quantitative PCR analysis can be performed using DNA extracted from fixed material. Relative to the expression of GAPDH or ß-actin, we demonstrate that RNA extracted from fixed tissue can be used for gene expression analysis with confidence, depending upon the size of the amplicon. Furthermore, in comparing the microarray results from fixed rat prostate RNA to fresh rat prostate RNA, we show <1% drop in signal specificity and <4% increase in background.
In conclusion, the ease of our technique makes it suitable for gene expression analysis in both research and clinical applications.
25
Direct Flow-Through Hybridisation Device: An Effective and Lost Cost Apparatus for Rapid DNA Analysis. J.W.O. Tam, Department of Biochemistry, The University of Hong Kong, HKSAR, China.
DNA analysis is emerging into every corner of bioscience and is being used in basic as well as applied research and development. One of the most frequently used processes in molecular biology involving nucleic acid research is undoubtedly the hybridisation, DNA annealing process. Unfortunately most of the current procedures are still very time consuming and require large amount of expensive chemicals or equipment. We would like to present a rapid as well as very inexpensive procedure- the direct flow-through hybridisation process and a simple device. This is a membrane-based hybridisation procedures consisting: (1) a membrane immobilized with target DNA/RNA (e.g. the conventional Southern Blot) or capturing probe such as the Reversed Dot Blot, RDB, (2) an accurately regulated temperature chamber and (3) the hybridisation and signal developing reagents. Since in most conventional processes, the annealing process, under similar nucleic acid concentration , the annealing process is mainly controlled by the diffusion rate of the two complementary strands getting in close contact for reaction. The flow-through process on the other hand directs the flow of the probe towards the target thereby increasing the effective local concentration to achieve faster reaction. Furthermore the sensitivity increases as the analytes rapidly flow inside the membrane to capture those bound inside the membrane pore to produce quantitative result. We are consistently able to achieve more than 90% intensity within 1 minute and 95% in 5 minutes of those obtained in overnight incubation by conventional Southern blot hybridisation. We are able to perform multiple sample and /or multiple loci analyses in a single strip and are able to adopt a format similar to the microchip but more versatile. Using this device we have performed DNA diagnoses for genetic diseases e.g. Thalassemia, G6PD; infectious diseases such as HBV, HCV, HGV and parasites. Currently we are using it for SNP studies. Since the device is very inexpensive and the procedure simple that can have a much wider circulation. (This investigation is funded by the Industrial Support Fund of the Hong Kong SAR)
26
Hybrigel Purification: A Novel Technique for Accelerated Preparation of DNA Sequence Products for Capillary Electrophoresis and Multiplexing. Lawrence Weir, Rahul K. Dhanda, T. Christian Boles, Christopher P. Adams, Mosaic Technologies, Inc. Boston, MA.
To obtain high quality data, automated DNA sequencing requires substantial purification of sequence products prior to analysis. When using dideoxy-dye terminators it is essential to remove unincorporated dye terminators. In some cases, especially when using capillary electrophoresis, it may also be desirable to remove salt, template DNA and excess primers. Several methods are currently used for purification of DNA sequencing products, none of which are entirely satisfactory. Precipitation with ethanol or isopropanol is time-consuming and requires care to achieve consistent product yields. Spin columns are more efficient at removing dye-terminators but they are costly, labor-intensive and also time-consuming.
We have developed a new electrophoretic technique for purifying and concentrating the products of DNA sequencing reactions based on Mosaic's AcryditeTM technology. This technology enables researchers to make gels containing immobilized DNA probes. 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, termed Hybrigels. During electrophoresis, complementary targets are themselves immobilized whereas non-complementary molecules migrate through the gel unimpeded. AcryditeTM capture probes were designed with sequences complementary to products of Mp18 sequencing reactions. These probes efficiently capture sequencing products in miniature Hybrigel purification devices. Primers, salt, unincorporated nucleotides and dye terminators pass through the gel into the buffer reservoir. The captured material may then be released by temperature and recovered in a small volume ready for loading on a capillary or sequencing gel, while template DNA remains trapped in the Hybrigel. The current protocol takes 5 to 10 minutes and is ideally suited for automation using the microtiter format.
We demonstrate that high quality sequence may be obtained from Hybrigel-purified material. We also show the feasibility of a multiplexing strategy whereby multiple sequencing reactions performed in the same vessel may be separated using specific capture probes.
27
Oligo Tag-PHFA: A Method for Multiplex Analysis of SNPs. Takanori Oka, Takahiro Ogawa, Hironari Matsunaga, and Akio Yamane, Institute for Medical Research, Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan.
The system to detect human genetic mutations and/or polymorphisms is one of the most important tools in molecular genetic analysis of disease. As most of DNA sequence variations are single nucleotide change, we established a simple and powerful method to detect multiple SNPs (Single Nucleotide Polymorphisms). This system is based on PCR-PHFA (PCR-Preferential Homoduplex Formation Assay), a method to detect single base substitutions in a PCR amplicon by competitive hybridization between double-labeled amplicon (standard DNA) and unlabeled amplicon (sample DNA) under precisely controlled annealing condition with temperature gradient. Standard DNA has two labels attached on PCR amplicon at 5' terminal of each strand; one label is biotin for capturing onto solid support and another is hapten for binding of enzyme conjugated antibody. After annealing, when a sample DNA is identical to standard DNA, the population of double-labeled amplicon is decreased due to strand exchange. This decrease, namely the presence of particular polymorphic sequence, is monitored by coloring reaction in the microtiter plate.
Since repertoire of combinations between oligonucleotide tag and its
complementary nucleotide is nearly unlimited, we modified this system
for multiplex analysis of SNPs by using oligonucleotide tag for
capturing to polymer support. A standard DNA containing corresponding
polymorphic sequence carries a unique oligonucleotide tag on one strand
and a common detection label, biotin, on another strand. Target regions
containing particular polymorphic sequence are amplified by multiplex
PCR and mixed with corresponding standard DNAs. After heat
denaturation, the specific annealing reaction is taken place under the
temperature gradient. In case of microtiter plate format, the annealing
mixture is dispensed into microtiter wells and the tagged DNAs in the
mixture are captured on corresponding microtiter well on which
oligonucleotide having complementary sequence to the tag is
immobilized. In case of membrane format, all tagged DNAs are
simultaneously delivered to the spots containing corresponding
complementary nucleotide. After coloring reaction with streptavidin
conjugated enzyme, the presence of each polymorphic sequence is
identified. We report here, the SNP analysis data of Human Platelet
Antigen (HPA) 1-8 presented on GPIIIa, GPIa, GPIb
, and GPIIb.
28
Enhancements to the VIDAS PROBE Mycobacterium tuberculosisTest. K. Clark-Dickey1, M. Longiaru1, T. Quigley1, A. Sitay1, G. McKinley2, 1Gen-Probe Incorporated, San Diego, CA, 2bioMérieux, Incorporated, Rockland, MA.
The World Health Organization has predicted that by the year 2020 nearly 1 billion people will be infected with Mycobacterium tuberculosis (Mtb), with 70 million deaths. A rapid, simple and automated diagnostic method is needed to stop the spread of tuberculosis. The VIDAS PROBE MTB Test is developed to identify Mtb in respiratory samples within 4 hours. Processed sample is added to a disposable VIDAS PROBE strip containing all reagents necessary for amplification and detection. The strip is placed in a new instrument (AMPstation), which automates Transcription-Mediated Amplification (TMA) inside the enclosed strip. Both Mtb rRNA, if present in the sample, and an internal control are co-amplified using Gen-Probe's isothermal TMA. The amplified products are sequentially detected in the strip by the bioMérieux VIDAS Immunoassay System instrument using fluorescent-labeled reporter probes. The internal control monitors amplification failure. As part of an ongoing study, three hundred nine clinical respiratory specimens were tested at Gen-Probe by this method and compared to fluorescent acid-fast stain and culture (Bactec 12B broth and Lowenstein-Jensen agar) methods for Mtb. Two hundred nine of 211 (99% specificity) respiratory specimens smear and culture negative for Mtb were also negative by the VIDAS PROBE MTB Test, including 41 specimens that grew non-tuberculous mycobacteria. The internal control successfully detected amplification failure in 7 instances (2.3%). Forty-six of 46 smear-positive and Mtb culture positive samples were positive by the VIDAS Probe method (100% sensitivity). Thirty-nine of 50 smear-negative and Mtb culture positive specimens were positive by the new test (78%); upon repeat testing of the sediment, some of these were positive, suggesting specimen heterogeneity. Two specimens were positive by the VIDAS test, but negative by culture for Mtb; however, these specimens were from previously diagnosed Mtb patients currently on therapy for Mtb. The automated VIDAS PROBE MTB Test, co-developed by Gen-Probe and bioMérieux, will provide a rapid and convenient method to aid in the diagnosis of tuberculosis.
29
Homogeneous Detection of HCV RNA by Multi-fluorescence Real-time Monitoring of Isothermal Sequence Amplification with INAF DNA Probes. Juichi Saitoh, Toshiki Taya, Ryuichi Horie, Toshinori Hayashi, Takahiko Ishiguro, Tokyo Research Laboratories, Tosoh Corporation, Kanagawa 252-1123, Japan.
The real-time monitoring of isothermal sequence amplification is a key technology to realize large-scale screenings and full automation.
Recently, we introduced a novel fluorescent DNA probe (named INAF probe) to enable to construct the homogeneous detection of a nucleic acid sequence without any post-amplification analysis. The probe is equipped with an fluorescent intercalator through a linker arm, which would intercalate into adjacent base pairs of formed double stranded complex with a target sequence to emit enhanced fluorescence.
We demonstrated homogeneous simultaneous detection of multi targets, HCV RNA and internal standard, in one tube, by two-color fluorescence real-time monitoring of isothermal sequence amplification in the presence of INAF DNA probes, oxazole yellow (YO) linked- and ethidium bromide (EtBr) linked- probes.
In blue excitation (488 nm), YO linked-probe showed enhanced green fluorescence, while ethidium bromide (EtBr) linked-one brought red fluorescence, upon binding to complementary target sequences for each.
The present isothermal amplification of the RNA segment was performed at 50° on the basis of the cooperative two-enzyme feed-back reaction (TRC 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 fluorescence intensity (515 nm, 600 nm) in the reaction tubes was monitored with a dedicated instrument, TRC monitor, in the course of the reaction.
The fluorescence intensity in the reaction tube of 104 copies, starting RNA copies, showed marked increase with the progress of the reaction in 30-minute incubation. The time required for the fluorescence enhancement reach up to the cut-off value depended on starting copies of RNA ranged from 102 to 106 copies. HCV RNA in clinical specimen was quantified with sufficient precision on the basis of the calibration curve obtained from the profile.
30
An RT-PCR ELISA Assay for the Detection of Flavivirus Nucleic Acids in Viral Cultures. Deema Al-Sheikhly1, Peter Devine2, John Aaskov1 and Ross Barnard1, 1School of Life Sciences, CRC for Diagnostic Technologies, Queensland University of Technology, GPO QLD 4001, Australia, 2PanBio Pty Ltd., Windsor, QLD 4030, Australia.
The clinical diagnosis of flaviviruses is difficult due to the non-specific nature of the symptoms, which are often confused with malaria, measles or other childhood diseases. While serological diagnosis is the simplest and fastest method of confirming a clinical diagnosis, a significant number of patients are viraemic and have not produced detectable amounts of antibody when they first seek medical assistance.
We have developed a multiplex reverse transcriptase/polymerase chain reaction (RT-PCR) assay for the rapid and specific early detection of dengue (serotypes 1 to 4), Yellow fever and Japanese encephalitis viruses (Flaviviruses). The protocol was developed using two different sets of oligonucleotide primers. Dengue (serotypes 1 to 4) specific primers (from the core-membrane genes) and the Yellow fever and Japanese encephalitis specific primers (from the NS3 gene) along with their respective consensus primers were used in the multiplex RT-PCR. The procedures was achieved using 35 cycles of PCR at an annealing temperature calculated from the average of Tm values of each primer used. The multiplex RT-PCR yielded fragments of characteristic size from DEN, YF and JE viruses. This technology was then transferred to a colourimetric ELISA-based assay.
Supported by the Australian Government Co-operative Research Centres Program.
31
Noninfectious Chlamydia trachomatis and Neisseria gonorrhea Combination Control for Use in Evaluating Clinical Laboratory Testing Precision. Gratiano, T., Crush-Stanton, S., Chorley, M., Bradley, H., Lawrence, N., Manak, M., BBI Biotech Research Laboratories, Inc., Gaithersburg, MD.
BBI has developed a dual Chlamydia/Neisseria control suitable for use with laboratory precision tests and for detecting errors in laboratory procedures for commercially available Chlamydia and Neisseria kits. This dual control is designed to mimic an actual clinical sample (urine or swab) and will control for all aspects of testing, including sample processing. The control has been formulated for use in genetic amplification based IVD test procedures that detect Chlamydia trachomatis (CT) and Neisseria gonorrhea (NG) DNA. To prepare the CT stock, high titer purified elementary bodies were inactivated and diluted to a working concentration of 1.0 x 108 EB/ml. Infectivity of the inactivated stock was determined by counting Inclusion Forming Units (IFUs) after passage on McCoy cells. The inactivation protocol of this stock yielded <0.1 IFU/ml in the final product concentration. To prepare the NG stock, high titer cultured cells were inactivated and dilutions (0, 1:10, and 1:100) were plated out on Chocolate agar plates to determine the number of remaining viable cells. No viable cell count was detected even at the lowest dilution (1:10) tested. The relative sensitivity of the Roche Amplicor Microwell Plate, Roche COBAS Amplicor, Abbott LCx, and Becton-Dickinson SDA assays was then determined using serial dilutions for the individual CT and NG preparations. In a series of experiments, the CT and NG preparations were blended so that the individual analytes could be successfully amplified and detected without the presence of one analyte affecting the sensitivity of the other and regardless of which commercially available assay was used. Data from accelerated stability studies at 37°C extrapolate to 2–3 years stability at 4°C for this product. For the final formulations, the concentration of both CT and NG were adjusted to provide low, on-scale signal in the indicator amplification assays such that significant deterioration of assay performance can be readily detected. This dual control with intact non-viable cells offers the industry a convenient format for monitoring the performance of either CT of NG amplification assays on actual urine or swab clinical specimens.
32
Enrichment of Mutant Alleles by Chromatographic Removal of Wild-Type Alleles: A New Approach for the Detection of Alleles with Unknown Point Mutations in Excess of Wild-Type Alleles. P. Nollau, C. Fischer, P. Tschentscher, and C. Wagener, Department of Clinical Chemistry, Medical Clinic, University Hospital Eppendorf, 20251 Hamburg, Germany.
In human carcinomas, mutations that alter tumor genes such as the KRAS, P53, or APC genes, are mostly point mutations. The detection of mutant alleles in specimens such as urine, pancreatic juice, sputum, and stool holds great promise for an early diagnosis of cancer. In addition, the detection of mutant tumor genes in tissue samples such as lymph nodes or resection margins may allow a sensitive diagnosis of residual malignant disease. However, the reliable detection of mutant alleles in excess of wild type alleles remains an unresolved problem when the mutations are not known a priori. Here we describe a new approach, which makes possible the detection of unknown point mutations in tumor genes at excess of wild type alleles. The method is based on the removal of wild type alleles by hybridization to immobilized complementary oligonucleotides. An enrichment of alleles with KRAS and APC mutations of one mutant in 102 normal alleles has been achieved. For the detection of mutations in the P53 gene, the enriched mutant allele was reamplified and submitted to a second round of enrichment. In this way, the sensitivity was increased to the detectability of one mutant allele in at least 103 normal alleles. By iterating this approach, the sensitivity will be limited by errors of the DNA polymerase only. Parallel miniaturized separation units with oligonucleotides complementary to defined sequences of a wild type allele should allow the detection of unknown point mutations as well as small insertions or deletions which occur in the sequence range covered by the oligonucleotides.
33
Superior Kinetic Performance of DNA Hairpin Probes in Solid Phase Hybridization. Leung, K.T.1 (Merante, F.1, Zastawny, R.L.1, Janeczko, R.1, Riccelli, P.1, and Benight, A.S.1,2), 1Tm Bioscience, Corp., Toronto, Ontario, Canada; 2Dept. of Chemistry, Univ. of Illinois at Chicago, IL.
Efficient capture of nucleic acid targets by oligonucleotide probes attached to a solid surface is crucial for detection of hybridization events on microarrays and other solid phase based assays. In our assay systems, we use dangling ended DNA hairpin probes rather than linear probes to capture target DNA molecules. In this study, we investigated and compared the kinetics of target capture by hairpin and linear probes as a function of target concentration and temperature. Hairpin probes consist of a 16 base pair duplex stem, linked by a penta-thymidine loop. The third thymidine in the loop was biotinylated for coupling to avidin coated microtiter wells. The target capture region of the hairpin was a 32-base, 3'dangling end complementary to a unique sequence of the porcine malignant hyperthermia gene. The target molecule was a 33P-labelled 65-base single-stranded oligonucleotide. Capture assays were conducted in tandem under identical conditions for the hairpin and linear capture probes. Under all conditions examined, hairpins consistently displayed higher rates of hybridization and higher total amounts of captured target. At 35°C, the hairpins showed the greatest advantage over the linear probes. Rates of hybridization at this temperature were at least four times greater for the hairpin compared to the linear capture probes at all target concentrations examined. These results clearly demonstrate that hairpins offer substantial physical advantages to nucleic acid capture in solid support systems. Hence, hairpin probes can significantly enhance the performance of nucleic acid assays, such as, high throughput diagnostics, single nucleotide polymorphism (SNP) detection and microarray based gene expression profiling, where the current challenge is to develop more rapid and sensitive detection methods.
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