Chemiluminescent detection of DNA in low- and medium-density arrays.

Abstract

DNA microarray technology is revolutionizing the analysis of genetic information by enabling the high-throughput analysis of gene mutations and gene expression. This has been made possible by the development of technologies that can synthesize thousands of oligonucleotide probes in situ (1) , and high-speed robotic “printing” of cDNA probes (2) on a glass chip coupled with fluorescent labeling and detection of hybridized nucleic acids. The technologies involved in such analyses are out of the reach of low-budget research and clinical laboratories. Here we demonstrate the use of chemiluminescent detection in a simple and inexpensive method of detecting plasmid DNA and oligonucleotide arrays on nylon membranes and glass slides. A new method for rapid chemiluminescent detection of DNA arrays on nylon membranes and glass slides was developed using an ultrasensitive chemiluminescent reagent, LumigenTM APS-5 (Fig. 1⇓ A). The speed, sensitivity, and resolution of DNA spotted in low- and medium-density arrays was assessed with this reagent using several solid supports. Lumigen APS-5 Chemiluminescent Substrate (Lumigen, Inc.) is supplied as an optimized formulation containing the substrate Lumigen APS-5 and lucigenin in pH 8.8 Tris buffer. DNA was spotted manually with fine-tipped pipet prepared by drawing out a Pasteur pipet over a flame. Higher density arrays were applied under a dissecting microscope. Nylon membranes from several commercial suppliers were evaluated, and polylysine-coated glass slides were from Sigma Chemical Co. The spotting was done in rows or in 1 × 1 …