title: Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance.
abstract: Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes; TaqMan-probes) of variable genomes, such as HIV-1, can result in underestimation of viral copy numbers due to mismatches in the FH-probe's target sequences. Therefore both target conservation and physical properties of FH-probes, such as melting temperature, baseline fluorescence and secondary structure, should be considered in design of FH-probes. Analysis of a database of 1242 near full-length HIV-1 sequences with a novel computational tool revealed that the probability of target and FH-probe identity decreases exponentially with FH-probe length. In addition, this algorithm allowed for identification of continuous sequence stretches of high conservation, from which FH-probes with global HIV-1 clade coverage could be chosen. To revise the prerequisites of physical FH-probe function, properties of 30 DNA and 21 chimeric DNA locked nucleic acid (DLNA) HIV-1 FH-probes were correlated with their performance in qPCR. This identified the presence of stable secondary structures within FH-probes and the base composition and thermal stability of the 5' proximal end as novel predictors of FH-probe performance. Thus, empirically validated novel principles of FH-probe design regarding conservation and qPCR-performance were identified, which complement and extend current rules for FH-probe design.
authors.affiliation: Division of Infectious Diseases, University Hospital Zurich, University of Zurich, Zurich, Switzerland. claudia.althaus@usz.ch
authors.forename: Claudia F
authors.initials: CF
authors.lastname: Althaus
authors.forename: Sara
authors.initials: S
authors.lastname: Gianella
authors.forename: Philip
authors.initials: P
authors.lastname: Rieder
authors.forename: Viktor
authors.initials: V
authors.lastname: von Wyl
authors.forename: Roger D
authors.initials: RD
authors.lastname: Kouyos
authors.forename: Barbara
authors.initials: B
authors.lastname: Niederöst
authors.forename: Adrian
authors.initials: A
authors.lastname: Schmid
authors.forename: Karin J
authors.initials: KJ
authors.lastname: Metzner
authors.forename: Beda
authors.initials: B
authors.lastname: Joos
authors.forename: Huldrych F
authors.initials: HF
authors.lastname: Günthard
authors.forename: Marek
authors.initials: M
authors.lastname: Fischer
mesh_terms: D001483:Base Sequence; D005456:Fluorescent Dyes / Q000737:chemistry*; D014644:Genetic Variation; D016679:Genome, Viral / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; C477371:locked nucleic acid