DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA.
DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA.
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DOI：
10.1099/00207713-50-3-1095
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发表时间：
2000-05
影响因子：
2.8
通讯作者：
Henrik Christensen;Ø. Angen;Reinier Mutters;John Elmerdahl Olsen;Magne Bisgaard
中科院分区：
文献类型：
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作者：
Henrik Christensen;Ø. Angen;Reinier Mutters;John Elmerdahl Olsen;Magne Bisgaard
本研究的目的是减少在物种水平上进行DNA-DNA杂交以进行细菌分类所需的时间和人力。建立了一种微孔型DNA杂交方法，并进行了验证。DNA提取是通过小规模的方法进行的，DNA被机械地剪切成400到700个碱基的片段。根据用分光光度法获得的DNA相似性，对杂交条件进行校正。最佳条件是每孔添加300 ng DNA，并通过共价结合到NucleoLink上。用500ngDNA进行杂交，其中5%(w/w)的DNA被光激活生物素(竞争杂交)标记，在65℃下在2x SSC中杂交2.5h，然后在相同温度下用2x SSC严格洗涤。接受结果的标准是最大15%的标准偏差，以四个重复微孔的平均值的百分比计算，至少在两个独立的实验中DNA相似性没有显著差异。微孔法(Y)与分光光度法(X)的DNA相似性关系为y=0.534x+30.6，用于23株放线杆菌、禽类溶血杆菌和曼氏杆菌。通过交换用于共价结合的菌株和杂交获得的DNA相似性之间的相关性(皮尔森)为0.794。与微孔法相比，分光光度法对三对杂交的DNA相似性显著降低。去除这些数据后，微孔法和分光光度法获得的DNA相似性之间的关系改善到y=0.855x+11.0。结果表明，微孔法的准确度和精密度与分光光度法相当，但大大减少了人工和分析时间。微孔杂交的使用将允许在为特定分类研究选定的所有菌株之间进行DNA-DNA杂交，以便构建完整的数据矩阵和改进物种定义。
The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.