Dynamic Allele Specific Hybridization

Dynamic allele-specific hybridization (DASH) is a temperature-dependent, real-time variation of ASOH.26 Dynamic allele-specific hybridization begins with amplification of polymorphic DNA regions by PCR in which one primer is biotinylated. The biotinylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated strand is washed away. An oligonucleotide probe complementary to one allele is annealed to a bound PCR product strand, forming a DNA duplex that interacts with a double-strand-specific intercalation dye. On excitation, the dye emits fluorescence proportional to the amount of double-stranded DNA (probe-target duplex) present. The duplex is heated through a temperature range while the fluorescent signal is continually monitored. A rapid fall in fluorescent signal indicates denaturation of the DNA duplex. DNA duplexes with nonhomologous regions denature at a lower temperature than completely homologous DNA. The melting-temperature profile distinguishes homozygosity for either allele alone or a heterozygous mixture of the two.

0 0

Post a comment