Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts
Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts
复制标题
DOI：
10.1128/aem.69.1.320-326.2003
复制
发表时间：
2003-01-01
影响因子：
4.4
通讯作者：
Friedrich, MW
中科院分区：
文献类型：
--
作者：
Lueders, T;Friedrich, MW
末端限制性片段长度多态性 (T-RFLP) 分析是一种广泛使用的方法，通过针对小亚基 (SSU) rRNA 和功能标记基因来分析不同生境中的微生物群落结构。然而，尚不清楚各个群落成员的相对基因频率是否在 T-RFLP 所示的 PCR 后扩增子频率中得到充分代表。在本研究中，制备了精确定义的人工模板混合物，其中含有不同比例的四种不同产甲烷菌的基因组 DNA，用于随后的 T-RFLP 分析。 PCR 扩增子由既定混合物生成，不仅针对 SSU rRNA，还针对产甲烷菌的甲基辅酶 M 还原酶 (mcrA/mrtA) 基因。通过比较特征末端限制性片段的荧光强度来量化微生物的相对扩增子频率。通过 PCR-T-RFLP 分析完全恢复了四成员群落定义模板混合物中的 SSU 核糖体 DNA (rDNA) 模板比例，这表明所评估的 T-RFLP 分析可以给出模板库的定量视图。发现自然群落的 SSU rDNA 靶向 T-RFLP 分析具有高度重现性，与 PCR 退火温度无关，并且不受 PCR 循环次数增加的影响。 mcrA 靶向 T-RFLP 分析的比率存在偏差，很可能是由于所用引物的简并性而通过 PCR 选择造成的。因此，对于微生物群落分析，应仔细评估所使用的每个引物系统是否存在可能的 PCR 偏差。事实上，可以通过使用 T-RFLP 分析作为 PCR 产物库精确定量的工具来检测这种偏差。
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.