Gene digging
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Abstract
A method termed “gene digging” has been developed based on our observation of stretches of highly conserved nucleotide sequence in the coding region of many genes across related species. Rabbit-specific nucleotide sequences corresponding to desired coding segments of 14 different genes were obtained with primers that were designed based on conserved nucleotide stretches. Our success in gene digging could be attributable to the method’s inherent ability to reduce the degeneracy of primers by more than two orders of magnitude (sometimes by more than three orders of magnitude) compared to primers designed from conserved amino acids. Our results not only demonstrate the value of the method, but also hint at a thus far unknown functional significance of conserved nucleotide stretches in the coding region of various genes. In our hands the method worked 14 out of 14 times indicating generality of the concept.
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Gene digging conserved cross-species degenerate PCR open reading frame sequence rabbitPreview
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References
- 1.Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M., and Caskey, C. T. (1988) Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase.Science 239, 1288–1291.PubMedCrossRefGoogle Scholar
- 2.Girgis, S. I., Alevizaki, M., Denny, P., Ferrier, G. J. M., and Legon, S. (1988) Generation of DNA probes for peptides with highly degenerate codons using mixed primer PCR.Nucleic Acids Res. 16, 10,371.CrossRefGoogle Scholar
- 3.Compton, T. (1990) Degenerate primers for DNA amplification, inPCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, CA, pp. 39–45.Google Scholar
- 4.Sarkar, G., Koeberl, D. D., and Sommer, S. S. (1990) Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species.Genomics 6, 133–143.PubMedCrossRefGoogle Scholar
- 5.Metsaranta, M., Kujala, U. M., Pelliniemi, L., Osterman, H., Atto, H., and Vuorio, E. (1996) Evidence for insufficient chondrocyte differentiation during repair of full-thickness defects of articular cartilage.Matrix Biol. 15, 39–47.PubMedCrossRefGoogle Scholar
- 6.Brill, J. A. and Weigel, J. (1997) Differentiation between spore-forming and asporogenic bacteria using a PCR and Southern hybridization based method.J. Microbial Methods 31, 29–36.CrossRefGoogle Scholar
- 7.Nichols, R., Andrews, P. C., Zhang, P., and Bergstrom, D. E. (1994) A universal nucleoside for use at ambiguous sites in DNA primers.Nature 369, 492,493.CrossRefGoogle Scholar
- 8.Loakes, D. and Brown, D. M. (1994) 5-Nitroindole as an universal base analogue.Nucleic Acids Res. 22, 4039–4043.PubMedCrossRefGoogle Scholar
- 9.Sanyal, A., O’Driscoll, S. W., Bolander, M. E., and Sarkar, G. (1997) An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR.Mol. Biotechnol. 8, 135–137.PubMedGoogle Scholar
- 10.Maniatis, T., Fritsch, E. F., and Sambrook, J. (1992) Isolation of high-molecular weight, eukaryotic DNA from cells grown in tissue culture, inMolecular Cloning: A Laboratory Manual (Maniatis, T., Fritsch, E. F., and Sambrook, J., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 280,281.Google Scholar
- 11.Haqqi, T. M., Sarkar, G., David, C. S., and Sommer, S. S. (1988) Specific amplification with PCR of a refractory segment of genomic DNA.Nucleic Acids Res. 16, 11,844.CrossRefGoogle Scholar
- 12.Sarkar, G. and Bolander, M. E. (1995) Semi exponential cycle sequencing.Nucleic Acids Res. 23, 1269,1270.PubMedCrossRefGoogle Scholar
- 13.Sarkar, G. and Bolander, M. E. (1997) Direct sequencing of unpurified PCR-amplified DNA by Semi-Exponential Cycle Sequencing (SECS).Mol. Biotechnol. 8, 269–277.PubMedGoogle Scholar