毛细管电泳快速检测限制性内切酶酶切产物

Rapid Detection on the Enzyme-digested Products of Restriction Endonuclease by Using Capillary Electrophoresis

[目的]建立1种利用毛细管电泳快速、高效检测限制性内切酶酶切产物的方法。[方法]以甲基纤维素(MC)为筛分介质,用PBR322/BsuRⅠDNA Marker为试验对象,研究筛分介质浓度、pH值、毛细管柱温度和电场强度对毛细管电泳法分离小片段双链DNA的影响,寻求最佳电泳条件,并将此方法用于检测MspⅠ内切酶的酶切产物。[结果]MC浓度对双链小片段DNA的分离度有较大影响。随MC溶液浓度的增加,分离度呈先增后减的趋势。毛细管电泳的最佳条件为:MC浓度为2.0%,pH值为8.0,毛细管柱温度15℃,电场强度为275 V/cm。在此条件下,对MspⅠ内切酶的酶切产物进行检测,在20 min内检测到3个酶切片段。[结论]与平板凝胶电泳相比,运用毛细管电泳检测小片段限制性内切酶酶切产物更高效。

[ Objective] The research aimed to establish a kind of rapid and effective methed for detecting the enzyme-digested products of restriction endonuclease by using capillary electrophoresis. [Method] With methyl cellulose（MC） as sieving matrix, PBR322[BsuR]DNA Marker was taken as test object to study the effects of the concn, of the sieving matrix, pH value, the capillary column temperature and electric field intensity on separating small double-chain DNA fragment by using capillary electrophoresis and seek the optimum electrophoresis conditions. The enzyme-digested products of Msplendonuclease was detected by this method. [Result] MC concn, had greater effects on the separation degree of small double-chain DNA fragment. With the increasing of MC solution concn., the separation degree showed the trend of first increasing and then decreasing. 3he optimum condition for capillary electrophoresis was as 2.0% MC, pH value of 8.0,the capillary column temperature of 15℃ and electric field intensity of 275 V/cm. Under these conditions, the enzyme-digested products of Msplendonuclease were detected and 3 enzyme-digested fragments were detected in 20 rain. [ Conclusion] Compared with flat gel electrophoresis, it was more effective to detect the enzyme-digested products of small restriction endonuclease by using capillary electrophoresis