SNP Detection Using DNA Microarrays
There is increasing interest in the development of reliable and cost-effective methods for clinical diagnosis of genetic disorders. In this work, DNA microarray technology and solid phase minisequencing was applied in the development of an inexpensive assay for multiplex detection of single nucleotide polymorphisms. 11 samples from healthy heterozygote carriers of beta-thalassemia were correctly classified as heterozygote or homozygote for mutations at one of four loci in the beta globin gene.
Agarose coated glass slides were used as support for arrays of immobilized capture probes modified with a tail of thymine and cytosine residues. Target DNA was amplified by PCR and prepared by fragmentation with uracil-N-glycosylase (UNG), which resulted in better discrimination compared to other target preparation methods as asymmetric PCR, fragmentation with DNAse I or hydroxyl radicals, or separation of strands with streptavidin coated magnetic beads.
Minisequencing of target prepared by UNG fragmentation resulted in twice as high discrimination ratios compared to allele specific oligonucleotide hybridization of target prepared by asymmetric PCR. The use of inexpensive Tempase DNA polymerase for PCR and only 1 unit of Thermo Sequenase per reaction reduced reagent costs. Agarose coated slides containing microarrays of capture probes were produced at a cost of approximately € 1.78 per slide, and the total cost of the assay were minimized to approximately € 8.48 per sample.