溴化乙锭在毛细管DNA传感器中的应用

Application of ethidium bromide in capillary DNA biosensor

目的:构建一种新型毛细管的DNA生物传感器。方法:实验于2004-10/2005-12在四川大学华西基础医学与法医学院生物化学实验室完成。毛细管内壁通过poly-l-lysine将20-mer-ssDNA(探针)固定,与互补靶核苷酸杂交后,通过溴化乙锭染色后检测与0.033 mmol/L靶核苷酸杂交后的荧光强度;同样浓度的靶核苷酸序列连续测定3次,进行重现性比较;及对不同杂交时间的荧光强度进行检测。结果:通过溴化乙锭对毛细管固定探针与互补靶DNA杂交进行染色,用RF5000荧光光度仪测定荧光值,测定组高于试剂空白组,差异有显著性意义[分别为(203.15±19.41),(133.05±11.82)s-1,t=4.1106,P<0.05]。杂交14h的荧光强度明显高于杂交12h,差异有显著性意义[分别为(203.06±17.36),(101.54±25.63)s-1,P<0.01],重现性较好。结论:在毛细管DNA传感器构建中,溴化乙锭可作为荧光标记物对模式寡核苷酸进行检测。

AIM： To construct a new DNA biosensor of capillary, METHODS： The experiment was conducted in the Biochemical Laboratory of West China College of Basic Medicine and Forensic Medicine of Sichuan University from October 2004 to December 2005. Probes （20-mer-ssDNA） were fixed to the inner wall of capillary by poly-I-lysine, hybridized with full complementary target oligonucleotide, and dyed by ethidium bromide （EB）. The fluorescent intensity after hybrldlzaticn with 0.033xmmol/L target oligonucleotide were detected. The sequence of target oligonucleotide of the same concentration was detected for three times and compared of the reduplication. The fluorescent intensity was determined at different hybridization time. RESULTS： The probes fixed on the inner wall of capillary and complementary target DNA hybridization were stained with EB, and the fluorescent intensity was detected by RF5000 fluorometry. The fluorescent intensity was higher in the test group than that In the control group, and the differences ware significant [respectively ware （203.15±19.41）,（133.05 ±11.82）s^-1, t =4.110 6, P 〈 0.05]. The fluorescent Intensity at the 14^th hour of hybridization was obviously stronger that that at the 12^th hour, and the differences ware significant [which ware respectively （203.06±17.36）, （101.54±25.63） s^-1, P 〈 0.01] with good reproducibility. CONCLUSION： In the construction of capillary DNA biosensors, EB can be taken as the fluorescent marker to detect the target oligonucleotide.