Abstract
A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
- Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clemens P. R., Fenwick R. G., Chamberlain J. S., Gibbs R. A., de Andrade M., Chakraborty R., Caskey C. T. Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Genet. 1991 Nov;49(5):951–960. [PMC free article] [PubMed] [Google Scholar]
- Edwards A., Civitello A., Hammond H. A., Caskey C. T. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum Genet. 1991 Oct;49(4):746–756. [PMC free article] [PubMed] [Google Scholar]
- Hearne C. M., Ghosh S., Todd J. A. Microsatellites for linkage analysis of genetic traits. Trends Genet. 1992 Aug;8(8):288–294. doi: 10.1016/0168-9525(92)90256-4. [DOI] [PubMed] [Google Scholar]
- Koenig M., Hoffman E. P., Bertelson C. J., Monaco A. P., Feener C., Kunkel L. M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987 Jul 31;50(3):509–517. doi: 10.1016/0092-8674(87)90504-6. [DOI] [PubMed] [Google Scholar]
- Thacker J., Chalk J., Ganesh A., North P. A mechanism for deletion formation in DNA by human cell extracts: the involvement of short sequence repeats. Nucleic Acids Res. 1992 Dec 11;20(23):6183–6188. doi: 10.1093/nar/20.23.6183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vanin E. F., Henthorn P. S., Kioussis D., Grosveld F., Smithies O. Unexpected relationships between four large deletions in the human beta-globin gene cluster. Cell. 1983 Dec;35(3 Pt 2):701–709. doi: 10.1016/0092-8674(83)90103-4. [DOI] [PubMed] [Google Scholar]
- Weber J. L., May P. E. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet. 1989 Mar;44(3):388–396. [PMC free article] [PubMed] [Google Scholar]
- Weissenbach J., Gyapay G., Dib C., Vignal A., Morissette J., Millasseau P., Vaysseix G., Lathrop M. A second-generation linkage map of the human genome. Nature. 1992 Oct 29;359(6398):794–801. doi: 10.1038/359794a0. [DOI] [PubMed] [Google Scholar]