gene expression is well regulated by the epigenetic modification of dna and histone tails independent of their primary sequences .
in particular , cytosine methylation , in which the c5 position of the cytosine base is methylated enzymatically , plays a crucial role in the regulation of chromatin stability , gene regulation , parental imprinting , and x - chromosome inactivation in females [ 14 ] .
therefore , detection of cytosine methylation is very important , and much effort has gone into developing a simple reaction for 5-methylcytosine ( c ) detection . for the evaluation of the methylation status of genes ,
several conventional methods have so far been used , such as a cleavage assay with methylation - insensitive restriction enzymes [ 57 ] , hydrolysis and sequencing with a bisulfite salt [ 810 ] , and immunofluorescence with anti-5-methylcytosine antibody [ 11 , 12 ] .
although the conventional methods have many merits , there are many disadvantages , and methylation detection assays must be further improved through another approach . the existence of a more rapid and selective chemical reaction capable of distinguishing between methylcytosine and unmethylated cytosine on a chip has promise as a good method for efficiently analyzing the status of cytosine methylation at a specific site in a gene .
the sequence - selective dna methylation - detection probe , icon ( interstrand complexes formed by osmium and nucleic acids ) , may be effective for the development of an on - chip analysis of dna methylation [ 1315 ] . in the presence of osmium oxidants and a bipyridine ligand ,
5-methylcytosine forms a stable osmium - centered complex , in contrast to unmethylated cytosine ( figure 1 ) [ 1619 ] .
icon probes form a crosslink with a specific 5-methylcytosine in the probe - hybridizing dna mediated by osmium - centered complex formation .
this function will be effective for the capture of methylated dna on a chip for sequence - selective methylation analysis . in this paper , development of an on - chip analyzing method for typing of dna methylation at a specific cytosine
icon probes fixed to the bottom of microwells assisted the on - chip detection of the methylation status of a specific cytosine in the target dna .
artificial dna was synthesized by the conventional phosphoramidite method using an applied biosystems 392 dna / rna synthesizer or an nts h-6 dna / rna synthesizer .
the phosphoramidite form of bipyridine - modified adenine ( b ) was prepared according to the synthetic protocol described in a previous paper .
the 5-amino end was attached using the phosphoramidite of 5-aminomodifier c12 ( glen research ( http://www.glenresearch.com/index.php ) ) . synthesized dna was purified by reverse phase hplc on a 5-ods - h column ( 10  mm  150  mm , elution with a solvent mixture of 0.1  m triethylammonium acetate ( teaa ) , ph 7.0 , linear gradient over 30 min from 5% to 20% acetonitrile at a flow rate of 3.0  ml / min ) .
a 100  l solution of synthetic dna ( 100  nm ) in te buffer ( ph 7.0 ) or deionized water in the presence of 10  mm 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ,   and 10  mm 1-methylimidazole was put into each well of nucleolink strips ( nalge nunc ( http://www.nalgenunc.com/ ) ) . after incubation at 50c for 5  h , the reaction mixture was removed from the well , and the well was rinsed three times with a solution of 100  mm tris - hcl ( ph 7.5 ) , 150  mm sodium chloride , and 0.1% tween20 , and then three times with deionized water . the target dna sequence p53(n 
n ) was 5-tgt gca gct gtg g gtt gat t cga cac ccc c gcc cgg cac c cgc gtc cgc g cca tgg cca t cta caa gca g tca cag cac a tga ngg agg t tgt gag g ng c tgc ccc cac c-3 ( n ,
a 25  l solution of dna ( 8  nm ) in 50  mm tris - hcl buffer ( ph 7.7 ) , 0.5  mm edta , and 1  m sodium chloride was added to each probe - attached well at 0c .
the reaction mixture was incubated at 0c for 5  min , and then the solution was removed from the wells .
a 25  l solution of 5  mm potassium osmate(vi ) and 100  mm potassium hexacyanoferrate(iii ) in 50  mm tris - hcl buffer ( ph 7.7 ) , 0.5  mm edta , and 1  m sodium chloride was incubated at 0c for 5  min or at 25c for 10  min .
the wells were rinsed seven times with 0.4  m sodium hydroxide , 0.25% tween20 ( 130  l / well ) .
after further rinsing with deionized water twice , the wells were coated with 10  mg / ml bsa in 100  mm tris - hcl ( ph 7.5 ) , 150  mm sodium chloride , and 0.1% tween20 .
the process of pcr amplification was performed in a reaction solution of 1  u takara ex taq hs , 10 buffer , 2.5  mm dntp mix , and 1  m primer mix ( forward , 5-agctgd514gggttgattc-3 for the exciton primer method , 5-tgtgcagctgtgggttgattc-3 for the sybr green i method ; reverse , 5-actgcttgtagatggccatg-3 ; d514 in an exciton primer is a hybridization - sensitive fluorescent nucleotide ( figure 1 ) ) . in the case of using the stain method with sybr green
i fluorescence for monitoring the amplification , sybr green i dye was also added to the reaction mixture in advance .
amplifications were performed in microwells as follows : after heating at 95c for 60  s , 35 cycles of denaturation at 95c for 5  s , annealing with fluorescence monitoring at 52c for 20  s , and extension at 72c for 20  s on the corbett rotor - gene , the amplification process was monitored by the fluorescence of d514 or sybr green i through an sybr green i ( 470  nm/510  nm ) filter .
for the on - chip study , we adopted a nucleolink strip , because it is a microwell strip in which the amino - modified icon probe can be attached to well bottoms with covalent bonds .
the target dna was a fragment of the human p53 gene exon 5 including mutation hotspots at cpg dinucleotides .
the sequence of the icon probe was designed to form a crosslink with one of the methylated cytosines in the target dna .
the icon probe was modified with an amino end for attaching to the plate and an alkyl linker and a poly c sequence for introducing enough distance between the probe and the plate .
this icon probe was synthesized by the conventional phosphoramidite method by using a dna autosynthesizer .
the phosphoramidite form of bipyridine - modified adenine ( b ) was prepared according to the synthetic protocol described in a previous paper .
synthetic dna was fixed to the bottom of each well of nucleolink strips in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-methylimidazole ( figure 2 ) .
oxidative osmium complexation using icon probes is a rapid and mild reaction for detection of methylated dna , and it is not accompanied with nonspecific strand damage , in contrast to conventional bisulfite methods . therefore , this reaction would be effective for on - chip analysis of dna methylation .
the target dna was put into the wells and hybridized with the icon probes fixed in the wells .
the dna samples in the wells were incubated at 0c for 5 min then at 25c for 5  min in the presence of 5  mm potassium osmate(vi ) and 100  mm potassium hexacyanoferrate(iii ) in 50  mm tris - hcl buffer ( ph 7.7 ) , 0.5  mm edta , and 1  m sodium chloride . after reaction
, the wells were rinsed with 4  m sodium hydroxide and then coated with bsa .
the fixed dna was detected using pcr amplification of a part of the crosslinking dna strand .
the pcr primers were designed for the region without icon binding . for one of the primers ,
a hybridization - sensitive fluorescent dna was used , containing a fluorescent nucleotide d514 ( figure 1 ) [ 2124 ] .
this fluorescent dna shows very weak fluorescence in the unhybridized state , whereas it shows strong fluorescence after hybridization with the complementary nucleic acids .
this fluorescence switching is controlled by an intramolecular excitonic interaction between dyes tethered to the dna .
this dna shows weak fluorescence emission , whereas the pcr mixture emits strong fluorescence after pcr amplification .
this system has been applied to the detection of single nucleotide polymorphisms in genome dna samples .
the process of pcr amplification was performed in a reaction solution of takara ex taq hs polymerase in the presence of a mixture of dntp and primer mix .
amplifications were performed in microwells and the change in the fluorescence intensity monitored using a real - time pcr system . in the experiment for p53(c  c ) and p53(c  c ) , the methylation of the target cytosine was determined from the increase in the fluorescence signal associated with the exponential growth of the pcr product .
c ) started first , and then the amplification of p53(c  c ) started several cycles later ( figure 3 ) .
washingout of uncrosslinked sample dna brought about this lag in the starting point of amplification .
the amplification curve observed for p53(c  c ) almost overlapped that for the osmium - untreated p53(c  c ) , suggesting that the amplification curve for unmethylated dna is due to amplification of the dna nonspecifically adsorbing to the well surface . on - chip capture of methylated dna by the icon probe at the methylation site made possible the sequence specific detection of methylation through pcr amplification .
a prototype for icon - based on - chip methylation analysis makes possible sequence - specific amplification .
we prepared four dna strands with different methylation sites , p53(c  c ) , p53(c  c ) , p53(c  c ) , and p53(c  c ) .
the sample dna was added to the wells , in which the icon probe targeting only the c of the sample dna 5 side was fixed .
after osmium treatment and bsa coating , the crosslinked dna was amplified by pcr in the presence of unlabeled primers and sybr green i. after 15 cycles of the amplification reaction , the fluorescence intensity of sybr green i was quantified on a microplate reader ( figure 4 ) .
c ) and p53(c  c ) exhibited higher fluorescence intensities compared with those from wells containing p53(c 
the icon probe fixed on the well bottom distinguished 5-c from 3-c and detected only methylation of 5-c regardless of methylation of 3-c .
we have described a new , high - value aspect of on - chip methylation analysis through osmium - dna complexation . an icon probe fixed onto a microwell formed a crosslink with the target 5-methylcytosine and assisted the detection using pcr amplification .
although there remain further aspects to be examined toward realizing an easier - to - use methylation analysis , such as optimization of pcr conditions suitable for icon , this on - chip assay supported by the chemical basis could be an important component of the next generation of high - throughput methylation analyses .
 the development of a reaction for detecting the presence / absence of one methyl group in a long dna strand is a chemically and biologically challenging research subject . 
 a newly designed chemical assay on a chip for the typing of dna methylation has been developed . 
 a methylation - detection probe fixed at the bottom of microwells was crosslinked with methylated dna mediated by osmium complexation and contributes to selective amplification of methylated dna . 