Hybridization parameters revisited: solutions containing SDS.
Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson equation. High concentrations of SDS are used in some common protocols, 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 PCR- or transcription-generated probes, we report that the 50% dissociation temperature (Tm*) in the absence of SDS was 15 degrees C-17degrees C lower than the calculated Tm. SDS had only modest effects on Tm* [1% (w/v) equating to 8 mM NaCl]. RNA/DNA hybrids were approximately 11 degrees C more stable than DNA/DNA hybrids. Incomplete homology (69%) significantly reduced the Tm* for DNA/DNA hybrids (approximately /4degrees C; 0.45 degrees C/% nonhomology) but far less so for RNA/DNA hybrids (approximately 2.3 degrees C; approximately 0.07 degrees C/% non-homology); incomplete homology also markedly reduced the extent of hybridization. On these nylonfilters, SDS had a major effect on nonspecific binding. Buffers lacking SDS, or with low salt concentration, gave high hybridization backgrounds; buffers containing SDS, or high-salt buffers, gave reproducibly low backgrounds.
University of Edinburgh, UK.
Ken
K
Rose
John O
JO
Mason
Richard
R
Lathe
D015342:DNA Probes / Q000187:drug effects*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000187:drug effects* / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012965:Sodium Chloride / Q000494:pharmacology*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D015342:DNA Probes; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride