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.
Read more about this topic: Oligomer Restriction
Famous quotes containing the word problems:
“I conceive that the leading characteristic of the nineteenth century has been the rapid growth of the scientific spirit, the consequent application of scientific methods of investigation to all the problems with which the human mind is occupied, and the correlative rejection of traditional beliefs which have proved their incompetence to bear such investigation.”
—Thomas Henry Huxley (1825–95)
“The man who is forever disturbed about the condition of humanity either has no problems of his own or has refused to face them.”
—Henry Miller (1891–1980)
“An interesting play cannot in the nature of things mean anything but a play in which problems of conduct and character of personal importance to the audience are raised and suggestively discussed.”
—George Bernard Shaw (1856–1950)