在75℃下使用单链特异性核酸酶测定脱氧核糖核酸同源性。
Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.
作者信息
Barth P T, Grinter N J
出版信息
J Bacteriol. 1975 Feb;121(2):434-41. doi: 10.1128/jb.121.2.434-441.1975.
Abstract
We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.
摘要
我们研究了核酸酶S1粗制品在何种条件下能使变性脱氧核糖核酸（DNA）实现最大程度的水解，同时使天然DNA的水解程度降至最低。通过过滤并使用³H标记的底物测定酸不溶性反应产物来衡量水解情况。我们还研究了进行该测量时的各种参数。在适当条件下（在pH 4.8的1 mM硫酸锌、0.168 M氯化钠中），变性DNA在反应完成3%以内被水解，而天然DNA基本不受影响。该反应被用于检测质粒DNA同源双链体和异源双链体，结果证明该方法简单、快速且可重复。
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