Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer
Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer
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DOI：
10.3390/microorganisms8040532
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发表时间：
2020-04-01
期刊：
影响因子：
4.5
通讯作者：
Karlovsky, Petr
中科院分区：
文献类型：
--
作者：
Guerra, Victor;Beule, Lukas;Karlovsky, Petr
随着我们对土壤生物学的理解不断加深，人们越来越需要对表层土壤（称为底土）下的微生物过程进行调查。底土中的高粘土含量往往阻碍了足够数量的DNA的回收，因为粘土颗粒结合核酸。在这里，一个有效的和可重复的DNA提取方法为200毫克干燥土壤的基础上十二烷基硫酸钠（SDS）裂解在磷酸盐缓冲液的存在下，已开发。通过定量从通过裂解方法和磷酸盐缓冲液洗涤的不同组合获得的提取物扩增的细菌16S和真菌18S rRNA基因来优化提取方案。发现珠粒撞击一分钟，然后在含有0.5%SDS的IM磷酸盐缓冲液存在下在65 ° C下孵育十分钟的组合产生最佳结果。优化的协议进行了比较，与常用的十六烷基三甲基溴化铵（CTAB）的方法，使用黑土从60厘米深的常规农业领域，并验证5个底土。通过实验室间比较证实了方案的重现性和耐用性。DNA提取方案为基于DNA的底土生物学研究提供了一种可重复且具有成本效益的工具。
As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65 degrees C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.