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Nucleic Acids Research 2005 33(2):e19; doi:10.1093/nar/gni020
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Published online 1 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Diagnosis of HNF-1{alpha} mutations on a PNA zip-code microarray by single base extension

Jae Yang Song, Hyun Gyu Park*, Sung-Ouk Jung1 and JaeChan Park1

Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea 1 Samsung Advanced Institute of Technology San 14-1, Nongseo-Ri, Kiheung, Kyunggi-Do, 449-712, Republic of Korea

*To whom correspondence should be addressed. Tel: +82 42869 3932; Fax: +82 42869 3910; Email: hgpark{at}kaist.ac.kr

Received October 15, 2004. Revised January 13, 2005. Accepted January 13, 2005.

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1{alpha} (HNF-1{alpha}) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1{alpha} with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.


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