ZHAN Cheng, MAO Qiang, WANG Sheng-hua, CHEN Fang
China Biotechnology. 2013, 33(7): 64-70.
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.
