title: Measurements of the binding of a large protein using a substrate density-controlled DNA chip.
abstract: The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.
authors.affiliation: Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Chuo-ku, Kobe, Japan. shuichi@center.konan-u.ac.jp
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Nakano
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kanzaki
authors.forename: Mariko
authors.initials: M
authors.lastname: Nakano
authors.forename: Daisuke
authors.initials: D
authors.lastname: Miyoshi
authors.forename: Naoki
authors.initials: N
authors.lastname: Sugimoto
mesh_terms: D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis / Q000379:methods; D057166:High-Throughput Screening Assays; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics*; D012680:Sensitivity and Specificity; D013046:Specific Gravity; D020349:Surface Plasmon Resonance; D013824:Thermus / Q000737:chemistry
chemical_list: D009841:Oligonucleotides; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein