Abstract
The limiting factor in the presently available techniques for the detection of DNA sequence variation in the human genome is the low resolution of Southern blot analysis. To increase the analytical power of this technique, we applied size fractionation of genomic DNA restriction fragments in conjunction with their sequence-dependent separation in denaturing gradient gels; the two-dimensional separation patterns obtained were subsequently transferred to nylon membranes. Hybridization analysis using minisatellite core sequences as probes resulted in two-dimensional genomic DNA fingerprints with a resolution of up to 625 separated spots per probe per human individual; by conventional Southern blot analysis, only 20-30 bands can be resolved. Using the two-dimensional DNA fingerprinting technique, we demonstrate in a small human pedigree the simultaneous transmission of 37 polymorphic fragments (out of 365 spots) for probe 33.15 and 105 polymorphic fragments (out of 625 spots) for probe 33.6. In addition, a mutation was detected in this pedigree by probe 33.6. We anticipate that this method will be of great use in studies aimed at (i) measuring human mutation frequencies, (ii) associating genetic variation with disease, (iii) analyzing genomic instability in relation to cancer and aging, and (iv) linkage analysis and mapping of disease genes.
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.
- Colb M., Yang-Feng T., Francke U., Mermer B., Parkinson D. R., Krontiris T. G. A variable tandem repeat locus mapped to chromosome band 10q26 is amplified and rearranged in leukocyte DNAs of two cancer patients. Nucleic Acids Res. 1986 Oct 24;14(20):7929–7937. doi: 10.1093/nar/14.20.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Delehanty J., White R. L., Mendelsohn M. L. International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC Meeting Report No. 2. Approaches to determining mutation rates in human DNA. Mutat Res. 1986 May;167(3):215–232. doi: 10.1016/0165-1110(86)90031-x. [DOI] [PubMed] [Google Scholar]
- Donis-Keller H., Green P., Helms C., Cartinhour S., Weiffenbach B., Stephens K., Keith T. P., Bowden D. W., Smith D. R., Lander E. S. A genetic linkage map of the human genome. Cell. 1987 Oct 23;51(2):319–337. doi: 10.1016/0092-8674(87)90158-9. [DOI] [PubMed] [Google Scholar]
- Fischer S. G., Lerman L. S. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1579–1583. doi: 10.1073/pnas.80.6.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer S. G., Lerman L. S. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell. 1979 Jan;16(1):191–200. doi: 10.1016/0092-8674(79)90200-9. [DOI] [PubMed] [Google Scholar]
- Fischer S. G., Lerman L. S. Two-dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods Enzymol. 1979;68:183–191. doi: 10.1016/0076-6879(79)68013-8. [DOI] [PubMed] [Google Scholar]
- Jeffreys A. J., Royle N. J., Wilson V., Wong Z. Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature. 1988 Mar 17;332(6161):278–281. doi: 10.1038/332278a0. [DOI] [PubMed] [Google Scholar]
- Jeffreys A. J., Wilson V., Thein S. L. Hypervariable 'minisatellite' regions in human DNA. Nature. 1985 Mar 7;314(6006):67–73. doi: 10.1038/314067a0. [DOI] [PubMed] [Google Scholar]
- Jeffreys A. J., Wilson V., Thein S. L. Individual-specific 'fingerprints' of human DNA. Nature. 1985 Jul 4;316(6023):76–79. doi: 10.1038/316076a0. [DOI] [PubMed] [Google Scholar]
- Jeffreys A. J., Wilson V., Thein S. L., Weatherall D. J., Ponder B. A. DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees. Am J Hum Genet. 1986 Jul;39(1):11–24. [PMC free article] [PubMed] [Google Scholar]
- Nakamura Y., Leppert M., O'Connell P., Wolff R., Holm T., Culver M., Martin C., Fujimoto E., Hoff M., Kumlin E. Variable number of tandem repeat (VNTR) markers for human gene mapping. Science. 1987 Mar 27;235(4796):1616–1622. doi: 10.1126/science.3029872. [DOI] [PubMed] [Google Scholar]
- Reeders S. T., Breuning M. H., Davies K. E., Nicholls R. D., Jarman A. P., Higgs D. R., Pearson P. L., Weatherall D. J. A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature. 1985 Oct 10;317(6037):542–544. doi: 10.1038/317542a0. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Thein S. L., Jeffreys A. J., Gooi H. C., Cotter F., Flint J., O'Connor N. T., Weatherall D. J., Wainscoat J. S. Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br J Cancer. 1987 Apr;55(4):353–356. doi: 10.1038/bjc.1987.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vijg J., Uitterlinden A. G. A search for DNA alterations in the aging mammalian genome: an experimental strategy. Mech Ageing Dev. 1987 Nov;41(1-2):47–63. doi: 10.1016/0047-6374(87)90053-4. [DOI] [PubMed] [Google Scholar]
- Wyman A. R., White R. A highly polymorphic locus in human DNA. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6754–6758. doi: 10.1073/pnas.77.11.6754. [DOI] [PMC free article] [PubMed] [Google Scholar]