title: Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.
abstract: DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.
authors.affiliation: Sumitomo Bakelite Co., Ltd. 1-1-5 Muroya, Nishi-ku, Kobe 651-2241, Japan. kenji_k@mukogawa-u.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Kinoshita
authors.forename: Kentaro
authors.initials: K
authors.lastname: Fujimoto
authors.forename: Toru
authors.initials: T
authors.lastname: Yakabe
authors.forename: Shin
authors.initials: S
authors.lastname: Saito
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Hamaguchi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kikuchi
authors.forename: Ken
authors.initials: K
authors.lastname: Nonaka
authors.forename: Shigenori
authors.initials: S
authors.lastname: Murata
authors.forename: Daisuke
authors.initials: D
authors.lastname: Masuda
authors.forename: Wataru
authors.initials: W
authors.lastname: Takada
authors.forename: Sohei
authors.initials: S
authors.lastname: Funaoka
authors.forename: Susumu
authors.initials: S
authors.lastname: Arai
authors.forename: Hisao
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Kanehisa
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Fujiwara
authors.forename: Kenichi
authors.initials: K
authors.lastname: Matsubara
mesh_terms: D017931:DNA Primers / Q000737:chemistry*; D007700:Kinetics; D008689:Methacrylates / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D011092:Polyethylene Glycols / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism*; D013696:Temperature
chemical_list: D017931:DNA Primers; D008689:Methacrylates; D015345:Oligonucleotide Probes; C513779:poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate); D011092:Polyethylene Glycols; D019914:Taq Polymerase