RAPID-DETERMINATION OF 16S RIBOSOMAL-RNA SEQUENCES FOR PHYLOGENETIC ANALYSES
RAPID-DETERMINATION OF 16S RIBOSOMAL-RNA SEQUENCES FOR PHYLOGENETIC ANALYSES
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DOI：
10.1073/pnas.82.20.6955
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
PACE, NR
中科院分区：
文献类型：
--
作者：
LANE, DJ;PACE, B;PACE, NR
虽然小亚基核糖体RNA（16 S rRNA）序列用于细菌分类的适用性现在被广泛接受，但这些分子的一般使用受到获得其序列的技术困难的阻碍。描述了一种用于快速产生大块16 S rRNA序列数据而不分离16 S rRNA或克隆其基因的方案。通过使用逆转录酶和与普遍保守的16 S rRNA序列互补的合成寡脱氧核苷酸引物，选择性靶向批量细胞RNA制剂中的16 S rRNA进行双脱氧核苷酸终止测序。三个特别有用的引发位点提供了进入三个主要16 S rRNA结构域的途径，通常产生800-1000个核苷酸的16 S rRNA序列。该方法的准确性，灵敏度的模板RNA中的修饰的核苷酸，和系统发育的有用性，通过检查几个16 s rRNA的基因序列是已知的。这种方法的相对简单，应有利于快速扩展的16 S rRNA序列收集系统发育分析。
Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16s rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.