The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

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.

Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

“…Our overall aim was to capture distantly related exogenous retroviral sequences in complex samples from humans. Even short complementary regions may mediate hybridization between capture probes and targets 18 19 within the host genome e.g. orthologous genes and human endogenous retrovirus (HERV) sequences.…”

“…Most methods are dependent on stringent reaction conditions for discrimination between correct target and competing irrelevant sequences with varying similarity. Kane et al established that cross hybridization may happen if nucleotide sequence similarity exceeds around 75% 18 , unless carefully controlled 19 . Matching stretches of as little as 12–15 complementary nucleotides are sufficient to mediate unspecific cross-hybridization of 50-bp oligonucleotides 18 19 .…”

“…Kane et al established that cross hybridization may happen if nucleotide sequence similarity exceeds around 75% 18 , unless carefully controlled 19 . Matching stretches of as little as 12–15 complementary nucleotides are sufficient to mediate unspecific cross-hybridization of 50-bp oligonucleotides 18 19 . The risk of cross-hybridization has prompted researchers and manufacturers to maximize stringency during capture, including tightly controlled reaction conditions involving denaturing compounds (e.g.…”

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

“…Such duplexes formed when only 12 bp of complementary sequence were present. Surface hybridization experiments have confirmed that a signal indistinguishable from a full-strength signal arising from a low copy number PM can be obtained from sequences that form only a partial duplex (63). …”

Sequence characteristics define trade-offs between on-target and genome-wide off-target hybridization of oligoprobes

“…A number of studies have analyzed factors that influence genome-wide cross-hybridization levels of microarray probes. Duplexes of 10–16 nucleotides that are complementary to targets may be sufficient to generate a cross-hybridization signal [ 27 – 28 ]. For 50-nt probes in particular, it was noted that “a complementary stretch of nucleotides as short as 12 nucleotides may result in the appearance of significant signal from an unintended binding partner” [ 28 ].…”