Oligomer Restriction - Problems

Problems

The Oligomer Restriction method was beset by a number of problems:

• It could be applied only to the small set of DNA polymorphisms which alter a restriction site, and only to those sites for which sequence information was known. Many of the known RFLP assays detected polymorphisms which were far away from their probe locations.

• It is difficult to label oligonucleotides to a level high enough to use them as probes for genomic DNA. This problem also plagued the development of ASO probes.

• It is difficult to design oligonucleotides and use them in a way that they become hybridization probes for just a single site within a genome. Binding to non-specific locations can often obscure the effect of the probe at the target location.

• Not all restriction enzymes have the desired specificity for their recognition sequence. Some can recognize and cut single-stranded DNA, and some show a low level of cleavage for mismatched sites. Even a small amount of non-specific cleavage can swamp the weak signal expected from the target sequence.

• It was difficult to design an OR method that included controls for both of the alleles being tested. In part 2 of the simplified example described above, the probe was not cleaved when hybridized to a mutant target. But the same (non-) result would occur for the large excess of unhybridized probe, as well as if any problem occurred preventing the complete digestion by restriction enzyme. In the actual method reported, a second non-polymorphic restriction site was used to cut all of the hybridized probe, and a second unlabeled oligonucletide was used to 'block' the unhybridized probe. These controls would not have been available for other targets.