Oligomer restriction
Encyclopedia
Oligomer Restriction is a procedure to detect an altered DNA sequence in a genome
. A labeled oligonucleotide
probe
is hybridized to a target DNA, and then treated with a restriction enzyme
. If the probe exactly matches the target, the restriction enzyme will cleave the probe, changing its size. If, however, the target DNA does not exactly match the probe, the restriction enzyme will have no effect on the length of the probe. The OR technique, now rarely performed, was closely associated with the development of the popular polymerase chain reaction
(PCR) method.
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 Sickle Cell Anemia, or SCA). The mismatched hybrid no longer acts as a recognition site for the restriction enzyme, and the probe remains at its original length.
(RFLP) assay method, with the hope of avoiding the laborious Southern blot
ting step used in RFLP analysis. OR was conceived by Randall Saiki and Henry Erlich in the early 1980s, working at Cetus Corporation
in Emeryville, California
. It was patented in 1984 and published in 1985, having been applied to the genomic mutation responsible for Sickle Cell Anemia. OR was soon replaced by the more general technique of Allele Specific Oligonucleotide
(ASO) probes.
, who also worked at Cetus, had synthesized the oligonucleotide probes being tested by Saiki and Erlich. Aware of the problems they were encountering, he envisioned an alternative method for analyzing the SCA mutation that would use components of the Sanger
DNA sequencing technique. Realizing the difficulty of hybridizing an oligonucleotide primer to a single location in the genome, he considered using a second primer on the opposite strand. He then generalized that process and realized that repeated extensions of the two primers would lead to an exponential increase in the segment of DNA between the primers - a chain reaction
of replication
catalyzed by DNA polymerase
.
As Mullis encountered his own difficulties in demonstrating PCR
, 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 thus became the first publication of PCR, and for several years would become the report most cited by other researchers.
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. A labeled oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
is hybridized to a target DNA, and then treated with a restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
. If the probe exactly matches the target, the restriction enzyme will cleave the probe, changing its size. If, however, the target DNA does not exactly match the probe, the restriction enzyme will have no effect on the length of the probe. The OR technique, now rarely performed, was closely associated with the development of the popular polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) method.
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 human β-hemoglobin gene), as shown on the next line. Part of the probe includes the Recognition site for the restriction enzyme Dde I (underlined).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
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
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 Sickle Cell Anemia, or SCA). The mismatched hybrid no longer acts as a recognition site for the restriction enzyme, and the probe remains at its original length.
History
The Oligomer Restriction technique was developed as a variation of the Restriction Fragment Length PolymorphismRestriction fragment length polymorphism
In molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...
(RFLP) assay method, with the hope of avoiding the laborious Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
ting step used in RFLP analysis. OR was conceived by Randall Saiki and Henry Erlich in the early 1980s, working at Cetus Corporation
Cetus Corporation
Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California in 1971, but conducted most of its operations in nearby Emeryville. Before merging with another company in 1991, it developed several significant pharmaceutical drugs as well as a...
in Emeryville, California
Emeryville, California
Emeryville is a small city located in Alameda County, California, in the United States. It is located in a corridor between the cities of Berkeley and Oakland, extending to the shore of San Francisco Bay. Its proximity to San Francisco, the Bay Bridge, the University of California, Berkeley, and...
. It was patented in 1984 and published in 1985, having been applied to the genomic mutation responsible for Sickle Cell Anemia. OR was soon replaced by the more general technique of Allele Specific Oligonucleotide
Allele specific oligonucleotide
An allele-specific oligonucleotide is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay...
(ASO) probes.
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.
Relationship to PCR
Despite its limitations, the OR technique benefited from its close association with the development of the polymerase chain reaction. Kary MullisKary Mullis
Kary Banks Mullis is a Nobel Prize winning American biochemist, author, and lecturer. In recognition of his improvement of the polymerase chain reaction technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and earned the Japan Prize in the same year. The process was first...
, who also worked at Cetus, had synthesized the oligonucleotide probes being tested by Saiki and Erlich. Aware of the problems they were encountering, he envisioned an alternative method for analyzing the SCA mutation that would use components of the Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...
DNA sequencing technique. Realizing the difficulty of hybridizing an oligonucleotide primer to a single location in the genome, he considered using a second primer on the opposite strand. He then generalized that process and realized that repeated extensions of the two primers would lead to an exponential increase in the segment of DNA between the primers - a chain reaction
Chain reaction
A chain reaction is a sequence of reactions where a reactive product or by-product causes additional reactions to take place. In a chain reaction, positive feedback leads to a self-amplifying chain of events....
of replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
catalyzed by DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
.
As Mullis encountered his own difficulties in demonstrating PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
, 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
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
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 thus became the first publication of PCR, and for several years would become the report most cited by other researchers.