ELBA SUMMER SCHOOL The Eurosterone DNA Array
نویسندگان
چکیده
Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson (1965) equation. High concentrations of sodium dodecyl sulfate (SDS) are used in some common protocols (Church and Gilbert, 1984; Virca et al., 1990) but the effects of SDS on hybridization stringency have not been reported. We investigated hybridization parameters in solutions containing SDS. With targets immobilized on nylon membranes and PCRor transcription-generated probes we report that the 50% dissociation temperature (Tm*) in the absence of SDS was 15-17C lower than the calculated melting temperature (Tm). SDS had only modest effects on Tm* (1% w/v equating to 8 mM NaCl). RNA/DNA hybrids were ~11C more stable than DNA/DNA hybrids. Incomplete homology (69%) significantly reduced the Tm* for DNA/DNA hybrids (~14C; ~0.45C per % nonhomology) but far less so for RNA/DNA hybrids (~2.3C; ~0.07C per % non-homology); incomplete homology also markedly reduced the extent of hybridization. On these nylon filters, SDS had a major effect on non-specific binding. Buffers lacking SDS, or with low salt concentration, gave high hybridization backgrounds; buffers containing SDS, or high salt buffers, gave reproducibly low backgrounds.
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