Improved accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standard definition
Improved accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standard definition
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DOI：
10.1111/j.1399-302x.2007.00384.x
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发表时间：
2007-12-01
影响因子：
--
通讯作者：
Yamashita, Y.
中科院分区：
文献类型：
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作者：
Takeshita, T.;Nakano, Y.;Yamashita, Y.
背景资料：末端限制性片段长度多态性（T-RFLP）分析通常用于分析微生物群落，包括口腔微生物群落。然而，精确鉴定末端限制性片段（T-RF）的来源是由不可预测的错误大小阻止，从而需要克隆文库分析。为了最大限度地减少尺寸误差，我们提出了优化的大小定义的内部standards.Methods：GeneScan-1000 ROX再生作为内标，通过重新定义的片段大小的分子量（MW）的基础上，其流动性相对于6-羧基荧光素（FAM）标记的限制性片段来自16 S重组RNA基因的牙龈卟啉单胞菌。应用新的粒度定义，对6门8种口腔细菌的平均粒度误差进行了估计，并与传统方法进行了比较。使用新的MW大小定义分析从唾液中分离的微生物群落。使用TRFMA，一个基于Web的工具T-RFLP分析，并与那些在克隆库analysis.Results：使用新的大小定义，40个T-RFs的平均大小的错误被分配到峰的细菌物种被大幅减少，从2.42到0.62碱基，和大的尺寸误差（超过两个碱基）被消除。超过90%的克隆文库分析检测到的总细菌克隆被分配T-RFLP.Conclusion：新构建的内标物的大小定义减少了片段大小的错误，并允许准确的分配细菌峰的T-RFLP分析。这为研究微生物群落，包括口腔微生物区系提供了更有效的手段。
Background: Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used to analyze microbial communities, including oral microflora. However, accurate identification of terminal restriction fragment (T-RF) origins is prevented by unpredictable errors in sizing, thus necessitating the clone library analysis. To minimize sizing errors, we proposed optimizing the size definition of internal standards.Methods: GeneScan-1000 ROX was regenerated as an internal standard by redefining the fragment sizes in terms of molecular weight (MW) based on their mobility relative to 6-carboxyfluorescein (FAM) -labeled restriction fragments derived from the 16S recombinant RNA gene of Porphyromonas gingivalis. Using the new size definition, the average sizing error among eight oral bacteria from six phyla was estimated and compared with that of the conventional method. Microbial communities isolated from saliva were analyzed using the new MW size definition. Bacterial species were assigned to peaks using TRFMA, a Web-based tool for T-RFLP analysis, and compared with those identified in a clone library analysis.Results: Using the new size definition, the average sizing error for 40 T-RFs was drastically reduced from 2.42 to 0.62 bases, and large sizing errors (more than two bases) were eliminated. More than 90% of the total bacterial clones detected by the clone library analysis were assigned by T-RFLP.Conclusion: The size definition of the newly constructed internal standards reduced fragment sizing errors and allowed for accurate assignment of bacteria to peaks by the T-RFLP analysis. This provided a more effective means for studying microbial communities, including the oral microflora.