Oligomer restriction
Oligomer Restriction (abbreviated OR) is a procedure to detect an
Example
In part 1a of the schematic the oligonucleotide probe, labeled on its left end (asterisk), is shown on the top line. It is fully complementary to its target DNA (here taken from the
In part 1b, the restriction enzyme has cleaved the probe and its target (Dde I leaves three bases unpaired at each end). The labeled end of the probe is now just 8 bases in length, and is easily separated by Gel electrophoresis from the uncut probe, which was 40 bases long.
In part 2, the same probe is shown hybridized to a target DNA which includes a single base mutation (here the mutation responsible for
History
The Oligomer Restriction technique was developed as a variation of the
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,[2] a second non-polymorphic restriction site was used to cut all of the hybridized probe, and a second unlabeled oligonucleotide was used to 'block' the unhybridized probe. These controls would not have been available for other targets.
Relationship to PCR
Despite its limitations, the OR technique benefited from its close association with the development of the polymerase chain reaction.
As Mullis encountered his own
he joined an existing group of researchers that were addressing the problems with OR. Together, they developed the combined PCR-OR assay. Thus, OR became the first method used to analyze PCR-amplified genomic DNA.Mullis also encountered difficulties in publishing the basic idea of PCR (scientific journals rarely publish concepts without accompanying results). When his manuscript for the journal Nature was rejected, the basic description of PCR was hurriedly added to the paper originally intended to report the OR method (Mullis was also a co-author there). This OR paper[2] thus became the first publication of PCR, and for several years would become the report most cited by other researchers.
References
- ^ Saiki RK, Erlich, HA "Method for detection of polymorphic restriction sites and nucleic acid sequences." U.S. Patent 4683194.
- ^ PMID 2999980. Archived from the originalon 19 December 2008.
- ^ Saiki RK, Bugawan TL, Mullis KB, and Erlich HE "Analysis of enzymatically amplified beta-globin and HLA-DQa DNA with allele-specific oligonucleotide probes" Nature vol. 324(6093) pp. 163-166 (1986).
- ^ Mullis K "The Unusual Origin of the Polymerase Chain Reaction" Scientific American vol. 262(4): pp. 56-65 (1990).
- ^ Mullis K "The Polymerase Chain Reaction", Nobel Lecture, December 8, 1993.
- ^ Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996).