Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidium.
A two-stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucaryotes. The method was evaluated using the ciliate protozoon Spathidium 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 Spathidium. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background autofluorescence. The Spathidium sequence is closely related to that from the haptorid Homalozoon vermiculare.
Dyal
P L
PL
Department of Zoology, Natural History Museum, London, UK.
Hope
S
S
Roberts
D M
DM
Embley
T M
TM
D000818:Animals; D001483:Base Sequence; D016798:Ciliophora / Q000166:cytology / Q000235:genetics*; D005456:Fluorescent Dyes; D017403:In Situ Hybridization / Q000379:methods; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D016053:RNA, Protozoan; D012335:RNA, Ribosomal