title: Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension.
abstract: 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-1alpha (HNF-1alpha) 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-1alpha 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.
authors.affiliation: 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.
authors.forename: Jae Yang
authors.initials: JY
authors.lastname: Song
authors.forename: Hyun Gyu
authors.initials: HG
authors.lastname: Park
authors.forename: Sung-Ouk
authors.initials: SO
authors.lastname: Jung
authors.forename: JaeChan
authors.initials: J
authors.lastname: Park
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D003924:Diabetes Mellitus, Type 2 / Q000175:diagnosis*; D005838:Genotype; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D020135:Peptide Nucleic Acids; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1