Impact of point-mutations on the hybridization affinity of surface-bound DNA-DNA and RNA-DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulgesReportar como inadecuado




Impact of point-mutations on the hybridization affinity of surface-bound DNA-DNA and RNA-DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Biotechnology

, 8:48

First Online: 13 May 2008Received: 10 January 2008Accepted: 13 May 2008

Abstract

BackgroundThe high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch MM discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.

ResultsWe performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths in situations without competitive hybridization in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect MM or bulge type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA-DNA and RNA-DNA duplexes shows, that RNA-DNA purine-purine MMs are more discriminating than corresponding DNA-DNA MMs. In DNA-DNA MM discrimination the affected base pair C·G vs. A·T is the pertinent parameter. We attribute these differences to the different structures of the duplexes A vs. B form.

ConclusionWe have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-8-48 contains supplementary material, which is available to authorized users.

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Autor: Thomas Naiser - Oliver Ehler - Jona Kayser - Timo Mai - Wolfgang Michel - Albrecht Ott

Fuente: https://link.springer.com/article/10.1186/1472-6750-8-48



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