Construction of oligonucleotide microarrays (biochips) via thioether linkage for the detection of bacterial meningitis.
Oligonucleotide-based arrays are increasingly becoming useful tools for the analysis of gene expression and single-nucleotide polymorphism. Here, we report a method that allows the direct immobilization of thiolated oligonucleotides onto an epoxy-activated glass surface via a stable thioether linkage under microwaves. The described chemistry efficiently immobilizes the probes via terminal thiol groups with uniform spot morphology. The thioether linkage could endure repeated PCR-like heat cycling with only 2.5% loss after 20 cycles, indicating that the chemistry can be used in integrated PCR/microarray devices. The highlighting feature of the proposed method is that the detection limit for the probe concentration can be reduced to 0.25 microM with 20-mer oligonucleotides. The efficiency of the projected method (approximately 33%) indicates its advantage over the existing standard methods, viz., NTMTA (approximately 9.8%), epoxide-amine (approximately 9.8%) and disulfide (approximately 1.7%). The constructed microarrays were validated through the detection of base mismatches and bacterial meningitis. These features make the projected strategy ideal for manufacturing oligonucleotide arrays and detection of mismatches and bacterial diseases.
Mahajan
S
S
Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi University Campus, Delhi-110 007, India.
Sethi
D
D
Seth
S
S
Kumar
A
A
Kumar
P
P
Gupta
K C
KC
D004867:Equipment Design; D005898:Glass; D016920:Meningitis, Bacterial / Q000175:diagnosis*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry*
D009841:Oligonucleotides; D013440:Sulfides