The forgotten variables of DNA array hybridization

Carletti, Erminia; Guerra, Emanuela; Alberti, Saverio

doi:10.1016/j.tibtech.2006.07.006

Cited by 24 publications

(15 citation statements)

References 52 publications

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“…Besides platform-specific issues, which require experimental validation, reduced tiling densities may represent a platform-independent approach to efficiently capture long templates for NGS technologies that generate long sequence reads. Because of their lower diffusion rates and increased intramolecular base pairing, the hybridization of long DNA fragments to capture probes is expected to be inferior to that of short fragments (30 ). Use of a high tiling density for target enrichment may therefore produce a general bias toward shorter captured fragments, reducing the overall sequence read length.…”

Section: Reduced Tiling Density and The Advantage Of Long Read Lengthsmentioning

confidence: 99%

Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

Amstutz

Andrey-Zürcher

Suciu³

et al. 2011

Clinical Chemistry

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BACKGROUND Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples. METHODS We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample. RESULTS Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples. CONCLUSIONS The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.

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Section: Reduced Tiling Density and The Advantage Of Long Read Lengthsmentioning

confidence: 99%

Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

Amstutz

Andrey-Zürcher

Suciu³

et al. 2011

Clinical Chemistry

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“…In bulk solution the reaction may be viewed as being second order where the rate is dependent on the solvent, length of strand and complexity of the sequence (Wetmur, 1991). For larger DNA sequences the time for hybridisation equilibrium (T 1/2 ) can be very long with T 1/2 for a target human genome being 41 days (Carletti et al, 2006). Even with shorter probe molecules the time required to reach equilibrium may be in the region of 1-2 days (Halperin et al, 2006).…”

Section: Thermodymanic and Kinetic Propertiesmentioning

confidence: 99%

The physicochemical aspects of DNA sensing using electrochemical methods

Batchelor‐McAuley

Wildgoose

Compton

2009

Biosensors and Bioelectronics

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“…DNA probes showed a more dramatic response to the ionic effect, and duplex formation decreased rapidly at rather high (150 mM) sodium concentration. Other factors influencing microarray hybridization signal include spot size (25), probe length and G + C content (2,26,27), target length (28), concentration (29) and complexity (30), labeling of target (31), temperature and composition of hybridization buffer (2,26) and/or stringency wash temperature (32) and buffer (Poulsen et al , unpublished data). Whereas signal intensities generally increase with probe length (2,29), there is an inverse relation between probe length and specificity (27,29).…”

Section: Introductionmentioning

confidence: 99%

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Poulsen

Søe

Snakenborg

et al. 2008

Nucleic Acids Research

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Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis.

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The forgotten variables of DNA array hybridization

Cited by 24 publications

References 52 publications

Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

The physicochemical aspects of DNA sensing using electrochemical methods

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

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