Nonradioactive Method for Restriction Mapping of lambda Phage DNA

摘要

The method of Rackwitz et al. (2) is in wide use for the mapping of restriction sites in lambda phages. Lambda phages contain 5' extending cohesive ends of 12-nucleotide length. Hybridizing a labeled oligonucleotide complementary to one of the overhangs allows the selective visualization of (partial) restriction fragments that encompass this end In the original protocol, a primer, labeled at its 5' end with [gamma-~(32)P]ATP, is annealed to a partial restriction digest. The mixture of fragments is separated on an agarose gel, which is subsequently dried on DEAE paper to ensure retention of the oligonucleotide. We adapted this protocol to nonra-dioactive detection of end-containing fragments. For this purpose, the oligonucleotide has to be covalentlylinked to one of the recessed ends of lambda. The covalent attachment allows the efficient retention of the biotinylated marker on the blot membrane, where fragments that carry the labeled oligonucleotide at their 3' ends can be visualized by any of the many available nonradioactive detection methods. Without ligation, the retention of the small oligonucleotide is poor.