Microarray oligonucleotide probes.
Oligonucleotide probes are increasingly the method of choice for many modern DNA microarray applications. They provide higher target specificity, probe selection gives improved experimental control of hybridization properties, and targeting of specific gene subsequences allows better discrimination of highly similar targets such as splice variants or gene families. Only recently has there been substantial progress in dealing with the complexities of probe set design and probe-specific signal interpretation. After a discussion of advantages and disadvantages of oligonucleotide probes in comparison to amplicons, this chapter focuses on recent advances and remaining key challenges in probe design and computational data analysis for spotted and in situ-synthesized oligonucleotide microarray technologies. Both experimental questions and computational aspects are addressed. Experimental issues discussed include the choice of an optimal number of probes per target and probe lengths and their influence on bias and random measurement noise, effects of different probe or substrate modifications, and laboratory protocols on signal specificity and sensitivity. Computational topics include practical considerations and a case study in probe sequence design, the exploitation of probing multiple target regions, and the modeling of probe sequence-specific signals. The current state of the art of the field is examined, and principled thermodynamic probe design criteria are proposed that are based on the free energy of the probe-target complex at the hybridization temperature rather than its melting temperature. Finally, this chapter notes and discusses an emerging trend in recent computational work toward a focus on signal interpretation rather than probe sequence design.
Kreil
David P
DP
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Russell
Roslin R
RR
Russell
Steven
S
D000818:Animals; D006801:Humans; D015335:Molecular Probes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides
D015335:Molecular Probes; D009841:Oligonucleotides