Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA.
We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.
Barr
F G
FG
Division of Human Genetics and Molecular Biology, Children's Hospital of Philadelphia, Pennsylvania 19104.
Emanuel
B S
BS
D001386:Azides; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives*; D004247:DNA / Q000032:analysis*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D019809:Streptavidin; D013382:Subtraction Technique
D001386:Azides; D001426:Bacterial Proteins; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C045549:photobiotin