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Lutgarde Raskin - One of the best experts on this subject based on the ideXlab platform.
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Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes
Microbial ecology, 2000Co-Authors: D. Zheng, Lutgarde RaskinAbstract:A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide Hybridization Probes were developed and experimentally characterized. The Probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and Hybridization conditions for fluorescence in situ Hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed Probes together with previously designed Probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.
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Desulfotomaculum genus- and subgenus-specific 16S rRNA Hybridization Probes for environmental studies.
Environmental microbiology, 2000Co-Authors: Krassimira R. Hristova, Margit Mau, Dandan Zheng, Rustam Aminov, Roderick I. Mackie, H. Rex Gaskins, Lutgarde RaskinAbstract:Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested Hybridization Probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans ), and five specific Probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through Hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe ‘nesting’ using samples obtained from four different environments. Fixation and Hybridization conditions for fluorescence in situ Hybridizations were also optimized. The Probes were used in quantitative membrane Hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3–2.1% of the total rRNA in the digesters, 2.6–6.6% in soil, 1.5–3.3% in human faeces and 2.5–6.2% in pig colon samples.
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Characterization of Filamentous Foaming in Activated Sludge Systems using Oligonucleotide Hybridization Probes and Antibody Probes
Water Science and Technology, 1998Co-Authors: Francis L. De Los Reyes, Daniel B. Oerther, Ma. Fiorella De Los Reyes, Mark Hernandez, Lutgarde RaskinAbstract:A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ Hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona . The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and Hybridization Probes. The results of this technique were compared to those from membrane Hybridization studies using radioactively-labeled oligonucleotide Probes. Quantitative membrane Hybridizations, in situ Hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleotide Probes provide a definitive identification for Gordona , and represents a new approach for in situ studies of this organism9s role in foaming.
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characterization of universal small subunit rrna Hybridization Probes for quantitative molecular microbial ecology studies
Applied and Environmental Microbiology, 1996Co-Authors: Dandan Zheng, David A Stahl, Elizabeth W Alm, Lutgarde RaskinAbstract:Universal oligonucleotide Hybridization Probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal Probes also serve to normalize results obtained with Probes targeting specific phylogenetic groups of microorganisms. In this study, six universal Probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all Probes. This could lead to a significant bias when these Probes are used to quantify microbial populations in environmental samples. We suggest lowering the postHybridization wash stringency for two of the universal Probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two Probes were evaluated with traditional and modified Hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.
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group specific 16s rrna Hybridization Probes to describe natural communities of methanogens
Applied and Environmental Microbiology, 1994Co-Authors: Lutgarde Raskin, J Stromley, Bruce E Rittmann, David A StahlAbstract:Eight oligonucleotides which are complementary to conserved tracts of 16S rRNA from phylogenetically defined groups of methanogens were designed and characterized for use as Hybridization Probes for studies in environmental and determinative microbiology. The target-group specificity and temperature of dissociation for each probe were characterized. In general, the Probes were very specific for the target methanogens and did not hybridize to the rRNAs of nontarget methanogens. Together, the eight Probes circumscribe methanogens now represented in pure culture (with the exception of members of the family Methanothermaceae). Three Probes are order specific; two identify members of the order Methanobacteriales, and one is specific for the order Methanococcales. The fourth probe encompasses three families belonging to the order Methanomicrobiales, the third order within the current classification. The fifth probe is specific for the remaining family within this order (Methanosarcinaceae). Three additional Probes encompass different genera within the Methanosarcinaceae. Images
Nicholas J Turro - One of the best experts on this subject based on the ideXlab platform.
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fluorescent Hybridization Probes for nucleic acid detection
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Jingyue Ju, Nicholas J TurroAbstract:Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent Hybridization Probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary Probes (BPs) are two very important Hybridization Probes that are designed based on well-established photophysical principles. These Probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide Probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary Probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These Probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various Hybridization Probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.
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fluorescent Hybridization Probes for sensitive and selective dna and rna detection
Accounts of Chemical Research, 2007Co-Authors: Angel A Marti, Jingyue Ju, Steffen Jockusch, Nathan Stevens, Nicholas J TurroAbstract:We outline the different approaches taken by our group in the design of fluorescent Hybridization sensors. Molecular beacons (MBs) and binary Probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the Probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene Probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.
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fluorescent Hybridization Probes for sensitive and selective dna and rna detection
Accounts of Chemical Research, 2007Co-Authors: Angel A Marti, Steffen Jockusch, Nathan Stevens, Nicholas J TurroAbstract:We outline the different approaches taken by our group in the design of fluorescent Hybridization sensors. Molecular beacons (MBs) and binary Probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the Probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene Probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.
David A Stahl - One of the best experts on this subject based on the ideXlab platform.
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characterization of universal small subunit rrna Hybridization Probes for quantitative molecular microbial ecology studies
Applied and Environmental Microbiology, 1996Co-Authors: Dandan Zheng, David A Stahl, Elizabeth W Alm, Lutgarde RaskinAbstract:Universal oligonucleotide Hybridization Probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal Probes also serve to normalize results obtained with Probes targeting specific phylogenetic groups of microorganisms. In this study, six universal Probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all Probes. This could lead to a significant bias when these Probes are used to quantify microbial populations in environmental samples. We suggest lowering the postHybridization wash stringency for two of the universal Probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two Probes were evaluated with traditional and modified Hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.
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group specific 16s rrna Hybridization Probes to describe natural communities of methanogens
Applied and Environmental Microbiology, 1994Co-Authors: Lutgarde Raskin, J Stromley, Bruce E Rittmann, David A StahlAbstract:Eight oligonucleotides which are complementary to conserved tracts of 16S rRNA from phylogenetically defined groups of methanogens were designed and characterized for use as Hybridization Probes for studies in environmental and determinative microbiology. The target-group specificity and temperature of dissociation for each probe were characterized. In general, the Probes were very specific for the target methanogens and did not hybridize to the rRNAs of nontarget methanogens. Together, the eight Probes circumscribe methanogens now represented in pure culture (with the exception of members of the family Methanothermaceae). Three Probes are order specific; two identify members of the order Methanobacteriales, and one is specific for the order Methanococcales. The fourth probe encompasses three families belonging to the order Methanomicrobiales, the third order within the current classification. The fifth probe is specific for the remaining family within this order (Methanosarcinaceae). Three additional Probes encompass different genera within the Methanosarcinaceae. Images
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Genus- and group-specific Hybridization Probes for determinative and environmental studies of sulfate-reducing bacteria
Systematic and Applied Microbiology, 1992Co-Authors: Richard Devereux, Matthew D. Kane, Janet Winfrey, David A StahlAbstract:Summary A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as Hybridization Probes in determinative and environmental microbiology. Four Probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two Probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus mutlivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by Hybridizations against closely related nontargeted species. In addition, each probe was screened by using a “phylogrid” membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The application of these Probes to studies in environmental microbiology was evaluated by Hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.
Carl T. Wittwer - One of the best experts on this subject based on the ideXlab platform.
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genotyping of human platelet antigens 1 to 6 and 15 by high resolution amplicon melting and conventional Hybridization Probes
The Journal of Molecular Diagnostics, 2006Co-Authors: Michael Liew, Elaine Lyon, Lesa Nelson, Rebecca L Margraf, Sheri Mitchell, Maria Erali, Rong Mao, Carl T. WittwerAbstract:High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled Probes. Conventional melting analysis with Hybridization Probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods.
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Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes
Clinical chemistry, 2000Co-Authors: Philip S. Bernard, Carl T. WittwerAbstract:Over the past 5 years, there has been substantial progress in sequencing the human genome and identifying clinically significant genes (1). Genes that are clinically significant are diagnostic or prognostic for disease and/or helpful in guiding treatment. Unknown gene mutations, resulting from germline or somatic DNA alterations, are initially defined by direct sequencing. Other methods that detect specific mutations can then be used for higher throughput. Recently developed instrumentation and techniques for genotyping combine PCR and fluorescent Hybridization Probes for homogeneous amplification and product analysis within 1 h (2)(3)(4)(5). The target is amplified from genomic DNA by rapid-cycle PCR (6) with all the reagents needed for genotyping present from the beginning of the reaction. After 15–20 min, PCR is completed and the instrument automatically begins a melting curve protocol. Fluorescence is acquired continuously as the reaction is slowly heated and genotypes are identified by their characteristic melting curves. Because amplification and genotyping occur in the same instrument in a closed tube format, there is no concern of contamination by previously amplified product. Hybridization Probes are oligonucleotides that are singly labeled with a donor or acceptor fluorophore. During probe/target Hybridization, these fluorophores are brought into close proximity and fluorescence resonance energy transfer occurs. Two Hybridization probe schemes for fluorescent resonance energy transfer have been developed (3)(5). One method uses a 3′-labeled Hybridization probe designed to anneal to a PCR strand …
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Color multiplexing Hybridization Probes using the apolipoprotein E locus as a model system for genotyping.
Analytical biochemistry, 1999Co-Authors: Philip S. Bernard, Gregory Pritham, Carl T. WittwerAbstract:Abstract Fluorescent Hybridization Probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent Hybridization of 3′-fluorescein-labeled “donor” Probes paired with 5′-labeled “acceptor” Probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled Probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the Probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization Probes of the same length, but different GC content are Tm shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1°C/s) annealing phase. Although each site preferred different concentrations of Mg2+ and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.
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Rapid F508del and F508C Assay Using Fluorescent Hybridization Probes
Genetic testing, 1999Co-Authors: Cameron N. Gundry, Philip S. Bernard, Mark G. Herrmann, Gundrun H. Reed, Carl T. WittwerAbstract:Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The Hybridization of adjacent fluorescent Probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent Hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein Hybridization Probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator ge...
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homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent Hybridization Probes
American Journal of Pathology, 1998Co-Authors: Philip S. Bernard, Richard S Ajioka, James P Kushner, Carl T. WittwerAbstract:Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature ( T m ) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by T m for the first time. The new probe design uses a 3′-fluorescein-labeled probe and a 5′-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.
Elaine Lyon - One of the best experts on this subject based on the ideXlab platform.
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genotyping of human platelet antigens 1 to 6 and 15 by high resolution amplicon melting and conventional Hybridization Probes
The Journal of Molecular Diagnostics, 2006Co-Authors: Michael Liew, Elaine Lyon, Lesa Nelson, Rebecca L Margraf, Sheri Mitchell, Maria Erali, Rong Mao, Carl T. WittwerAbstract:High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled Probes. Conventional melting analysis with Hybridization Probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods.
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Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of Hybridization Probes
Clinical chemistry, 2005Co-Authors: Alison Millson, Genevieve Pont-kingdon, Sam Page, Elaine LyonAbstract:Background: Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with Hybridization Probes. Methods: We designed a LightCycler assay that uses fluorescence resonance energy transfer Hybridization Probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele. Results: Analysis of 132 samples revealed 9 unique derivative melting temperatures ( T ms); the lowest was 42.4 °C and the highest was 63.6 °C. The lowest T ms were in the TGn-9T group, the intermediate T ms in the TGn-7T group, and the highest T ms in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T. Conclusions: Different combinations of polymorphisms under the reporter Hybridization probe had unique and characteristic T ms. This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene.
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Mutation detection using fluorescent Hybridization Probes and melting curve analysis.
Expert review of molecular diagnostics, 2001Co-Authors: Elaine LyonAbstract:The LightCycler is a real-time PCR instrument that combines a thermocycler and a micro-volume fluorimeter. LightCycler technology is gaining popularity due to its ability to detect mutations quickly and accurately. Multiple base alterations are discriminated using Hybridization Probes and fluorescent melting curves. This review focuses on mutation detection and base discrimination by fluorescent Hybridization Probes. Assay designs for single base mutation detection and complex multiplex reactions are discussed. Types of mutations detected and reported applications are reviewed. Guidelines using melting curve analysis for the clinical laboratory are presented.
