Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four res...

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Bibliographic Details
Published in:Electrophoresis 1999-06, Vol.20 (6), p.1274-1279
Main Authors: Triga, Dimitra, Pamjav, Horolma, Vellai, Tibor, Fodor, András, Buzás, Zsuzsanna
Format: Article
Language:English
Subjects:
Animals
DNA, Helminth - analysis
Electrophoresis, Agar Gel - methods
Electrophoresis, Polyacrylamide Gel - methods
Individual nematodes
Internally transcribed spacer sequence
Molecular taxonomy
PhastSystem
Polymorphism, Restriction Fragment Length
Rhabditoidea - genetics
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Summary:The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions (Pamjav et al., Electrophoresis 1999, 20, 1264—1271.)
ISSN:0173-0835
1522-2683
DOI:10.1002/(SICI)1522-2683(19990101)20:6<1274::AID-ELPS1274>3.0.CO;2-E