Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.
- 1 March 1989
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 86 (6) , 1934-1938
- https://doi.org/10.1073/pnas.86.6.1934
Abstract
The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (218) and 8192 (213) unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.This publication has 18 references indexed in Scilit:
- Conservation of short patches of amino acid sequence amongst proteins with a common function but evolutionarily distinct origins: implications for cloning genes and for structure-function analysisNucleic Acids Research, 1988
- Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA PolymeraseScience, 1988
- Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family.Proceedings of the National Academy of Sciences, 1987
- Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaScience, 1985
- The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identificationFEBS Letters, 1985
- THE MITOCHONDRIAL ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION SYSTEMAnnual Review of Biochemistry, 1985
- [12] One-step gene disruption in yeastPublished by Elsevier ,1983
- Assignment of a gene for succinate dehydrogenase to human chromosome 1 by somatic cell hybridizationCytogenetic and Genome Research, 1980
- [53] Proteins, polypeptides, prosthetic groups, and enzymic properties of complexes I, II, III, IV, and V of the mitochondrial oxidative phosphorylation systemPublished by Elsevier ,1979
- Mammalian cells with defective mitochondrial functions: a Chinese hamster mutant cell line lacking succinate dehydrogenase activityCell, 1977