"RFLP subtraction": a method for making libraries of polymorphic markers.

Abstract

We developed a method, "RFLP subtraction," that isolates large numbers of unique sequence restriction fragment length polymorphisms (RFLPs) in a single experiment. The technique purifies small restriction fragments from one genome containing sequences that reside on large fragments in a related genome. We first isolate samples containing the small restriction fragments from two polymorphic strains. Subtractive hybridization then removes the fragments that are present in both samples. The remaining sequences are RFLPs: they occur on small fragments in one strain but not in the other. Here we use RFLP subtraction to make a library of hundreds of unique sequence RFLPs from two inbred mouse strains. We analyze and map a subset of the RFLPs and show that the genetic linkage of these markers can be rapidly determined by an efficient dot blot mapping technique. Several other potential applications of RFLP subtraction, including isolating region specific markers, are discussed.