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San Diego Conference |
H. Akhavan-Tafti, R. DeSilva, W.G. Cripps, R.A. Eickholt, R.S. Handley, K.S. Lauwers, M.D. Sandison, S. Siripurapu, and A.P. Schaap.
Lumigen Inc., Southfield, MI.
A new type of solid phase material for binding and releasing nucleic acids has been developed. The materials bind DNA and RNA with a strength far exceeding conventional materials and feature a novel mechanism to release bound nucleic acids. Unlike silica particle based materials and ion exchange polymeric resins, both of which elute bound nucleic acid through alteration of buffer ionic strength, neither high nor low ionic strength buffer is required to release nucleic acid. Bound nucleic acid is, however, readily released intact under controlled conditions with a 5 minute protocol. The solid phase materials have been fabricated as both magnetic and non-magnetic microparticles. No chaotropic salts are required to bind or release nucleic acids. DNA and RNA can be captured from water or a variety of buffers and media over a range of pH and salt concentrations. Binding of DNA and RNA from solution results in immobilization of nucleic acids that resists reversal under all commonly used release conditions. Repeated washing of the particles with water, low or high ionic strength buffers and surfactants does not remove captured nucleic acid. Nucleic acids of a wide range of molecular weight were isolated with the new materials. DNA ranging in size from oligonucleotides up to genomic fragments of several kilobases were captured quantitatively and released in high yield. Both magnetic and non-magnetic particles have been used in methods of isolating or purifying nucleic acids from several different sources. The particles were used to isolate the plasmid pUC18 from an overnight E. coli culture after pelleting, SDS lysis and potassium acetate precipitation. Human genomic DNA was extracted from whole blood with a conventional alkaline lysis procedure. DNA in the supernatants was captured on 10 mg of particles and released to produce DNA of MW 
20kb. In addition, a simple and rapid protocol for extraction of high MW DNA from whole blood without lysis has been devised using the new particles. Nucleic acids captured, purified and released using the solid supports are useful in numerous downstream applications. A linearized pUC18 target DNA was immobilized on non magnetic particles, released and amplified by PCR to produce a 285 bp amplicon. Confirmation of product was performed by agarose gel electrophoresis. Similarly, pUC18 was amplified by LMO (ligation of multiple oligonucleotides) to produce a 68 bp amplicon.
Diagnosis of Aneuploidies within 6 Hours Using Flow-Through PamChip Microarrays.
R. van Beuningen,1 L. Vahlkamp,1 M.E. Kalf,1,2 S.J.White,2 M.Kriek,2 T. den Dunnen,2 M.H. Breuning,2 and A. Chan.1
1 PamGene International B.V. ’s Hertogenbosch, The Netherlands; 2Leiden University Medical Center, Leiden, The Netherlands.
Diagnosis of syndromes caused by chromosomal abnormalities requires cytogenetic analysis by karyotyping on a metaphase spread. Since cells need to be cultured, a diagnosis takes at least 3 days. In some cases, for example Edwards Syndrome (trisomy 18) and Patau Syndrome (trisomy 13) the prognosis is such that treatment is usually withheld. Consequently it is important to know as rapidly as possible whether these chromosomal aberrations might be involved or not. If not, the appropriate treatment can be arranged in a timely manner. We have tested Multiplex Ligation-dependent Probe Amplification (MLPA) in combination with a porous micro array substrate (PamChip®) as a tool for detection of such aneuploidies. Compared with planar arrays, the PamChip array has several advantages, including the larger surface area, the possibility to vary hybridization stringency during analysis and most interestingly a decreased hybridization time of about 10 minutes. Two diagnostic systems are available for processing PamChip arrays, PamStationTM4 and PamStationTM96 for processing 4 or 96 samples simultaneously. For each of the chromosome to be tested (13, 18, 21, X) six probes were designed, as well as control probes derived from other chromosomes (15, Y). To facilitate array-based analysis, each MLPA probe contains a 20 nucleotide non-hybridizing tag sequence. For analysis, oligos complementary to the tag sequences were spotted on the PamChip. By comparing spot intensities between controls and patients it was possible to detect different aneuploidies within 6 hours from blood sample until result. Using the methodology developed, we performed a blind study on 25 patients and typed them all correctly. Preliminary results showed that even mosaic cases (>50%) could be detected. To facilitate wide-spread applications several different protocols were tested successfully, including MLPA performed on buccal swabs, amniotic fluid samples and 1 ng amplified genomic DNA. The multiplicity of this method was tested by using a larger number of probes (200) in one assay; excellent results were obtained. This allows the potential for a universal diagnostic PamChip array system for screening for the most common genetic imbalances in a single reaction, with a resolution at the gene level.
The NCI-Funded Standardized Expression Measurement (SEM) CenterTM Based on StaRT-PCRTM Enables Multi-Institutional Clinical Trials and Facilitates Development of Drugs and Molecular Diagnostic Tests.
Terry W. Osborn, James C. Willey, Charles R. Knight, Cheryl A. Motten, Bradley J. Austermiller, Elizabeth A. Herness Peters, Robert J. Zahorchak, and Andy Bass.
Medical College of Ohio, Toledo, OH and Gene Express, Inc., Toledo, OH.
Measuring expression of multiple genes in cell or tissue samples holds the promise of augmenting morphologic analysis and thereby facilitating development of drugs and molecular diagnostic tests through more meaningful classification. Advances in this area will depend on expression measurement methods that are standardized, reproducible, gene-specific, and sensitive. These properties are necessary for comparison of data across studies, analysis of small clinical diagnostic specimens, development of a reference data base necessary for meaningful diagnostic testing, and compliance with requirements established by regulatory agencies. Through NCI-funding (CA 95806), we have established the Standardized Expression Measurement (SEM) Center. The SEM Center uses a robotic liquid handler to prepare standardized RT (StaRT)-PCR reactions in 96-well microplates, followed by PCR-amplification in multi-block thermocyclers, then high throughput electrophoretic separation and quantification in a Caliper LabChip 90 microfluidic capillary electrophoresis device. The system is linked through proprietary software and programs in Microsoft (MS) Access, MS Excel, and MS Visual Basic for Applications. With implementation of these programs and establishment of standard operation procedures over the last year the throughput in gene expression assays/day has increased from 100 to over 1,000 and CV has decreased from 35% to less than 10%. Further, the SEM Center has successfully completed projects for multi-institutional trials, drug development and diagnostic testing. Moreover, the reference database is growing rapidly and now includes gene expression data from normal and/or malignant human lung, liver, kidney, spleen, uterus, ovary, bladder, blood and breast, as well as the Stratagene Reference RNA.
Development of a Fully Automated Protocol of MagaZorb Nucleic Acid Isolation Reagents on PSS Magtration System 12GC.
Jagdish Saini, Nnamdi Uche, Shiva Pourkaveh, Dokhi Nargessi, Dan Maffeo, and Chris Kusumoto.
Cortex Biochem, San Leandro, CA.
MagaZorb Nucleic Acid Isolation Reagents are based on a proprietary magnetic separation technology that is readily adaptable to automation. We have developed protocols for the application of these reagents to Precision System Science Company Limited’s Magtration System 12GC. Magtration System 12GC can process up to 12 samples per run. It allows the complete automation of nucleic acids isolation using the prepackaged unit reagents. Sample lysis, binding, wash and elution steps as well as all mixings are performed by the instrument. Following generic procedure describes the isolation of DNA from Whole Blood or Buffy Coat using MagaZorb DNA Isolation Kit. User pipets appropriate volume of sample (100 or 200 µl) in the sample tube and places it on the sample rack along with a fresh tip, a pre-filled reagent cartridge and empty tube to collect purified DNA; and presses START key. Instrument mixes the sample with Proteinase K and Lysis Buffer to lyse the cells. Lysed sample is then mixed with Binding Buffer and MagaZorb Reagent where nucleic acids selectively bind to the magnetic particles. Particles are then washed in Wash Buffer and finally nucleic acids are eluted in Elution Buffer. Total time per run is 27 and 29 minutes for 100 µl and 200 µl protocols respectively. Eluted DNA is ready for downstream applications such as PCR, sequencing or blotting procedures. Results: Based on absorbance at 260 nm, using 100 µl protocol; the average DNA recovery was 3.77 (2.51-4.80) µg from 18 whole blood samples tested compared with 1.49 (0.74-2.52) µg using another supplier’s reagents on the same instrument. Based on PCR quantitation, the DNA recovery from 9 blood samples when compared with the same supplier’s kit was 141-653%. Similar results were obtained with 200 µl protocol using the same 18 whole blood samples. Average DNA recovery by 260 nm absorbance was 8.16 (5.32-13.92) ug. PCR recovery was 100-523% compared with another supplier’s reagents. All 260 nm/280 nm ratios were >1.7. DNA integrity was confirmed by agarose gel electrophoresis. Similar protocol is also applicable for purification of total DNA from other human body fluids (e.g. seminal fluid, saliva, CSF), tissues, bacteria and viruses. The MagaZorb application described above allows the Nucleic acids isolation to be fully automated, consistent and cost effective with minimum risk of cross contamination.
Opportunity and Application of Innovated Quantitative Fluorescent PCR (IQF PCR) in Fetal DNA Analysis in Non-Invasive Prenatal Diagnosis.
Radek Vodicka,1 Radek Vrtel,1 Ladislav Dusek,2 Dita Svabova,1 and Jiri Santavy.1
1Department of Clinical Genetics and Fetal Medicine, University Hospital Olomouc, Czech Republic; 2Centre of Biostatistics and Analyses, Masasyk University, Brno, Czech Republic.
Introduction: Pre-implantation diagnosis and non-invasive prenatal diagnosis of fetal nucleic acid in maternal tissues are in the forefront of taking prenatal diagnosis to early stage of pregnancy. Then fetal nucleated cells or free extra-cellular DNA or bound RNA in maternal plasma are used. The concentration of free fetal DNA is approximately thousand times higher than DNA amount in intact fetal cells circulating in maternal peripheral blood. For diagnostic purposes precise diagnosis and quantification of fetal DNA is crucial. The quantitation of fluorescently labeled PCR products on capillary electrophoresis (QF PCR) has limits primarily in possibilities of more sensitive analyses to detect minority cell lines and small variations depending on the time. PCR efficiency and human factor affect measuring error and reproducibility of results in these cases. Determination of fetal sex from maternal serum by real-time PCR of Y specific sequences in male fetuses is relatively well developed and reliable, but detection of fetal aneuploidies from maternal serum is complicated. The main objective of this study was to assess and optimize quantitative possibilities of IQF PCR in detection of free fetal DNA in maternal serum by examination of STRs and Y sequences. Material and methods: Artificial prepared DNA mosaics, DNA from plasma, amniotic fluid, and maternal and paternal peripheral blood were analyzed by IQF PCR (which replaces real-time PCR) in D21S1411, D21S1446, AMELY and TSPY loci. Results: Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on the graphs. Calibration curve for Y sequences quantitation was set by the analyses of ratio of Y/X fluorescent signals. For rare mosaic calculation was created empirical formula. Variance assessment of mean RFU values of alleles ratio proved increase or decrease of those values compare to the normal lineage. In one case free fetal DNA was proven in TSPY and twice in D21S1446 loci. Conclusion: IQF PCR enables mosaics differentiation on level of several percentages. To decide if fetal DNA diagnostics is feasible for distinguishing disomy/trisomy by quantification STRs from desired chromosome the curve of normal fetal DNA concentrations during normal pregnancies must be compiled. This technique also eliminates limits of QF PCR and specifies quantitative analyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantitation of DNA mosaics. Further application is in examination of chimerisms after bone marrow transplantation and TSPY repetition assessment. Supporting grants:IGA MZ CR NR7817-3 and NR/7821-3
Appearance of a Novel Antibody Against Insulin Activated Nitric Oxide Synthase (IANOS) in the Circulation in Acute Coronary Syndrome (ACS).
U. Ray,1,3G. Khan, K. Chakraborty,1 S. Chakraborty, G. Reddy, and A.K. Sinha.1,2
1Sinha Institute of Medical Science & Technology, Calcutta, India; 2Mount Sinai Medical Center, NY; 3Royal Hobart Hospital Hobart, Tasmania, Australia.
Background: Studies by many investigators including us have demonstrated that insulin through its multifaceted antithrombotic effects could be an essential humoral factor for the prevention of CAD (Bioessay 2004;26:91-98). It is then possible that the thrombosis in CAD is pathophysiologically related to the failure of insulin, at least partly, to provide thromboprotection in this condition. Since insulin has been reported to inhibit platelet aggregation through the synthesis of NO by stimulating platelet IANOS, we studied the status of the enzyme and found its activity was severely impaired due to the presence of an inhibitor in CAD plasma. The properties of the purified inhibitor were studied. Methods: Blood was collected from the patients with CAD (M=10, F= 10, Age= 35-80 years) and from equal number of age and sex matched normal volunteers. None of the participants was smoker or had diabetes mellitus or taken aspirin at least 14 days before blood donation. Blood from CAD patients were collected before the initiation of antithrombotic therapy. The inhibitor was purified from the anticoagulated plasma from single donor by chromatography. The homogeneity and Mr of the inhibitor was determined by SDS-polyacrylamide gel electrophoresis. The inhibitor was quantitated by ELISA. Results:The purified inhibitor was determined to be the heavy chain of IgG with Mr 66 KD. While the amount of the inhibitor in CAD plasma was 7.5 µg/ml, its presence in normal plasma could not be detected. While the incubation of normal washed platelets in Tyrode’s buffer with 200 µunit insulin/ml produced 3.3 nmol NO /107 platelets/h, incubation of the suspension with 7.5 µg inhibitor/ml for 60 min at 37°C reduced the insulin induced NO synthesis to 1.3 nmol/107 platelets/h under similar condition. Incubation of normal platelet-rich plasma with the same amount of the inhibitor under identical conditions resulted in the blockade of insulin induced inhibition of platelet aggregation by 70% induced by different aggregating agents. Conclusion:We conclude that a novel antibody against IANOS in platelet membrane appears in the circulation in CAD, which through the blockade of NO production impairs the insulin-induced inhibition of platelet aggregation and may be involved in the development of CAD.
Stabilization of IFNß-Induced MxA Transcript in Post-Phlebotomy Whole Blood Using the PAXgeneTM Blood RNA System.
Lynne Rainen,1 Cynthia Ballas,2 Donna Dail,3 and Andrew Pachner.3
1PreAnalytiX Hombrechtikon, CH; 2BD, Franklin Lakes, NJ; 3University of Medicine and Dentistry, Newark, NJ.
IFNß-induced expression of the MxA gene is an accepted marker for response to IFNß therapy for multiple sclerosis (MS). A major impediment, however, to accurate quantitation of MxA transcripts in whole blood is the RNA degradation that occurs immediately after phlebotomy and continues during sample transport and processing. The PAXgene Blood RNA System is comprised of a blood collection tube and sample processing kit that stabilizes cellular RNA in whole blood and yields high quality total RNA. Blood was collected in PAXgene and EDTA tubes from 24 consenting MS patients receiving IFNß therapy 12 hours prior to phlebotomy. All samples were stored at ambient temperature. RNA from paired samples was extracted within two and at 72 hours of phlebotomy and analyzed for relative transcript levels by quantitative RT PCR analyzed by ABI Prism® 7700 (Applied Biosystems) chemistry and instruments. After 72 hours, average CT (cycle threshold) units increased by an average of 7.151 CT units in EDTA whole blood but less than 2.0 CT units in PAXgene tubes. Changes in EDTA tubes negatively affected reported transcript levels by 32- to 128-fold thus introducing technical variability that may underestimate the true transcript levels indicating response to therapy. These data provide evidence that the PAXgene blood RNA System stabilizes bioresponse gene transcripts in whole blood and provides RNA suitable for RT PCR analysis. The PAXgene System used in this study is for investigational use only, not for sale in the U.S.
Stabilization of Six Inflammatory Gene Transcripts in Post-Phlebotomy Whole Blood Using the PAXgeneTM Blood RNA System.
Lynne Rainen,1 Cynthia Ballas,2 David Trollinger,3 Danute Bankaitis-Davis,3 Shawn Lewis,3 David Gesink,3 and Michael Bevilacqua.3
1PreAnalytiX Hombrechtikon, CH; 2BD, Franklin Lakes, NJ; 3Source Precision Medicine, Boulder, CO.
A major impediment to accurate analysis of gene expression in whole blood is the change in cellular transcript patterns that occur immediately after phlebotomy and continue during sample transport and processing. The PAXgene Blood RNA System is an integrated system comprised of a blood collection tube and sample processing kit that stabilizes cellular RNA in whole blood and yields high quality total RNA. Blood was collected in PAXgene and EDTA tubes from 70 consenting donors and stored at ambient temperature. RNA from paired samples was extracted within two and at 72 hours of phlebotomy and analyzed for relative transcript levels of C1Qa, HSPA1A, IL-18, MMP9, SERPINE1, and TIMP1 by quantitative PCR analyzed by ABI Prism® 7700 (Applied Biosystems) chemistry and instruments. After 72 hours, average CT (cycle threshold) units changed by 0.925, 5.3, 2.0, 3.5, 0.09, and 1.8 CT units respectively, reflecting a reduction in the amount of mRNA detected (higher CT) in EDTA whole blood. In contrast, PAXgene tubes were observed to change by less than 0.8 CT units for all transcripts measured. The shifts observed in EDTA tubes introduce technical variability that may mask transcript changes that occur in clinical populations. The distribution of transcript levels was less than 7% coefficient of variability for the six transcripts measured across all 70 blood donors measured by PAXgene tubes, demonstrating less variability than samples collected by the EDTA tube. These data provide evidence that the PAXgene blood RNA System is superior to EDTA for stabilization of gene transcripts in whole blood, providing RNA suitable for RT PCR analysis. The PAXgene System used in this study is for investigational use only, not for sale in the U.S.
A Single-Tube Extraction, Amplification, and Detection Method for Real Time PCR Using Nanoporous Aluminum Oxide Membranes.
Shale Dames,1 Marc Elgort,1 Mark Herrmann,1 Jacob Durtschi,1 L. Kathryn Bromley,1 Maria Erali,1 Roger Smith,1,2 and Karl Voelkerding.1,2
1ARUP Laboratories, Salt Lake City, UT; 2University of Utah, Salt Lake City, UT.
Nucleic acid isolation is an essential component in molecular diagnostics. Current extraction techniques involving silica matrices require lysis, adsorption, and elution steps. We have developed a single PCR tube extraction, amplification, and detection method for use in real time PCR. Specially designed 200 µl PCR tubes with an embedded aluminum oxide membrane (AOM) have been fabricated to extract nucleic acids from biological samples. AOM provides a matrix that allows the localization of nucleic acid by filtration yet will not inhibit downstream molecular applications such as real time PCR. Samples are lysed at room temperature in the AOM tube, filtered using a custom vacuum block, and washed. After the final wash, the AOM PCR tubes are removed from the vacuum, the bottoms capped, and master mix is added prior to real time PCR detection. The extraction and amplification of herpes simplex virus-1 (HSV-1) DNA from cerebrospinal fluid (CSF) was chosen as a model system to compare AOM methodology to the clinical assay currently used in our laboratory. Experiments using HSV positive CSF and HSV-1 DNA spiked into CSF have generated amplification curves and crossing thresholds comparable to those in the reference assay. We are optimizing buffer formulations for lysis, nucleic acid localization on the AOM, and PCR amplification for different biological samples. The single tube character of the AOM method provides a significant handling advantage to current clinical techniques and data indicates that sensitivity and accuracy comparable to our reference clinical test will be achievable. We conclude that localization, amplification, and detection of HSV-1 from CSF is feasible by the AOM methodology. In the future, this technology will be extended to other nucleic acid targets that would benefit from a self contained reaction tube that eliminates sample transfer and minimizes handling steps.
Rapid Detection of Enterovirus and or HSV from a Single Patient Preparation: Implications for Improving Clinical Outcomes and Resource Allocations.
Brian Mariani, Beatrix Meltzer, and Lacy Fehrenback.
Genetics and IVF Institute, Fairfax, VA.
INTRODUCTION: HSV and Enterovirus (EV) are ideal models for demonstrating the value of molecular diagnostics in terms of patient management, resource allocation, and clinical outcomes. Although pathologically different, the symptoms for each infection overlap, confounding patient management, especially in the newborn. Rapidly distinguishing between these infections aids clinical decisions. Our clinical laboratory has recently migrated our in-house HSV and EV "nested" PCR and RT-PCR assays to real-time, multiplex formats, utilizing patient-derived RNA/DNA as internal amplification controls, with a 2
hour TAT. A single specimen processed for DNA and RNA is used for both assays. Both viruses present challenges for test design. EV is a widespread, single-strand RNA virus comprised of 
60+ serotypes found in diverse specimen types, with several serotypes problematic for the newborn. HSV, a large double-stranded DNA virus, is comprised of 2 serotypes and can be fatal in the neonate if untreated. The EV assay described here is designed to be comprehensive without serotype differentiation, whereas the HSV assay distinguishes between types 1 & 2. For EV, the initial concern of the treating physician is the present or absence of EV, regardless of serotype. Our assay is designed to react with all the major serotypes using a primer/probe set targeting the 5’UTR of the viral genome. For HSV, rapid and definitive detection is paramount, including serotype determination. Ruling-out HSV can eliminate unnecessary anti-viral treatment and ICU isolation, whereas HSV-positive results can help guide effective therapy. METHOD: EV Real-time RT-PCR: a primer/probe set specific for the conserved 5`-UTR is utilized. This IRBS region is critical for viral expression and replication. In particular, a highly conserved feature (stem #5) has been exploited for primer/probe placement. Validation studies demonstrate detection of the major serotypes in circulation in the U.S. from 1998-2004. A 90-minute, one-step RT-PCR amplification of 45 cycles yields a detection threshold is 10 viral genomes/assay with a Ct of 35. A primer/probe set specific for human B-actin mRNA is included in all reactions as a patient-derived inhibition control. HSV Real-time PCR: type-specific primer/probe sets (of different wavelengths) targeting the glycoprotein D & G genes are used for real-time HSV differentiation (without melting-point analysis). A 45-cycle amplification is utilized yielding a detection threshold is 10 viral genomes per assay with Cts of 35 (type 1) and 38 (type 2). The primer/probe set for a patient-derived DNA sequence is included as an inhibition control. All amplification reactions are performed on a SmartCycler (Cepheid, Inc.). Blood, CSF, swab specimens, body fluids, lesions, etc., are acceptable specimen types using the Qiagen Viral RNA Mini Kit. RESULTS: Since August 2000, 1,053 EV and 3,750 HSV assays have been performed on hospital specimens (399 for EV & HSV), and over 1,000 using the Real-time RT-PCR. Clinical correlation of these results has indicated excellent specificity. Selected EV specimens forwarded to the CDC have demonstrated concordance between labs. Cases are documented showing improved patient care and management through patient triage due to the self-limiting nature of EV infection (in most cases), compared to HSV. Rapid HSV-positive results have validated anti-viral treatment; whereas EV-positive results in the HSV-negative newborn have warranted NICU release, termination of anti-microbials, and shorter length of stay. CONCLUSION: Treating physicians and hospital administrators have benefited from the rapid EV and HSV assays described herein. Detection rates were 23.8%, 4.2%, and 2.2% for EV, HSV-1, and HSV-2, respectively. Timely diagnoses allow patients to be treated accordingly and moved in and out of intensive care in a more rational manner, improving clinical outcomes and better utilizing hospital resources (scenarios to be discussed). These results demonstrate that assays of clinical significance not yet incorporated into automated systems can be addressed using rapid, user-developed or ASR systems. This is pertinent for emerging pathogens and other lower volume tests that none-the-less affect patient care, resource allocation, and health care costs.
Multiplexed PCR/Microfluidics Chip Assay to Determine Copy Number of Parkin (Park2) Exons.
Brian Dukek, Matthew Farrer, and Dennis O’Kane.
Mayo Clinic, Rochester, MN.
Introduction: Knockout mutations to Parkin (Park2) lead to early onset Parkinsonism. A large number of disease causing mutations are due to insertions or deletions of whole exons ranging from exons 2-9. Quantitative PCR methods such as Light Cycler which use a different fluorophore for each amplicon are limited by the number of available channels. By instead differentiating amplicons by size this limitation can be circumvented. We performed quantitative multiplexed PCRs of exons 2-9 followed by analysis on the Agilent Bioanalyzer 2100 to determine copy number. Method: Using 24 cycles, four multiplexed PCRs were performed with different combinations of Park2 exons as well as Factor V (FV) as a normalizing control. Run 1 included FV, Ex2, Ex4, Ex8, and Ex9. Run 2 included FV, Ex3, Ex5, Ex6, and Ex7. Run 3 included FV, Ex2, Ex3, Ex7, and Ex9. Run 4 included FV, Ex2, Ex3, Ex4, Ex5, and Ex6. Using a strategy of combinations of exons run with and without each other we could account for changes in amplification efficiency of one exon caused by a deletion or duplication of another exon. After amplification, samples were run on the Agilent 2100 Bioanalyzer 1000 chip. Values for the corrected area under the curve were recorded followed by two rounds of normalization. To normalize for sample to sample differences in starting template concentration each sample peak’s area was divided by the area of the Factor V peak that served as an internal control. To normalize for differences in efficiencies of amplification for each exon the results from the first normalization were divided by the normalized results for a known diploid sample. Results: Six samples with known deletions and duplications were run. These deletions and duplications were previously detected using fluorescent primers following amplification in the log-linear range on either the ABI 377 or 3100. The ABI results for these samples include Ex 4-7 deletion, Ex 6 duplication, Ex 7 deletion, duplication of Ex 2-4 with Ex 3 deletion, Ex 3 deletion with Ex 7 duplication, and Ex 2 deletion. ABI and Agilent results were the same for all of these samples. 58 samples from patients aged 65 and older without a history of neurodegenerative disease were run as unaffected controls. All 58 patients had diploid results for all exons. Conclusions: This method can be used to accurately quantitate relative exon copy number. By identifying products based on amplicon length rather than by dye color the maximum number of exons analyzed per experiment can be increased.
Fluorescent SNP Genotyping by High-Resolution Melting Analysis without Probes.
Michael Liew, Mark Johnson, Robert Graham, Cindy Meadows, Maria Erali, Rong Mao, Elaine Lyon, and Carl Wittwer.
Institute of Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT.
Rapid, closed-tube genotyping assays for common clinical SNPs were developed using high-resolution fluorescent melting analysis of small amplicons. Small amplicons were rapidly amplified using the LightCycler® with temperature cycles of 20 sec in the presence of the fluorescent double stranded DNA binding dye, LCGreenTM PLUS. The tubes were then transferred to the high-resolution melting instrument, HR-1TM for melting curve acquisition, requiring an additional 2 min. Different homozygotes (a/a vs b/b) were distinguished by amplicon melting temperature. Heterozygotes (a/b) were detected by the presence of heteroduplexes, resulting in melting curve shapes that differed from the melting curves of homozygous amplifications. The method was applied to human platelet antigen SNPs (HPA 1-5 and Gov), important determinants of neonatal alloimmune thrombocytopenic purpura. As predicted by nearest neighbor stability calculations, the melting temperature differences between homozygotes varied from 0.4 to 1.3 °C. All heterozygotes and homozygotes were easily identified by high-resolution melting. In blinded studies, the melting method showed complete concordance to genotyping by allele-specific PCR. Additional studies with Factor V Leiden, prothrombin and MTHFR SNPs indicate that high-resolution melting can discriminate rare, heterozygous polymorphisms from the expected heterozygous mutation by subtle differences in curve shape. High-resolution melting allows rapid assay development and execution, requires only two primers as analyte-specific reagents, and eliminates exposure of amplicon to the laboratory.
Optimization of Immunoassays Utilizing Multiplexed Protein Arrays.
Luis Burzio, Jo Ann Kraycer, and Sam Conzone.
Schott North America, Duryea, PA.
The development of an immunoassay involves optimizing many steps of the process. Initially, the specificity of the antibodies should be determined to ensure no cross reactivity with other antigens, which could lead to false positive results. The amounts of antibodies that will be immobilized to obtain the maximum signal from samples and allow for the highest possible sensitivity must also be optimized. Finally, the washing, blocking and incubation steps must be optimized to ensure a method that is ready for validation. Traditionally the optimization is done in microtiter plates, but here we propose the use of protein microarray technology on multiplexed substrates that are in the format of a microscope slide. We used multiplexed epoxy coated substrates to optimize immunoassays to detect various proteins. This epoxy-coated substrate has a hydrophobic pattern that produces 48 distinct wells in a 1x3 inch footprint. As many as 100 antibodies can be printed for testing in each well. We printed a set of antibodies and assayed them for specificity and optimized the assays in terms of reagent concentration, immobilization and incubation times, blocking, and washing. The results demonstrate that the advantages over traditional microtiter plate are that the miniaturization of the assay reduces the amounts of antibodies and samples required for the optimization, as well as the ability to optimize the conditions for various assays simultaneously.
Simultaneous Genotyping of 36 Candidate Markers for Acute Myocardial Infarction Using Luminex®100TM XMAP Technology.
Andre Arellano, Bin Wang, David Lew, Wing Lee, Bradford Young, Dov Shiffman, Mary Luke, John Kane, Marc Short, and Lance Bare.
Celera Diagnostics, Alameda, CA.
Complex diseases are associated with multiple genetic and environmental risk factors. Thus, genetic tests assessing patient risk in such diseases will need to interrogate multiple polymorphisms. We have developed a genotyping assay that simultaneously detects 36 markers previously associated with risk of myocardial infarction. This assay combines PCR, oligonucleotide ligation, and hybridization to Luminex®100TM xMAP microspheres in a single tube assay. Allele-specific ligation products are captured by oligonucleotide tags attached to color-coded microspheres labeled with Streptavidin-Phycoerythrin conjugate and identified using the Luminex®100TM instrument. Analytical accuracy for genotyping in the assay is >99%. Allele frequencies of the 36 markers in various ethnic populations will be reported. Evaluating this assay in a 1500 sample case-control study replicated the association of a SNP in a taste receptor gene with risk for myocardial infarction. This assay technology is an accurate and useful method for assessing the genetic contribution to complex human diseases.
Detection of Trace Amounts of Mutant K-ras DNA by Peptide Nucleic Acid as Both PCR Clamp and Sensor Probe.
Chiuan-Chian Chiou and Ji-Dung Luo.
Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Tao-Yuan, Taiwan.
K-ras mutation occurs frequently in several types of cancer and may serve as a useful marker of malignancy. The major problem of using this marker is that the mutant template usually exists in background of excess amounts of wild-type DNA in the clinical samples. All methods developed to solve this problem today are either laborious or time-consuming and are not suitable as a regular test for a clinical laboratory. In the present study, we have developed a simple method to detect trace mutants of mutant K-ras by using peptide nucleic acid (PNA) as both PCR clamp and hybridization probe. In a capillary PCR reaction, a 17-mer PNA complementary to wild type sequence and spanning the codens 12 and 13 of K-ras oncogene was used to clamp PCR for the wild type allele but not mutant alleles. This allows the amplification of trace mutant templates in the existence of at least 1,000-fold wild-type templates. Moreover, the PNA was labeled with a fluorescent dye and used as a sensor probe. The mutant PCR products, with a mismatch to the PNA probe, have a Tm about 10• lower than that of the wild-type product. Using this novel design, all the 12 possible K-ras mutations in codens 12 and 13 can be easily identified in a single-step, one-tube reaction within 30 minutes. We believe this protocol has a great potential in a clinical laboratory and the experimental design can be adapted for detection of trace mutants of other diseases.
Thrombophilia NucleoSight DNA Chip.
Wesley Lindsey, Rob Jenison, Evelyn Woodruff, and Heather Avens.
ThermoElectron, Louisville, CO.
Thrombophilia is a multigenic disorder caused by inherited defects and environmental risk factors that lead to an increased risk of venous thrombosis. Inherited thrombophilia, a genetic predisposition to thrombosis, is caused by a number of known mutations, with the most common defect being a dual heterozygosity of the Factor V Leiden and prothrombin G20210A alleles. In addition, recent data suggests an association between inherited thrombophilia and recurrent miscarriages. The NucleoSightTM DNA chip detects the most recent prevalent mutations in a high-throughput, automated format with excellent accuracy. This technology utilizes hybridization-based discrimination of alleles followed by thin film formation and detection. The chip is coated with an optically active material and spotted with an array of probes specific for each target allele. PCR-amplified biotin-labeled patient DNA is incubated on the chip, and hybrid duplexes are detected with anti-biotin antibody-HRP conjugate and a precipitating substrate. Signal develops as a precipitate forms directly over probes that hybridize with target signal, resulting in a molecular thin film and a perceived color change. Chip images and genotype determinations are generated by an automated image analysis station that uses a sophisticated spot-finding algorithm followed by spot intensity ratio analyses. The thrombophilia assay is run in an automated 96-well format, allowing for hands-off processing of approximately 1300 patient samples in 10 hours. The Thrombophilia PortraitTM platform is an inexpensive, automated, high throughput, DNA chip-based assay capable of accurately classifying all thrombophilia mutations in the recommended screening panel. Moreover, the technology is amenable to other genetic panel tests requiring inexpensive, accurate, high-throughput testing.
Application: Use of Unlabeled Oligonucleotide Probe System in Molecular Diagnostics.
Lan-Szu Chou.1 Andrew Lyon,1 Cindy Meadows,1 Elaine Lyon,1,2 and Carl Wittwer.1,2
1ARUP Laboratories, Salt Lake City, UT; 2University of Utah, Salt Lake City, UT.
Fluorescent-labeled primers or hybridization probes are commonly used to detect known mutations (genotyping) in a molecular laboratory. However, synthesis of labeled primers or hybridization probes for multiple mutations takes time and is expensive. In this study, we designed specific unlabeled oligonucleotide probes (3` blocked), applied an intercalating dye (LCGreen I) and coupled with asymmetric PCR (forward primer to reverse primer = 1:10) to detect mutations in Factor V gene (G1691A) and CFTR gene (deltaF-508). 100 of each Factor V and CF (delta-F 508) samples were amplified and analyzed in a LightCycler, a LightTyper and a high-resolution melter HR-1 (Idaho Technology). Results from all three instruments showed 100% agreement with previously genotyped data. However, the best data quality (peak resolution) was obtained using HR-1 for both of the Factor V amplicon (151 bp) and the CF amplicon (292 bp). The LightCycler gave the least peak resolution, especially for a longer PCR fragment (CF, 292 bp). In comparison with the fluorescent labeled primers or probes, this homogeneous system genotyped both heterozygous and homozygous mutations correctly using low-cost (unlabeled) oligo-probes without sacrificing data quality. In addition to the oligo-probe melt, the melt from amplified product can be used as a secondary confirmation. To obtain the best melting resolution, primer design (shorter PCR product) and probe design (GC% and location of the SNP) are very important.
HepCgen® HCV Genotyping for Treatment (by Real Time PCR).
R. Cross, S. Jewell, and W. Rosenberg.
HepCGen Ltd., Southampton General Hospital, Southampton, UK.
Introduction:Hepatitis C virus (HCV) is an RNA virus that is responsible for liver fibrosis, cirrhosis and can lead ultimately to liver cancer and death. The HCV genome has high sequence variability, which has led to the identification of 6 major genotypes (with many more subtypes) found worldwide. It is well documented and recognised by the National Institute of Clinical Excellence in the UK (and in the USA) that the "genotype of the virus is the most important determinant of efficacy of treatment"2. Method: TaqMan real time PCR has been developed for this assay; initially validated for use on the ABI 7700. Primers for G1, G2/3 and G4 have been designed to sequence unique to those HCV genotypes. Regions of the HCV genome which are relatively well conserved between genotypes (and subject to little mutagenesis) but contain subtle sequence variation between genotypes (such as E1 and the 5` UTR) have proved suitable for genotype specific primer design. The HepCgen® Genotyping for Treatment1 assay consists of 4 simultaneous real-time PCR reactions. The first detects all HCV genotypes ("catch-all"), the second G1 (genotypes 1, 5 and 6), the third G4 and the final reaction will detect G2/3. Results: The HepCgen® HCV Genotyping for Treatment1 assay shows an excellent correlation with the expected genotypes of members of a performance assessed proficiency panel (Quality Control for Molecular Diagnostics) and two commercially available genotype panels (Boston Biomedica Inc. and Teragenix Corp.). Over 70 clinical samples have been genotyped (from G1, G2/3 and G4) and results compared to that obtained using the VERSANT® HCV Genotyping Assay (LiPA). The HepCgen® HCV Genotyping for Treatment1 assay has showed 100% correlation in the results obtained. Conclusions: The simple and rapid genotyping method will provide the clinician information required to make a judgment on Interferon based therapy as determined by the genotype of the HCV infection without the need for lengthy, expensive and unnecessary detailed genotyping and subtyping. Notes: 1International Patents filed and Pending. HepCgen® is a registered trademark of HepCgen Ltd.
A Biochip for Rapid K-ras Mutation Screening in Ovarian Cancer.
Robert Zeillinger,1,3 Gerhild Fabjani,1,2 Gernot Kriegshaeuser,3 Andreas Schuetz,4 and Lothar Prix.4
1Medical University of Vienna, Department of Obstetrics and Gynecology, Molecular Oncology Group, Vienna, Austria; 2Ludwig Boltzmann Institute for Gynecology and Gynecologic Oncology, Vienna, Austria; 3Vienna Lab Labordiagnostika, GmbH, Vienna, Austria; 4Biofocus GmbH, Recklinghansen, Germany.
Ovarian carcinoma is the most common cause of death from gynecological malignancies in the western world. The K-ras oncogene is frequently activated by point mutations in various neoplasms. Recently, a new medium-density biochip platform, called "GeneStiX System" was introduced. The detection of 10 common mutations of K-ras codons 12 and 13 is one of the first applications, combining the superior sensitivity of mutant-enriched amplification with highly specific hybridization. This biochip is especially suitable for the detection of mutations in specimens containing only a small portion of mutated DNA. In order to evaluate the GeneStick system’s compatibility with rapid mutation screening in tumor tissue we have analyzed 98 ovarian carcinoma specimens for K-ras gene. Nineteen samples were identified positive for K-ras mutations, 17 were positive for codon 12 and two were positive for codon 13 mutations. The data will be presented in respect to their clinical relevance.
Detection of Catalase Gene Mutations in Hungarian Acatalasemia.
L. Góth, P. Rass, A. Pay, and M. Vitai.
University of Debrecen, Departments of Clinical Biochemistry, Molecular Pathology and Clinical Analytical Chemistry, Debrecen, Hungary.
Enzyme catalase seems to be the main regulator of hydrogen peroxide metabolism. High concentration of hydrogen peroxide is a toxic agent, while its low concentration is required for some physiological processes such as signaling, platelet activation. Acatalasemia, the inherited deficiency of catalase has early (1947) detection and worldwide (11 countries) distribution. Its clinical features might be oral gangrene, altered lipid, carbohydrate, homocysteine, erythrocyte metabolisms and increased risk of diabetes mellitus. The Japanese, Swiss and Hungarian types of acatalasemia are characterized and have different in biochemical and genetic aspects. Contrary, there are only limited reports on the syndrome causing mutations i.e. two mutations for the Japanese acatalasemic patients. For detection of syndrome causing mutations of acatalasemia in one acatalasemic and 14 hypocatalasemic Hungarian families, we used mutation screening methods for PCR products of all calalase gene exons and exon-intron junctions. The polymorphisms detected by these methods were verified by nucleotide sequencing. We developed a simple PCR-heteroduplex and a PCR-SSCP method which with the nucleotide sequencing yielded five novel syndrome causing catalase mutations. These mutations include two nonsense mutations in exon 2, a splicing mutation in intron 7, and two missense mutations in exon 9. These mutations are associated with decreased (56.7%) blood catalase activity. These data are showing that acatalasemia is genetically a heterogeneous syndrome and our simple PCR-heteroduplex and PCR-SSCP methods are useful tools in screening of catalase gene mutations.
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