A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase.
A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase.
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DOI：
10.1371/journal.pone.0042149
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Buckley DH
中科院分区：
文献类型：
--
作者：
Gaby JC;Buckley DH
nifH基因是用于鉴定固氮细菌和固氮菌的最广泛测序的标记基因。已经设计了许多PCR引物来扩增nifH，但尚未对nifH PCR引物进行全面评价。我们通过使用23，847个nifH序列的比对数据库对51个通用和35个组特异性nifH引物的特异性和覆盖率进行了计算机分析。我们发现有15个通用nifH引物靶向90%或更多的固氮剂，但也有23个nifH引物靶向不到50%的nifH序列。我们评估的nifH引物在它们的系统发育偏倚和它们从通常采样的环境中恢复序列的能力方面各不相同。此外，这些引物中的许多引物将扩增不介导固氮的基因，因此研究人员在分析前筛选其测序结果以确定是否存在非靶基因是明智的。通用引物进行了测试，以及在硅片经验与土壤样品和基因组DNA从一个遗传多样性的固氮菌株。这一分析将是非常有用的那些从事nifH基因的分离物和环境样品的分子分析。
The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.
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