Experimental optimization of probe length to increase the sequence specificity of high-density oligonucleotide microarrays

Shingo Suzuki,Tetsuya Yomo,Naoaki Ono,Chikara Furusawa,Akiko Kashiwagi

doi:10.1186/1471-2164-8-373

Abstract

BackgroundHigh-density oligonucleotide arrays are widely used for analysis of genome-wide expression and genetic variation. Affymetrix GeneChips – common high-density oligonucleotide arrays – contain perfect match (PM) and mismatch (MM) probes generated by changing a single nucleotide of the PMs, to estimate cross-hybridization. However, a fraction of MM probes exhibit larger signal intensities than PMs, when the difference in the amount of target specific hybridization between PM and MM probes is smaller than the variance in the amount of cross-hybridization. Thus, pairs of PM and MM probes with greater specificity for single nucleotide mismatches are desirable for accurate analysis.ResultsTo investigate the specificity for single nucleotide mismatches, we designed a custom array with probes of different length (14- to 25-mer) tethered to the surface of the array and all possible single nucleotide mismatches, and hybridized artificially synthesized 25-mer oligodeoxyribonucleotides as targets in bulk solution to avoid the effects of cross-hybridization. The results indicated the finite availability of target molecules as the probe length increases. Due to this effect, the sequence specificity of the longer probes decreases, and this was also confirmed even under the usual background conditions for transcriptome analysis.ConclusionOur study suggests that the optimal probe length for specificity is 19–21-mer. This conclusion will assist in improvement of microarray design for both transcriptome analysis and mutation screening.

Highlights

High-density oligonucleotide arrays are widely used for analysis of genome-wide expression and genetic variation
To improve the measurement of target amounts using the pairs of perfect match (PM) and MM probes, one possible strategy is to enhance the specificity for single nucleotide mismatches, i.e., changes in signal intensity caused by a single nucleotide mismatch
Design of the array To investigate the effects of probe length and position of mismatch for target-specific hybridization comprehensively on a high-density oligonucleotide array, we designed a custom array on which a number of probes were arranged in length, mismatch position, and types of mismatched nucleotide, using Maskless Array Synthesizer

Summary

Introduction

High-density oligonucleotide arrays are widely used for analysis of genome-wide expression and genetic variation. Affymetrix GeneChips – common high-density oligonucleotide arrays – contain perfect match (PM) and mismatch (MM) probes generated by changing a single nucleotide of the PMs, to estimate cross-hybridization. It has been pointed out that around 30% of probe pairs consistently give negative signals, which means that the difference between PM and MM probe intensity does not always reflect the true target amounts [5,6] This contradiction of PM and MM probe intensities is the main factor making expression analysis unreliable, especially when the target concentration is low. Such contradictions will occur when, for example, the difference in the amount of target specific hybridization between PM and MM probes is smaller than the variance in the amount of cross-hybridization. The specificity is important for analysis of single nucleotide polymorphisms (SNPs) using microarray technology [10,11]

Methods

Results

Discussion

Conclusion