TITLE

Secondary structure in the target as a confounding factor in synthetic oligomer microarray design

AUTHOR(S)
Ratushna, Vladyslava G.; Weller, Jennifer W.; Gibas, Cynthia J.
PUB. DATE
January 2005
SOURCE
BMC Genomics;2005, Vol. 6, p31
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Background: Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment. Results: Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridization due to the formation of stable intramolecular secondary structure. For example, at 65°C, 28 ± 7% of the average cDNA target sequence is predicted to be inaccessible to hybridization. We also analyzed the specific binding sites of a set of 70mer probes previously designed for Brucella using a freely available oligo design software package. 21 ± 13% of the nucleotides in each probe binding site are within a double-stranded structure in over half of the folds predicted for the cDNA target at 65°C. The intramolecular structures formed are more stable and extensive when an RNA target is modeled rather than cDNA. When random shearing of the target is modeled for fragments of 200, 100 and 50 nt, an overall destabilization of secondary structure is predicted, but shearing does not eliminate secondary structure. Conclusion: Secondary structure in the target is pervasive, and a significant fraction of the target is found in double stranded conformations even at high temperature. Stable structure in the target has the potential to interfere with hybridization and should be a factor in interpretation of microarray results, as well as an explicit criterion in array design. Inclusion of this property in an oligonucleotide design procedure would change the definition of an optimal oligonucleotide significantly.
ACCESSION #
28858472

 

Related Articles

  • Expression profiling using cDNA microarrays. Duggan, David J; Bittner, Michael; Chen, Yidong; Meltzer, Paul; Trent, Jeffrey M. // Nature Genetics;Jan99 Supplement, Vol. 21, p10 

    cDNA microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment. DNA targets, in the form of 3´ expressed sequence tags (ESTs), are arrayed onto glass slides (or membranes) and probed with fluorescent? or radioactively?labelled cDNAs....

  • Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios. Dorris, David R.; Nguyen, Allen; Gieser, Linn; Lockner, Randall; Lublinsky, Anna; Patterson, Marcus; Touma, Edward; Sendera, Timothy J.; Elghanian, Robert; Mazumder, Abhijit // BMC Biotechnology;2003, Vol. 3, p6 

    Background: DNA microarrays are now routinely used to monitor the transcript levels of thousands of genes simultaneously. However, the array fabrication method, hybridization conditions, and oligodeoxyribonucleotide probe length can impact the performance of a DNA microarray platform. Results:...

  • In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip technology. Grigoryev, Dmitry N; Ma, Shwu-Fan; Simon, Brett A; Irizarry, Rafael A; Ye, Shui Q; Garcia, Joe GN // BMC Genomics;2005, Vol. 6, p1 

    Background: Genomic approaches in large animal models (canine, ovine etc) are challenging due to insufficient genomic information for these species and the lack of availability of corresponding microarray platforms. To address this problem, we speculated that conserved interspecies genetic...

  • Going global. Phimister, Bette // Nature Genetics;Jan99 Supplement, Vol. 21, p1 

    Editorial. Comments on the use of a systematic global strategy in DNA microarrays. Lack of appreciation on the part of researchers of the different types of microarray and their manufacture and processing; Factors critical to the success of microarray analysis.

  • Single feature polymorphisms (SFPs) for drought tolerance in pigeonpea ( Cajanus spp.). Saxena, Rachit; Cui, Xinping; Thakur, Vivek; Walter, Barbara; Close, Timothy; Varshney, Rajeev // Functional & Integrative Genomics;Nov2011, Vol. 11 Issue 4, p651 

    Single feature polymorphisms (SFPs) are microarray-based molecular markers that are detected by hybridization of DNA or cRNA to oligonucleotide probes. With an objective to identify the potential polymorphic markers for drought tolerance in pigeonpea [ Cajanus cajan (L.) Millspaugh], an...

  • Validation of the Agilent 244K oligonucleotide array-based comparative genomic hybridization platform for clinical cytogenetic diagnosis. Shihui Yu; Douglas C Bittel; Nataliya Kibiryeva; David L Zwick; Linda D Cooley // American Journal of Clinical Pathology;Sep2009, Vol. 132 Issue 3, p349 

    High-resolution microarray comparative genomic hybridization (aCGH) is being adopted for diagnostic evaluation of genomic disorders, but validation for clinical diagnosis has not yet been reported. We present validation data for the Agilent Human Genome Microarray Kit 244K for clinical...

  • Mathematical tools to optimize the design of oligonucleotide probes and primers. Noguera, Daniel; Wright, Erik; Camejo, Pamela; Yilmaz, L. // Applied Microbiology & Biotechnology;Dec2014, Vol. 98 Issue 23, p9595 

    The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with high specificity and sensitivity. The design of these oligonucleotides (or 'oligos' for short) shares many of the same...

  • Microarray probe selection strategies. Tomiuk, Stefan; Hofmann, Kay // Briefings in Bioinformatics;Dec2001, Vol. 2 Issue 4, p17 

    During recent years, DNA microarrays have become the method of choice to monitor the expression level of a large number of genes. Depending on the focus of the study and the method of microarray fabrication, a number of different strategies for probe selection may be most appropriate. One...

  • Widespread occurrence of antisense transcription in the human genome. Yelin, Rodrigo; Dahary, Dvir; Sorek, Rotem; Levanon, Erez Y.; Goldstein, Orly; Shoshan, Avi; Diber, Alex; Biton, Sharon; Tamir, Yael; Khosravi, Rami; Nemzer, Sergey; Pinner, Elhanan; Walach, Shira; Bernstein, Jeanne; Savitsky, Kinneret; Rotman, Galit // Nature Biotechnology;Apr2003, Vol. 21 Issue 4, p379 

    An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify...

Share

Read the Article

Courtesy of THE LIBRARY OF VIRGINIA

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics