The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species
The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species
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DOI：
10.1099/13500872-142-5-1107
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发表时间：
1996-05-01
期刊：
影响因子：
2.8
通讯作者：
Wang, Y
中科院分区：
文献类型：
--
作者：
Wang, GCY;Wang, Y
我们对微生物多样性的理解受到无法培养生物圈中多达99%的微生物群落的极大阻碍。基于基因序列鉴定和确定微生物遗传的方法，以及直接从不同环境样品中回收基因的方法的发展，使得研究微生物而不需要培养成为可能。PCR技术已经彻底改变了保守基因序列的检索。然而，众所周知，同源基因的共扩增可能产生嵌合序列，导致描述不存在的物种。为了定量16S rRNA基因PCR扩增中嵌合序列的频率，我们选择了几个已知序列的16S rDNA，并在各种条件下将它们混合进行PCR扩增。使用该模型系统，我们检测到30%的嵌合序列发生后，30个循环的两个几乎相同的16S rRNA基因的共扩增。当模板的相似性为82%和86%时，嵌合体的形成率分别下降到12.9%和14.7%。我们还研究了扩增循环数、延伸期长度和受损DNA的存在对嵌合体形成的影响。这些结果应该为微生物学家提供有用的信息，他们使用基于PCR的策略从混合基因组中检索保守基因。
Our understanding of microbial diversity is greatly hampered by the inability to culture as much as 99% of the microbial community in the biosphere. Development of methods for identification and determining microbial phylogenies based on gene sequences, and for recovering genes directly from diverse environmental samples has made it possible to study microbes without the need for cultivation. PCR techniques have revolutionized retrieval of conserved gene sequences. However, it is well known that co-amplification of homologous genes may generate chimeric sequences leading to descriptions of non-existent species. To quantify the frequency of chimeric sequences in PCR amplification of 16S rRNA genes, we chose several 16S rDNAs with known sequences and mixed them for PCR amplifications under various conditions. Using this model system, we detected 30% occurrence of chimeric sequences after 30 cycles of co-amplification of two nearly identical 16S rRNA genes. The frequency of chimera formation decreased to 12.9% and 14.7% for templates with 82% and 86% similarity, respectively. We also examined effects of the number of amplification cycles, length of elongation periods and presence of damaged DNA on chimera formation. The results should provide useful information for microbiologists who use PCR-based strategies to retrieve conserved genes from mixed genomes.