Abstract
The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients.
Highlights
Microarray analysis is generally performed using a single working condition, and all probes must exhibit the same thermodynamic behavior in order to operate optimally in the array
A slide hybridized with amplified and labeled RNA, which had been derived from a subject heterozygous at position CD8/9+G, was mounted in the multi-stringency array washer (MSAW), and the individual subarrays were washed for 30 min at 37uC with the eight buffers described in Materials and Methods
Our goal was to determine whether a sodium concentration gradient could be used instead of a temperature gradient to investigate probe characteristics in order to avoid numerous practical difficulties associated with the thermal approach
Summary
Microarray analysis is generally performed using a single working condition (i.e., one hybridization or stringency washing temperature), and all probes must exhibit the same thermodynamic behavior in order to operate optimally in the array. Besides being an invaluable decision tool for assay developers, melting curves can be of direct use in applications where high specificity is paramount (e.g., when analyzing single nucleotide polymorphisms, SNPs) [3,4]. Other applications that can benefit from relaxed restriction on Tm matching of probes are sequencing by hybridization, microRNA analysis [6,7], tiling arrays [8,9], ‘‘exon’’ arrays [10], and even gene expression profiling [11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
