Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

Abstract

BackgroundThe performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats.ResultsA low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface.ConclusionsThese results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves.

Development of a DNA Microarray-Based Assay for the Detection of Sugar Beet Root Rot Pathogens

“…Hybridization signals could be further improved by excluding the competition with the complementary strand via asymmetric PCR. However, Tomlinson et al (2014) could show that this approach can be less discriminatory when SNP detection is required.…”

“…Despite low target sequence variability, a single mismatch in principle is sufficient for discrimination of two amplicons as it was demonstrated for the probe Bot_G_2, differentiating B. cinerea from S. sclerotiorum. However, the success depends on many parameters like the kind of mismatch, mismatch position and probe length (Lievens et al 2006;Seringhaus et al 2008;Tomlinson et al 2014). According to the aforementioned studies, best differentiation can be expected when amplicons are short with staggered positioned mismatches, resulting in purine-purine mispairs.…”

“…Recently, Dobnik et al (2014) utilized this platform for the detection of two important quarantine plant pathogens, namely Potato spindle tuber viroid and Ralstonia solanacearum. In contrast to previously applied array technologies (Chen et al 2009(Chen et al , 2013Fessehaie et al 2003;Lievens et al 2003;Tambong et al 2006;Zhang et al 2007), all working steps are conducted in one vessel using standard laboratory equipment, and can be completed in less than 3 h including signal analysis (Tomlinson et al 2014). The workflow of the developed microarray comprised two main steps: (i) exponential amplification of different marker genes followed by (ii) a hybridization between species-specific oligonucleotide probes and target amplicons enabling multiplex detection.…”

The effect of unequal strand length on short DNA duplex hybridization in a model microarray system: A Monte Carlo simulation study

“…mutations) in the sequence, have variable ability to discriminate between targets depending upon how a longer (ca. 400 bases) target's dangling ends are oriented, seeing greater hybridization efficiency when the target's dangling end 2 is primarily at the surface proximal end of the probe-target duplex; more recently Tomlinson et al found similar results [13].…”

Dynamic range extension of hybridization sensors

“…The focus of this manuscript is on hybridization techniques which are appealing due to simplicity: a nucleic acid probe is designed to hybridize to the perfectly matching target molecule with a high efficiency while having a low efficiency for targets 10 containing sequence variations. The principle is open to many technical implementations and miniaturization for use in biosensor devices [1,2,3,4,5,6]. However, hybridization has challenges on the side of probe design and dynamic range due to hybridization stringency conditions [7,8,9] which are not easy to optimize especially when parallelization is aimed for.…”