Chromosome walking in Dictyostelium: an application of the single specific primer PCR

We describe here an application of the polymerase chain reaction that sidesteps the necessity of knowing sequence information at both ends of a desired fragment of genomic DNA. The methodology is straightforward and works efficiently in organisms with relatively simple genomes. We have used it to wa...

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Bibliographic Details
Published in:Journal of microbiological methods 1997-02, Vol.28 (2), p.131-138
Main Author: Koonce, Michael P
Format: Article
Language:English
Subjects:
Biological and medical sciences
Dictyostelium discoideum
Diverse techniques
Fundamental and applied biological sciences. Psychology
Molecular and cellular biology
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Summary:We describe here an application of the polymerase chain reaction that sidesteps the necessity of knowing sequence information at both ends of a desired fragment of genomic DNA. The methodology is straightforward and works efficiently in organisms with relatively simple genomes. We have used it to walk more than 8 kb in six steps from a `starter' DNA sequence that was identified in a cDNA library screen. This technology complements existing procedures to walk in either direction along chromosomes, but because of the multiple levels of selection employed, it may prove to be more effective. Its primary utility will probably lie in extending sequence information in simple organisms that do not have pre-existing cDNA or genomic libraries, or for rapidly isolating genomic sequences that flank a cDNA clone.
ISSN:0167-7012
1872-8359
DOI:10.1016/S0167-7012(97)00974-3