Fabrication of DNA microarrays using unmodified oligonucleotide probes.
Microarrays printed on glass slides are often constructed by covalently linking oligonucleotide probes to a derivatized surface. These procedures typically require relatively expensive amine- or thiol-modified oligonucleotide probes that add considerable expense to larger arrays. We describe a system by which unmodified oligonucleotide probes are bound to either nonderivatized or epoxy-silane-derivatized glass slides. Biotinylated PCR products are heat denatured, hybridized to the arrays, and detected using an enzymatic amplification system. Unmodified probes appear to detach from the slide surface at high pH (> 10.0), suggesting that hydrogen bonding plays a significant role in probe attachment. Regardless of surface preparation, high temperature (up to 65 degrees C) and low ionic strength (deionized water) do not disturb probe attachment; hence, the fabrication method described here is suitable for a wide range of hybridization stringencies and conditions. We illustrate kinetics of room temperature hybridizations for probes attached to nonderivatized slides, and we demonstrate that unmodified probes produce hybridization signals equal to amine-modified, covalently bound probes. Our method provides a cost-effective alternative to conventional attachment strategies that is particularly suitable for genotyping PCR products with nucleic acid microarrays.
Pacific Northwest National Laboratory, Richland, WA, USA. drcall@vetmed.wsu.edu
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Call
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Chandler
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Brockman
D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes