Abstract
BackgroundHybridization 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.ResultsSolution 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.ConclusionsMicroarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative.
Highlights
Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target
DNA microarrays remain a popular technology for measuring gene expression and other global properties of the genome, with over 2200 experiments representing tens of thousands of samples published in ArrayExpress [1,2] so far in 2012
To generate a representative probe-target pair for this experiment, we designed a set of probes for the E. coli genome using commonly-used array design applications to screen sequences for uniqueness and thermodynamic uniformity, as described in the Methods
Summary
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. 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 crosshybridization. Cross-hybridization is defined as a specific side reaction between a probe and an unintended target to form a stable duplex, and microarray design pipelines generally attempt to avoid this either by screening for defined levels of sequence complementarity, or by using a thermodynamic cutoff, though in the latter cases, sequence complementarity is often used as a prescreen
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