An improved gel‐based DNA microarray method for detecting single nucleotide mismatch

Abstract

3-D polyacrylamide gel-based DNA microarray platforms provide a high capacity for nucleic acids immobilization and a solution-mimicking environment for hybridization. However, several technological bottlenecks still remain in these platforms, such as difficult microarray preparation and high fluorescent background, which limit their application. In this study, two new approaches have been developed to improve the convenience in microarray preparation and to reduce the background after hybridization. To control the polymerization process, solutions containing acrylamide-modified oligonucleotide, acrylamide, glycerol and ammonium persulfate are spotted onto a functionalized glass slide, and then the slide is transferred to a vacuum chamber with TEMED, so that TEMED is vaporized and diffused into the spots to induce polymerization. By applying an electric field across a hybridized microarray to remove the nonspecifically bound labeled targets, this approach can solve the problem of high fluorescent background of the gel-based microarray after hybridization. Experimental results show that our immobilization method can be used to construct high quality microarrays and exhibits good reproducibility. Moreover, the polymerization is not affected by PCR medium, so that PCR products can be used for microarray construction without being treated by commercial purification cartridges. Electrophoresis can improve the signal-to-noise significantly and has the ability to differentiate single nucleotide variation between two homozygotes and a heterozygote. Our results demonstrated that this is a reliable novel method for high-throughput mutation analysis and disease diagnosis.

Predictive Effect of XPA and XPD Polymorphisms on Survival of Advanced NSCLC Patients Treated with Platinum-Based Chemotherapy: A Three-Dimensional (3-D), Polyacrylamide Gel-Based DNA Microarray Method

“…The genotyping procedure involved polymerase chain reaction (PCR) and the three-dimensional (3-D) polyacrylamide gel-based DNA microarray method (23). Probes and primers were designed by Primer Premier 5.0 software as previously described (20,21).…”

XPA A23G polymorphism is associated with the elevated response to platinum-based chemotherapy in advanced non-small cell lung cancer

“…Solutions containing acrylamide-modified PCR products, glycerol, ammonium persulfate (APS), and acrylamide monomers were prepared, spotted, and polymerized onto the acryl-modified slide. In the process, TEMED (tetramethylethylenediamine) is introduced onto the spotted microarray to immobilize the modified nucleic acids [16,17]. Following the attachment, to obtain ssDNA for hybridization analysis, dsDNA on the slides was denatured in 0.1 M NaOH for 10 min.…”

Detection of single tumor cell resistance with aptamer biochip

“…The whole process was similar to the previous step. In previous studies (28,29), electrophoresis was used to detect SNP and DNA methylation. Single-base mismatches could be distinguished from complete matches.…”