Empirical establishment of oligonucleotide probe design criteria.
Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide 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 < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =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% 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.
Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038, USA.
Zhili
Z
He
Liyou
L
Wu
Xingyuan
X
Li
Matthew W
MW
Fields
Jizhong
J
Zhou
D020137:Base Pair Mismatch; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000235:genetics*; D012107:Research Design / Q000592:standards*; D020592:Shewanella / Q000235:genetics; D013045:Species Specificity
D015345:Oligonucleotide Probes