Oligonucleotide chip for the diagnosis of HNF-1 alpha mutations.
Mutations in HNF-1 alpha cause maturity-onset diabetes of the young (MODY) type 3, which is the most prevalent MODY subtype in most countries. In the present study, we investigated an oligonucleotide microchip for the detection of the known HNF-1 alpha mutations. We first optimized the coupling chemistries for covalent immobilization of allele-specific oligonucleotides on aldehyde (CHO)- and thiocyanate (NCS)-activated glass slides and compared their hybridization efficiencies. CHO-glass was found to provide a more favorable environment for hybridization than NCS-glass, whereas the binding capacity of NCS-glass for amine-activated oligonucleotide was much greater than with CHO-glass. We also investigated the effects of the length of the capture probes on the hybridized signals. To determine the presence of HNF-1 alpha mutations in a human sample, we prepared an oligonucleotide chip from selected mutation sites of exon2 from HNF-1 alpha. Cy3-labeled RNA target probes were obtained by in vitro transcription of promoter-tagged PCR products from a wild-type blood sample and subsequent fragmentation. Hybridization of the chip with the RNA target probes successfully identified all of the genotypes for the tested sites. This work demonstrates that oligonucleotide chip-based analysis is a good candidate for routine clinical testing for HNF-1 alpha mutations.
Park
Hyun Gyu
HG
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. hgpark@kaist.ac.kr
Ham
Hyun Ok
HO
Kim
Kyung Hee
KH
Huh
Nam
N
D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D003924:Diabetes Mellitus, Type 2 / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020022:Genetic Predisposition to Disease / Q000235:genetics; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000097:blood / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods
D051538:Hepatocyte Nuclear Factor 1-alpha