Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.
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.
Kinoshita
Kenji
K
Sumitomo Bakelite Co., Ltd. 1-1-5 Muroya, Nishi-ku, Kobe 651-2241, Japan. kenji_k@mukogawa-u.ac.jp
Fujimoto
Kentaro
K
Yakabe
Toru
T
Saito
Shin
S
Hamaguchi
Yuzo
Y
Kikuchi
Takayuki
T
Nonaka
Ken
K
Murata
Shigenori
S
Masuda
Daisuke
D
Takada
Wataru
W
Funaoka
Sohei
S
Arai
Susumu
S
Nakanishi
Hisao
H
Yokoyama
Kanehisa
K
Fujiwara
Kazuhiko
K
Matsubara
Kenichi
K
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
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