Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR
Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR
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DOI：
10.1016/j.mimet.2004.01.013
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发表时间：
2004-05-01
影响因子：
2.2
通讯作者：
Findlay, RH
中科院分区：
文献类型：
--
作者：
Mumy, KL;Findlay, RH
分子生物学技术通过分析核酸推进了微生物生态学领域。大多数使用DNA或RNA的技术需要从环境基质中提取它们，这可能是繁琐和低效的。虽然已经开发了许多基于实验室和商业上可用的提取方法，但这些方法都不包括确定提取效率的方便方法。我们开发了一种外部DNA回收标准品，即pBR 322中包含的λ DNA（靶DNA），可用于DNA回收效率的常规测定。将靶DNA作为全细胞加入到沉积物中，使用商业DNA提取/纯化试剂盒提取总DNA，并通过定量竞争性PCR（QC-PCR）定量回收的靶DNA的量。三种市售试剂盒（UltraClean（TM）Soil DNA. FastDNA SPIN R和Soil Master（TM）DNA Extraction）的回收率。三种试剂盒的回收率范围从不可检测到43.3%，平均回收率为14.9 +/-16.0%、28.3 +/- 10.5%和2.4 +/- 0.1%（UItraClean. FastDNA和Soil Master）。在被多环芳烃严重污染的沉积物中，目标DNA的定量证明是可靠的，在提取和扩增后，可以从加入0.5 g沉积物（湿重）的1x 10（3）个细胞中检测到外部回收标准。在提取前，还将外部DNA回收标准品作为纯化质粒DNA直接加入沉淀物中。当作为全细胞加入时，它以类似的效率回收，表明其在核糖体DNA研究中估计DNA回收的有用性。这些结果表明，虽然商业试剂盒提供了快速的样品处理，但提取效率因样品而异，并且<100%。因此，定量DNA研究需要估计DNA回收率。(C)2004 Elsevier B. V.保留所有权利。
Molecular biology techniques have advanced the field of microbial ecology through the analysis of nucleic acids. Most techniques that use DNA or RNA require their extraction from environmental matrices, which can be tedious and inefficient. While a number of extraction methods, both laboratory-based and commercially available, have been developed, none of these include a convenient method to determine extraction efficiency. We have developed an external DNA recovery standard, Lambda DNA (target DNA) contained within pBR322, allowing routine determinations of DNA recovery efficiency. Target DNA was added to sediments as whole cells, total DNA extracted using commercial DNA extraction/purification kits and the amount of target DNA recovered quantified by quantitative-competitive PCR (QC-PCR). Three commercially available kits (UltraClean(TM) Soil DNA. FastDNA SPIN R and Soil Master(TM) DNA Extraction) were evaluated for recovery efficiency. Recoveries for the three kits ranged from undetectable to 43.3% with average recoveries of 14.9 +/- 16.0%, 28.3 +/- 10.5% and 2.4 +/- 0.1% (UItraClean. FastDNA and Soil Master, respectively). Quantification of target DNA proved robust in sediments heavily polluted with polycyclic aromatic hydrocarbons and the external recovery standard could be detected following extraction and amplification from as few as 1 x 10(3) cells added to 0.5 g sediment (wet weight). The external DNA recovery standard was also added directly to the sediment as purified plasmid DNA prior to extraction. It was recovered with similar efficiency as when added as whole cells, suggesting its usefulness in estimating DNA recovery in ribosomal DNA Studies. These results show that, while the commercial kits offer expedited sample processing, the extraction efficiencies vary on a sample-by-sample basis and were < 100%. Therefore, quantitative DNA studies require an estimation of DNA recovery. (C) 2004 Elsevier B.V. All rights reserved.