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Molecular BioSystems Vol - 8 Issue - 3

Molecular BioSystems Vol - 8 Issue - 2

Molecular BioSystems Vol -7

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Molecular BioSystems Vol -5

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Molecular BioSystems Vol -3

Molecular BioSystems Vol -2

Analysis of crucial factors resulting in microarray hybridization failure

Content Provider	Royal Society of Chemistry (RSC)
Author	Blohm, Dietmar Liu, Jue Armstrong, Karen Wei, Ting Pearson, Mike N.
Copyright Year	2012
Abstract	The factors that affect the formation and stability of DNA/DNA duplexes are complicated and still mostly unknown. In this study attempts were made to look for the crucial factor affecting hybridization failure in DNA microarray assays. A comprehensive range of factors were investigated simultaneously using a 25-mer oligonucleotide Potyvirus microarray. These included steric hindrance, direct/indirect labelling types, distance of a probe to the fluorescent labelling end, target (the DNA fragment used to hybridize with microarray probes) strand types either single strand or double strand, probes without mismatch and with different numbers of mismatch nucleotides (up to 36%) and different mismatch locations (5′ end, centre and 3′ end), probe GC content and Tm, secondary structures of probes and targets, different target lengths (0.277 kb to ∼1.3 kb) and concentrations (0.1–30 nM). The results showed that whilst most of these known factors were unlikely to be the main causes of failed hybridization, there was strong evidence suggesting that the viral amplicon target structure is the most crucial factor. However, computing predicted target secondary structures by Mfold showed no correlation with the hybridization results. One explanation is that the predicted target secondary structures are different from the real structures. Here we postulate that the real target structure might be a combination of secondary structures resulting in a three-dimensional structure from exposure to three types of sub-structures: (1) a completely exposed linear structure to allow probes access for the successful hybridization and showing strong fluorescent signals; (2) a partially exposed structure to allow unstable binding and showing weak fluorescent signals; (3) a closed structure resulting in failed hybridization. These results are very important for microarray based studies as they not only provide an explanation for some current controversial results, but also provide potential resolution for the future studies. Due to the lack of available software for predicting the true target structure, development of microarrays should conduct an initial oligonucleotide probe selection procedure and those probes with capacity to hybridize with the target should be considered for the microarray development.
Starting Page	1325
Ending Page	1338
Page Count	14
File Format	HTM / HTML PDF
ISSN	1742206X
Volume Number	8
Issue Number	4
Journal	Molecular BioSystems
DOI	10.1039/c2mb05300d
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	DNA microarray Mfold Amplicon DNA Oligonucleotide Potyvirus Steric effects GC-content
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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