DNA recovery from soils of diverse composition
DNA recovery from soils of diverse composition
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DOI：
10.1128/aem.62.2.316-322.1996
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发表时间：
1996-02-01
影响因子：
4.4
通讯作者：
Tiedje, JM
中科院分区：
文献类型：
--
作者：
Zhou, JZ;Bruns, MA;Tiedje, JM
为了解决小模板DNA在后续PCR中形成嵌合体的风险，建立了一种简单、快速的细菌裂解和直接提取土壤DNA的方法，该方法基于高盐提取缓冲液裂解（1.5 M NaCl）和延长加热（2至3小时）的土壤悬浮液在十二烷基硫酸钠（SDS），十六烷基三甲基溴化铵，和蛋白酶K的存在下。该提取方法需要6 h，并在8种有机碳、粘粒含量和pH不同的土壤上进行了测试，包括难以提取DNA的土壤。所有土壤粗提物中的DNA片段大小均>23 kb。初步试验表明，从两种土壤接种革兰氏阴性细菌的DNA回收率为92%至99%。当该方法在所有8个未播种的土壤中进行测试时，提取前后土壤颗粒中的土著细菌的显微镜检查显示可变的细胞裂解效率（26%至92%）。8种土壤的DNA产量在2.5 ~ 26.9 μ gDNA g（-1）之间，与土壤有机碳含量呈正相关（r = 0.73），当高盐SDS加热法与研钵和研杵研磨和冻融结合时，来自纯培养物的革兰氏阳性菌的DNA产量高2至6倍，对4种纯化方法的回收率、片段大小、速度、酶切、PCR扩增和DNA-DNA杂交进行了评价。总的来说，所有方法都能产生足够纯度的DNA用于PCR扩增。由于土壤类型和微生物群落特征会影响DNA的回收，本研究为根据实验目的选择合适的提取和纯化方法提供了指导。
A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR, The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult, The DNA fragment size in crude extracts from all soils was >23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 mu g of DNA g(-1), and these were positively correlated with the organic carbon content in the soil (r = 0.73), DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight, Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification, Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.