Nucleic Acids Research Advance Access originally published online on November 28, 2006
Nucleic Acids Research 2007 35(1):e3; doi:10.1093/nar/gkl939
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Nucleic Acids Research, 2007, Vol. 35, No. 1 e3
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer
1 Sumitomo Bakelite Co., Ltd. 1-1-5 Muroya, Nishi-ku, Kobe 651-2241, Japan 2 DNA Chip Research Inc. 1-1-43 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan 3 School of Pharmaceutical Sciences, Mukogawa Women's University 11-68 Kyuban-cho, Koshien, Nishinomiya 663-8179, Japan
*To whom correspondence should be addressed. Tel: +81 798 45 9982; Fax: +81 798 41 2792; Email: kenji_k{at}mukogawa-u.ac.jp
Received August 8, 2006. Revised October 1, 2006. Accepted October 16, 2006.
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.