液相杂交快速检测特异DNA  

Fast Detection of Specific DNA by Liquid Hybridization

作　　者：詹诚[1] 毛强[1] 王胜华[1] 陈放[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065

出　　处：《中国生物工程杂志》2013年第7期64-70,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(31070276)

摘　　要：检测特异DNA片段的方法中,传统Southern blot技术由于其高度可重复性及能够显示条带大小的特性,一直是DNA检测的"黄金标准"。但是杂交时间长,步骤复杂,放射性污染等问题亟待解决。为了简化Southern blot,研究使用了一种液相杂交快速检测DNA的方法,即使用异硫氰酸荧光素(FITC)标记的dUTP掺入探针后,在溶液中与待检测DNA样本42℃下杂交,然后琼脂糖凝胶电泳检测荧光杂交信号。利用质粒为模板,优化了探针制作、杂交液组成、杂交时间和温度等参数。在FITC-dUTP:dTTP比例为1∶3、模板质粒浓度为50μg、1×杂交缓冲液(25mmol/LTris,10mmol/L EDTA,8mmol/L Nacl,PH=8.0)中95℃变性5～9 min和42℃杂交3h的实验条件下,可检出1.2μg的质粒,探针灵敏度为7.3ng/μl。这种方法不需要转膜,曝光,大大节约了时间,简化了操作,荧光检测也为该方法同时检测多色样本提供了可能,可广泛应用于核酸检测。Among the methods of DNA detection,Southern blot is the 'golden standard' because of its high repeatability and the specialty of displaying the size of DNA.However,problem should be solved like long hybridizing time,complex procedure and radioactive contamination.To simplify the traditional Southern blot,here is a fast way for DNA detection by liquid hybridization: Use fluorescein isothiocyanate(FITC) labeled dUTP to make probe by PCR,hybridize with the sample DNA in liquor at 42℃ for 3 hours,and detect the fluorescence after gel running.The optimization of probe making,hybridized buffer,hybridized time and temperature were illustrated.Optimized conditions of making probe was: dUTP∶dTTP=1∶3,50ng template DNA.The best degeneration time was 5~9min at 95℃.Sufficient hybridization occurs after 3 hours at 42℃ in liquor.Optimal PH of hybridization buffer was 8.0,hybridization band occurs when NaCl concentration was 8mmol/L.This method could detect a minimum of 1.2μg plasmid;the detection limit of probe is 7.3ng/μl.Signal could be detected under blue light source.Apparatus like Bio-rad Gel Doc could limit the background and detect higher signal.This method was verified by detect GUS gene in transgenic tobacco.There is no need for film transferring,exposure for this method,which reduced time,simplified the steps.Also,fluorescence detection raise the possibility of detect multi-color in one assay.This method could be used in nucleic acid detection widely.

关 键 词：SOUTHERN BLOT 液相杂交 FITC-dUTP DNA检测 方法优化 

分 类 号：Q523[生物学—生物化学]