Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region
Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region
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DOI：
10.1016/s0168-6496(98)00031-2
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发表时间：
1998-06-01
影响因子：
4.2
通讯作者：
Molin, S
中科院分区：
文献类型：
--
作者：
Hansen, MC;Tolker-Nielsen, T;Molin, S
16S rDNA的PCR扩增有很强的偏向性，因此四个物种中的一个物种的rDNA被优先扩增。我们提出的证据表明，观察到的PCR偏差很可能是因为某些物种的基因组DNA包含扩增序列之外的片段，这些片段抑制了最初的PCR步骤。试图通过使用降落式聚合酶链式反应程序或在变性剂或助溶剂(如乙酰胺、二甲基亚砜或甘油)存在的情况下进行聚合酶链式反应来克服这种偏见的尝试失败了。由于模板侧翼DNA片段的聚合酶链式反应抑制干扰明显依赖于引物点的位置，因此我们建议将基于16S rDNA扩增的群落多样性分析方法从只用一个引物组扩增的16S rDNA的比较分析扩展到用不同的引物组进行至少两个不同的16S rDNA扩增。(C)1998年，爱思唯尔科学公司出版。保留所有权利。
PCR amplification of 16S rDNA was found to be highly biased, so that the rDNA from one species out of four was preferentially amplified. We present evidence that the observed PCR bias most likely occurs because the genomic DNA of some species contains segments outside the amplified sequence that inhibit the initial PCR steps. Attempts to overcome this bias by use of a 'touch down' PCR procedure or by performing PCR in the presence of denaturants or cosolvents such as acetamide, DMSO, or glycerol were unsuccessful. Since the PCR inhibiting interference from template flanking DNA segments evidently is dependent on the position of the primer sites, we suggest that community diversity analysis based on PCR amplification of 16S rDNA can be improved by extending the procedure from comparative analysis of 16S rDNA amplified by use of only one primer set to a procedure involving at least two different 16S rDNA PCR amplifications performed with different primer sets. (C) 1998 Published by Elsevier Science B.V. All rights reserved.