Ref ID : 2847
Manfred Wanner, Jorg M. Nahring, and Rainer Fischer; Molecular Identification of Clones of Testate Amoebae Using Single Nuclei for PCR Amplification. Europ.J.Protistol. 33:192-199, 1997
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Culture growth and shell morphology of clonal cultures of terrestrial testate amoebae are highly variable in altering environmental conditions which is clearly reproducible and even reversible. Due to these taxonomical and ecological implications, a genetic approach based on RAPD-PCR was developed to supplement currently available data. Only when paired random oligonucleotide primers were used, most analyzed cultures of testate amoebae showed fingerprints that appeared to be taxon-specific. Further experiments demonstrated that a foreign DNA source was responsible for these culture-specific RAPD patterns, although no eukaryotic contamination was visible. The data obtained pointed out that this foreign DNA source is distinct from the food or the culture medium components and located within the amoebae casing, and therefore was inevitably transferred with the amoebae to each new subculture. This severe problem of coamplifying foreign but culture-specific DNA can be prevented by using single nuclei of testate amoebae as a reference. In the case of Euglypha strigosa, single cells were divide into a nucleus-free and nucleus-containing part. Comparing one to 63 cells of different cultures of the same clone of Euglypha strigosa always resulted in one or two primer-dependent, highly reproducible bands of amplified DNA which only appeared in the amoeba-nucleus containing sample. Specific primers derived from one sequenced RAPD-DNA fragment demonstrated its amoebae-specific nuclear origin. These RAPD-derived specific primers allowed highly specific amplification of testate amoebae DNA derived from one nucleus even in the presence of large amounts of contaminating foreign DNA and provided the basis for more detailed molecular identification studies in the future.