Oligomer restriction

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Oligomer Restriction (abbreviated OR) 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.

Contents

Mechanism of Oligomer Restriction. ORDemo.gif
Mechanism of Oligomer Restriction.

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 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 Polymorphism (RFLP) assay method, with the hope of avoiding the laborious Southern blotting 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 [1] and published in 1985, [2] 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. [3]

Problems

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

Relationship to PCR

Despite its limitations, the OR technique benefited from its close association with the development of the polymerase chain reaction. Kary Mullis, 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. [4] [5]

As Mullis encountered his own difficulties in demonstrating PCR, [6] 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.

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Inverse polymerase chain reaction

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History of polymerase chain reaction

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The cleaved amplified polymorphic sequence (CAPS) method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the Restriction Fragment Length Polymorphism (RFLP) method, using polymerase chain reaction (PCR) to more quickly analyse the results.

A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.

COLD-PCR is a modified Polymerase Chain Reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. Detection of mutations is important in the case of early cancer detection from tissue biopsies and body fluids such as blood plasma or serum, assessment of residual disease after surgery or chemotherapy, disease staging and molecular profiling for prognosis or tailoring therapy to individual patients, and monitoring of therapy outcome and cancer remission or relapse. Common PCR will amplify both the major (wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The capacity to detect a mutation in a mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can occur when provided with a heterogeneous sample – as is often the case with cancer biopsies. Currently, traditional PCR is used in tandem with a number of different downstream assays for genotyping or the detection of somatic mutations. These can include the use of amplified DNA for RFLP analysis, MALDI-TOF genotyping, or direct sequencing for detection of mutations by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for these downstream assays will increase the reliability in detecting mutations from mixed samples, including tumors and body fluids.

In silico PCR

In silico PCR refers to computational tools used to calculate theoretical polymerase chain reaction (PCR) results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome.

References

  1. ↑ Saiki RK, Erlich, HA "Method for detection of polymorphic restriction sites and nucleic acid sequences." U.S. Patent 4683194.
  2. 1 2 3 Saiki, RK; Scharf S; Faloona F; Mullis KB; Erlich HA; Arnheim N (20 December 1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science . 230 (4732): 1350–4. Bibcode:1985Sci...230.1350S. doi:10.1126/science.2999980. PMID   2999980. Archived from the original on 19 December 2008.
  3. ↑ 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).
  4. ↑ Mullis K "The Unusual Origin of the Polymerase Chain Reaction" Scientific American vol. 262(4): pp. 56-65 (1990).
  5. ↑ Mullis K "The Polymerase Chain Reaction", Nobel Lecture, December 8, 1993.
  6. ↑ Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996).
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