On-Chip Sequence-Specific Immunochemical Epigenomic Analysis Utilizing Outward-Turned Cytosine in a DNA Bulge with Handheld Surface Plasmon Resonance Equipment.
This paper reports a sequence-specific immunoassay chip for DNA methylation assessment by microfluidic-based surface plasmon resonance (SPR) detection. This was achieved by utilizing an affinity measurement involving the target, (methyl)cytosine, in a single-base bulge region and an anti-methylcytosine antibody in a microchannel, following hybridization with a biotinylated bulge-inducing DNA probe. The probe alters the target cytosine in a looped-out state because of the π-π stacking between flanking bases of the target. The probe design is simple and consists of the elimination of guanine paired with the target cytosine from a fragmented full-match sequence. We obtained the single methylation status in 6 amol (48 fg) of synthesized oligo DNA in 45 min, which is the fastest DNA methylation assessment yet reported, without employing a conventional bisulfite reaction, PCR, or sequencing. We also succeeded in discrimination of the methylation status of single cytosine in genomic λ DNA and HCT116 human colon cancer cells. The advantages of the proposed method are its small equipment, simple microfluidics design, ease of handling (two injections of DNA and antibody), lack of need for a methylation-sensitive enzyme, and neutral buffer conditions.
National Institute of Advanced Industrial Science and Technology , Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566.
Ryoji
R
Kurita
National Institute of Advanced Industrial Science and Technology , Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566.
Hiroyuki
H
Yanagisawa
National Institute of Advanced Industrial Science and Technology , Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566.
Kyoko
K
Yoshioka
National Institute of Advanced Industrial Science and Technology , Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566.
Osamu
O
Niwa
D010582:Bacteriophage lambda / Q000235:genetics; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D015342:DNA Probes / Q000737:chemistry; D057890:Epigenomics / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D020349:Surface Plasmon Resonance / Q000295:instrumentation*
D015342:DNA Probes; D003596:Cytosine; D004247:DNA