Abstract
A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.
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.
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lüdecke H. J., Senger G., Claussen U., Horsthemke B. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature. 1989 Mar 23;338(6213):348–350. doi: 10.1038/338348a0. [DOI] [PubMed] [Google Scholar]
- Montgomery E., Charlesworth B., Langley C. H. A test for the role of natural selection in the stabilization of transposable element copy number in a population of Drosophila melanogaster. Genet Res. 1987 Feb;49(1):31–41. doi: 10.1017/s0016672300026707. [DOI] [PubMed] [Google Scholar]
- Pardue M. L., Gall J. G. Nucleic acid hybridization to the DNA of cytological preparations. Methods Cell Biol. 1975;10:1–16. doi: 10.1016/s0091-679x(08)60727-x. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]