Empirical establishment of oligonucleotide probe design criteria

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 3753–3760 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.7.3753–3760.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 7

Empirical Establishment of Oligonucleotide Probe Design Criteria† Zhili He,1 Liyou Wu,1 Xingyuan Li,2 Matthew W. Fields,3 and Jizhong Zhou1* Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 378311; Harbin Institute of Technology, Harbin, Heilongjiang, China 1500012; and Department of Microbiology, Miami University, Oxford, Ohio 450563 Received 16 September 2004/Accepted 6 January 2005

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 15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (