The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization
1 Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Charlotte, NC, USA
2 Bioinformatics Services Division, University of North Carolina at Charlotte, North Carolina Research Campus, Kannapolis, NC, USA
3 Greenwood Genetic Center, Greenwood, SC, USA
Citation and License
BMC Research Notes 2013, 6:72 doi:10.1186/1756-0500-6-72Published: 27 February 2013
Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length.
Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide.
Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative.