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Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn
Author(s) -
DYAL P. L.,
HOPE S.,
ROBERTS D. M.,
EMBLEY T. M.
Publication year - 1995
Publication title -
molecular ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.619
H-Index - 225
eISSN - 1365-294X
pISSN - 0962-1083
DOI - 10.1111/j.1365-294x.1995.tb00244.x
Subject(s) - biology , ciliate , gene , ribosomal rna , microbiology and biotechnology , autofluorescence , oligonucleotide , sequence (biology) , fluorescence , genetics , physics , quantum mechanics
A two‐stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary‐otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto‐fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.