Empirical establishment of oligonucleotide probe design criteriausing perfect match and mismatch probes and artificial targets
Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via anoligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of 85 percent for both 50-mer and 70-mer probes. PM signal intensities (33 to 48 percent) were detected at a probe-target identity of 94 percent for 50-meroligonucleotides and 43 to 55 percent for 70-mer probes at a probe-target identity of 96 percent. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15bases) contributed an additional 9 percent of the PM signal intensity compared to a nonstretch probe (15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55 percent of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than 30 kcal/mol for 50-mer probes or 40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.
- Research Organization:
- OLLABORATION - Oak Ridge NationalLaboratory
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 860356
- Report Number(s):
- LBNL-58739; AEMIDF; TRN: US200524%%109
- Journal Information:
- Applied and Environmental Microbiology, Vol. 71, Issue 7; Related Information: Journal Publication Date: July, 2005; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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