Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.