Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-33618
Althaus, C F; Gianella, S; Rieder, P; von Wyl, V; Kouyos, R D; Niederöst, B; Schmid, A; Metzner, K J; Joos, B; Günthard, H F; Fischer, M (2010). Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance. Journal of Virological Methods, 165(2):151-160.
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.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||04 Faculty of Medicine > University Hospital Zurich > Clinic for Infectious Diseases|
|DDC:||610 Medicine & health|
|Deposited On:||22 Apr 2010 11:05|
|Last Modified:||27 Nov 2013 20:37|
|Citations:||Web of Science®. Times cited: 13|
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