Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension.
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.
Song
Jae Yang
JY
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.
Park
Hyun Gyu
HG
Jung
Sung-Ouk
SO
Park
JaeChan
J
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*
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