毛细管电泳快速检测限制性内切酶酶切产物

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。

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

Objective] The research aimed to establish a kind of rapid and effective method for detecting the enzyme-digested products of restriction endonuclease by using capillary electrophoresis.Method] With methyl cellulose(MC) as sieving matrix,PBR322/BsuRⅠDNA 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 MspⅠendonuclease 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.The 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 MspⅠendonuclease were detected and 3 enzyme-digested fragments were detected in 20 min.Conclusion] Compared with flat gel electrophoresis,it was more effective to detect the enzyme-digested products of small restriction endonuclease by using capillary electrophoresis.