Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide...

Efficiencies of mismatch discrimination using size-varied capture probes were examined at various hybridization temperatures. The probes were 17, 15, 13, 11, 9, and 7 nucleotides long and contained single-base mismatches at their 3' ends. The optimal signal intensity and efficiency of base stacking hybridization on mismatch discrimination were observed for capture probes with a melting temperature (Tm) value of 36 degrees C, in the detection of DNA sequence variations at 40 degrees C. We employed asymmetric PCR to prepare single-stranded target DNA labeled with a fluorescent dye, and the PCR product was hybridized on the DNA microarray with no further purification. Our efforts have enhanced the sensitivity and simplified the procedures of base stacking hybridization on mismatch discrimination. As a model experiment, this improved technology was used to identify plasmid templates of human leukocyte antigen (HLA)-A alleles 2601, 2902, and 0206 on oligonucleotide microarrays. It is now possible to apply this simple, rapid, sensitive, and reliable base stacking hybridization technology to detect DNA sequence variations on microarrays in clinical diagnosis and other applications.