Fluorotyping of HLA-A by sequence-specific priming and fluorogenic probing.
AuthorsSlateva K, Camps MA, Blasczyk R
JournalTissue Antigens
PubMed ID9864036
'The aim of our study was to develop a fluorotyping strategy for the HLA-A locus. In contrast to conventional sequence-specific primed PCR (PCR-SSP), which is based on an agarose gel electrophoresis, fluorotyping eliminates the drawback of low sample throughput, low potential for automation and problems related to contamination. Additionally, fluorotyping ... More
High-resolution typing for HLA-DRB1*15 amd -DRB1*16 by fluorescence-marked sequence-specific priming (TaqMan assay).
AuthorsTremmel M, Opelz G, Mytilineos J
JournalTissue Antigens
PubMed ID10599890
'Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories. The TaqMan method, which is described here for high-resolution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventional PCR-SSP and fluorescence detection of the amplicon with a doubly ... More
High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR).
'We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. ... More
Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing.
AuthorsAlbis-Camps M, Blasczyk R
JournalTissue Antigens
PubMed ID10203025
'Similar to our recently described HLA-A and -C fluorotyping strategies, the aim of this study was to develop a sequence-specific primed polymerase chain reaction (PCR-SSP)-based fluorotyping method for HLA-DRB. Applying the fluorogenic 5'' nuclease assay, it is possible to increase the sample throughput rate by abolishing all labor-intensive post-amplification steps. ... More
Fluorotyping of HLA-C: differential detection of amplicons by sequence-specific priming and fluorogenic probing.
AuthorsLuedeck H, Blasczyk R
JournalTissue Antigens
PubMed ID9458116
'Conventional PCR-SSP, which is based on an agarose gel-based read-out, has the disadvantages of time-consuming post-PCR steps and low potential for automation. The aim of our study was to sort out these drawbacks by establishing a fluorescence-based PCR-SSP system for HLA-C. The assay relies on the sequence-specific identification of amplicons ... More
Factor V Leiden genotyping using real-time fluorescent polymerase chain reaction.
AuthorsSanders Sevall J
JournalMol Cell Probes
PubMed ID10970729
'A fluorogenic probe-based PCR assay (Taqman; Perkin Elmer corp/Applied Biosystems, Foster City, CA, USA) was used for the detection of Factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for Deep Vein Thrombosis. This assay allows for the direct ... More
A novel method for real time quantitative RT-PCR.
AuthorsGibson UE, Heid CA, Williams PM
JournalGenome Res
PubMed ID8908519
'A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5'' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the ... More
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
AuthorsMarras SA, Kramer FR, Tyagi S
JournalNucleic Acids Res
PubMed ID12409481
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We ... More
Chromosome-specific panels of tri- and tetranucleotide microsatellite markers for multiplex fluorescent detection and automated genotyping: evaluation of their utility in pathology and forensics.
A set of 391 microsatellite markers (Weber set 6), 85% of which consist of tri- and tetranucleotide repeat markers, were used to design chromosome-specific panels that allowed for a high degree of multiplexing with respect to the fragment size range and fluorophore (FAM, HEX, TET). This marker set has an ... More
Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR.
AuthorsEun AJ, Seoh M, Wong S
JournalJ Virol Methods
PubMed ID10856762
Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan((R)) real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan((R)) probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat ... More
Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction--amplified short tandem repeats during denaturing capillary electrophoresis.
AuthorsHahn M, Wilhelm J, Pingoud A
JournalElectrophoresis
PubMed ID11545394
The determination of the length of polymerase chain reaction (PCR)-amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye-specific mobility anomalies exist for 5'-fluorophor-labelled single-stranded DNA (ssDNA) fragments in CE using the performance-optimized polymer 4 (POP4) buffer sieving ... More
Rapid identification of up to three Candida species in a single reaction tube by a 5' exonuclease assay using fluorescent DNA probes.
AuthorsShin JH, Nolte FS, Holloway BP, Morrison CJ
JournalJ Clin Microbiol
PubMed ID9854084
We used fungus-specific PCR primers and species-specific DNA probes to detect up to three Candida species in a single reaction tube by exploiting the 5' to 3' exonuclease activity of Taq DNA polymerase. Probes to the internal transcribed spacer region of the rRNA gene were labeled at the 5' end ... More