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Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem
Author(s) -
Triga Dimitra,
Pamjav Horolma,
Vellai Tibor,
Fodor András,
Buzás Zsuzsanna
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:6<1274::aid-elps1274>3.0.co;2-e
Subject(s) - restriction fragment length polymorphism , biology , agarose gel electrophoresis , restriction enzyme , agarose , microbiology and biotechnology , polymerase chain reaction , genomic dna , electrophoresis , gel electrophoresis , restriction digest , polyacrylamide gel electrophoresis , dna , amplified fragment length polymorphism , restriction fragment , genetics , gene , biochemistry , enzyme , genetic diversity , population , demography , sociology
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.)