New protocol for oligonucleotide microarray fabrication using SU-8-coated glass microslides.
Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).
Sethi
D
D
Indian Institute of Toxicology Research (CSIR), Mahatma Gandhi Marg, Lucknow, India.
Kumar
A
A
Gandhi
R P
RP
Kumar
P
P
Gupta
K C
KC
D020137:Base Pair Mismatch / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D001559:Benzhydryl Compounds; D004852:Epoxy Compounds / Q000737:chemistry*; D005898:Glass / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016920:Meningitis, Bacterial / Q000175:diagnosis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry; D010766:Phosphorylation; D013696:Temperature; D014435:Typhoid Fever / Q000175:diagnosis; D000098826:Bisphenol A Compounds
D001559:Benzhydryl Compounds; D004852:Epoxy Compounds; D009841:Oligonucleotides; D010636:Phenols; C020344:glycidyl ethers; C006780:bisphenol A; D000098826:Bisphenol A Compounds