THE CHARACTERIZATION OF ENZYMATICALLY AMPLIFIED EUKARYOTIC 16S-LIKE RRNA-CODING REGIONS
THE CHARACTERIZATION OF ENZYMATICALLY AMPLIFIED EUKARYOTIC 16S-LIKE RRNA-CODING REGIONS
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DOI：
10.1016/0378-1119(88)90066-2
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发表时间：
1988-11-30
期刊：
影响因子：
3.5
通讯作者：
SOGIN, ML
中科院分区：
文献类型：
--
作者：
MEDLIN, L;ELWOOD, HJ;SOGIN, ML
建立了聚合酶链反应条件以在真核小型亚基核糖体（16S样）rRNA基因的体外扩增。来自藻类，真菌和原生动物的编码区域是从含有RDNA基因的基因组DNA或重组质粒中扩增的。在5''和3'''真核16S样rRNA的末端辅助区域互补的寡脱氧核苷酸被用来在重复的变性，reannealing和DNA合成的重复循环中启用DNA合成。通过与先前报道的rDNA基因的序列或rRNA的引物扩展分析的序列进行比较来评估合成对振幅产物的保真度。在扩增的RRNA编码区域序列中，每个2000个位置少于一个误差。从其体外扩增的编码区的序列推断出，从海洋硅藻骨s骨Costatum的16S样rRNA的主要结构。
Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5'' and 3'' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplitication products was evaluated by comparisons with sequences of previously reported rDNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.