Frank van der Loop, Erika Timmer, Hubert Smeets.
Maastricht University, Department of Molecular Cell Biology and Genetics, Division Genetics, P.O. Box 1475, 6201BL Maastricht.
Microarrays are being used for gene expression profiling studies, but also for mutation detection and/or polymorphism screening. In the GenomeCenter Maastricht, a central microarray facility for the University of Maastricht and the Academic Hospital, protocols for both applications are being developed and implemented in ongoing research projects. High density DNA-CHIPs are yet available for mutation detection and polymorphism screening for a limited number of genes, but for most applications intermediate density microarrays have to be developed by the research group involved. We are currently comparing 3 strategies based on 5'-aminomodified oligonucleotides (catcher-probes), which are spotted on activated amino-silane-coated glass slides and hybridized to PCR amplified DNA.
The first approach involves spotted Allele Specific Oligonucleotides (ASO), which are hybridized with fluorescently labeled PCR-products. Hybridization will occur in case of a perfect match, with the middle position of the catcher-probe as the critical factor. The sensitivity of this method strongly depends on the conditions of hybridization and post-hybridization washing. The efficiency of direct incorporation of Cy3/5-labeled dNTP in the PCR product is being compared with the use of 5'-Cy3/5-labeled primers. Currently, we are developing protocols for the detection of point mutations, using microarrays consisting of 5'-aminomodified 15-mer oligonucleotides.
The second strategy is the Oligonucleotide Ligation Assay (OLA). Spotted catcher-probes are hybridized with unlabeled PCR-amplified target DNA, and are ligated to a (fluorescently) labeled reporter-probe. Enzymatic ligation only occurs in case of a perfect match of both the catcher-probe (with the 3' nucleotide as critical factor) and the reporter-probe. Preliminary results indicate that OLA at microarray level can be performed. The use of specific catcher-probes in combination with a mix of degenerated fluorescently labeled reporter-probes is now being investigated. The third strategy that we would like to develop is an Allele-specific Primer Extension protocol (APEX). This method is dependent on the availability of fluorescently labeled ddNTP's, which currently are being manufactured. In both the OLA and the APEX protocol the enzymatic step determines the sensitivity and the efficiency of the protocol.