PHYLOGENETIC GROUP-SPECIFIC OLIGODEOXYNUCLEOTIDE PROBES FOR IDENTIFICATION OF SINGLE MICROBIAL-CELLS
PHYLOGENETIC GROUP-SPECIFIC OLIGODEOXYNUCLEOTIDE PROBES FOR IDENTIFICATION OF SINGLE MICROBIAL-CELLS
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DOI：
10.1128/jb.170.2.720-726.1988
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发表时间：
1988-02-01
影响因子：
3.2
通讯作者：
PACE, NR
中科院分区：
文献类型：
--
作者：
GIOVANNONI, SJ;DELONG, EF;PACE, NR
对16 S rRNA序列集合的检查揭示了细胞谱系的三个主要谱系（古细菌、真细菌和真核细胞）所特有的（并且在其中不变的）序列结构域。合成与这些保守序列结构域互补的寡脱氧核苷酸并用作杂交探针。每个放射性标记的探针特异性杂交尼龙膜结合的16S rRNA从目标王国。使用与16S rRNA中普遍保守序列互补的探针作为阳性对照，而其互补物为非特异性结合提供阴性对照。在全细胞斑点印迹分析中测试探针特异性结合代表广泛的系统发育多样性的完整固定细胞的能力。同样，所有探针都特异性结合靶向基团。通过显微放射自显影，该方法被扩展到允许显微镜下的单细胞系统发育鉴定。
Examination of collections of 16S rRNA sequences revealed sequence domains that were unique to (and invariant within) the three primary lines of cellular descent: the archaebacteria, the eubacteria, and the eucaryotes. Oligodeoxynucleotides complementary to these conserved sequence domains were synthesized and used as hybridization probes. Each of the radiolabeled probes specifically hybridized to nylon membrane-bound 16S rRNA from the targeted kingdom. A probe complementary to a universally conserved sequence in 16S rRNAs was used as a positive control, while its complement provided a negative control for nonspecific binding. The abilities of the probes to bind specifically to whole, fixed cells representing a broad array of phylogenetic diversity were tested in whole-cell dot blot assays. Again, all of the probes specifically bound the targeted groups. By microautoradiography, the method was extended to permit phylogenetic identification of single cells microscopically.