DNA Microarray Probe Preparation by Gel Isolation Nested PCR

Abstract

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

DNA microarray analysis of the gene expression profile of kidney tissue in a type 2 diabetic rat model

“…The fluorescent-labeled cDNA probes were prepared through retro-transcription according to the method of Wang et al (5). The cDNA probes from the normal kidney tissue were labeled with Cy3-dCTP, while those from the T2D rat kidney tissue were labeled with Cy5-dCTP.…”