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

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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-1a (HNF-1a) mutations that cause type 3 maturity onset diabetes of the young (MODY).Amulti-epoxylinkercompoundwassynthesized 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. ThePCRproducts were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 30 complementarity to the specific mutation site and 50 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 muchhigher duplex specificity for thecomplementary 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-1a 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.
Publisher
Oxford University Press
Issue Date
2005
Citation

Nucleic Acids Research, Vol.33, No.2

DOI
10.1093/nar/gni020
URI
http://hdl.handle.net/10203/25211
Appears in Collection
CBE-Journal Papers(저널논문)

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