Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.