CAJ | 학술논문

目的探讨DNA经不同波长紫外线照射与经紫外线照射不同时间所致损伤对基因库构建中DNA连接转化效率的影响.方法经改造的质粒DNA使用Sfi Ⅰ酶切过夜,1%琼脂糖凝胶电泳分离,在不同波长不同时间紫外线照射的条件下回收目的片断,并进行目的片断的连接转化,计数菌落数,计算转化效率.结果经302nm波长紫外线照射后,其连接转化的菌落数极少(60个/ng DNA),365nm波长紫外线照射条件下回收的DNA仍可被有效连接转化.在365nm波长紫外线照射下,与未经照射进行比较,当照射时间在30 min,其连接转化成功率低于50%,而照射5 min、15 min的连接转化成功率仍高于70%,可满足大部分分子生物学实验的需要.回收的DNA连接转化的最大效率可超过20000个菌落/1 ng DNA.结论在构建大容量基因库回收目的片断时,应避免紫外线的照射;在需要时,应选择在365 mn波长紫外线照射条件下进行,且照射时间应尽量缩短,以不超过15 min为宜.
목적 탐토DNA경불동파장자외선조사여경자외선조사불동시간소치손상대기인고구건중DNA련접전화효솔적영향.방법 경개조적질립DNA사용Sfi Ⅰ매절과야,1%경지당응효전영분리,재불동파장불동시간자외선조사적조건하회수목적편단,병진행목적 편단적련접전화,계수균락수,계산전화효솔.결과 경302nm파장자외선조사후,기련접전화적균락수겁소(60개/ng DNA),365nm파장자외선조사조건하회수적DNA잉가피유효련접전화.재365nm파장자외선조사하,여미경조사진행비교,당조사시간재30 min,기련접전화성공솔저우50%,이조사5 min、15 min적련접전화성공솔잉고우70%,가만족대부분분자생물학실험적수요.회수적DNA련접전화적최대효솔가초과20000개균락/1 ng DNA.결론 재구건대용량기인고회수목적편단시,응피면자외선적조사;재수요시,응선택재365 mn파장자외선조사조건하진행,차조사시간응진량축단,이불초과15 min위의.
Objective To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells. Methods The expression vector was digested with the restriction enzyme Sfi[, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed. Results The transformation efficiency of the DNA with a 5-rain exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments. Conclusion In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.