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Microarray fabrication with covalent attachment of DNA using Bubble Jet technology

Abstract

We have developed a method for fabricating DNA microarrays that uses a Bubble Jet ink jet device to eject 5′-terminal-thiolated oligonucleotides to a glass surface. The oligonucleotides are covalently attached to the glass surface by heterobifunctional crosslinkers that react with the amino group on the substrate and a thiol group on the oligonucleotide probe. Using this method, we fabricated DNA microarrays that carried 64 groups of 18-mer oligonucleotides encoding all possible three-base mutations in the mutational “hot spot” of the p53 tumor-suppressor gene. These were screened with a fluorescently labeled synthetic 18-mer oligonucleotide derived from the p53 gene, or segments of the p53 gene that had been PCR amplified from genomic DNA of two cell lines of human oral squamous cell carcinoma (SCC). This allowed us to discriminate between matched hybrids and 1 bp-mismatched hybrids.

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Figure 1
Figure 2
Figure 3: Fluorescent images of the spot(s) on a homogeneous DNA microarray hybridized with 50 nM of tetramethylrhodamine-labeled oligonucleotides.
Figure 4: Arrangement and base sequences of 64 groups of 18-mer oligonucleotide probes arrayed for the detection of the p53 gene.
Figure 5: Fluorescent images of the microarrays for the detection of p53 gene hybridized with the tetramethylrhodamine-labeled synthesized oligonucleotides, complementary to the probes No. 42 (A) and No. 46 (B), ×40.
Figure 6: Fluorescent images of the microarrays for the detection of p53 gene hybridized with the p53 exons of HSC5 (A) and HSC4 (B), labeled with tetramethylrhodamine (×40).

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Acknowledgements

The authors express their sincere appreciation to Prof. Nobuo Tsuchida of Tokyo Medical and Dental University for kindly providing HSC4, HSC5, and the PCR primers, Drs. Fumie Hosoda and Hitoshi Ichikawa of National Cancer Research Center for invaluable advice.

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Correspondence to Nobuko Yamamoto.

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Okamoto, T., Suzuki, T. & Yamamoto, N. Microarray fabrication with covalent attachment of DNA using Bubble Jet technology. Nat Biotechnol 18, 438–441 (2000). https://doi.org/10.1038/74507

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