荧光原位杂交实验FluorescenceInSituHybridization的讲义的资料.pptVIP

荧光原位杂交实验 luorescence In Situ Hybridization  原位杂交( In situ hybridization) 1)同位素原位杂交( Isotopic in situ hybridization) 70年代 2)非同位素原位杂交( Non-isotopic in situ hybridization) 80年代中后期 (1)免疫酶联 (2)荧光原位杂交( Fluorescence in situ hybridization,FISH) 求*同位素原位杂交的不足之处: 1)不稳定; 2)高背景; 3)曝光时间长; 4)结果统计学处理繁琐 5)同位素的使用和处理  实验的基本原理 FISH is the detection of highly specific DNA probes which have been hybridized to either interphase or metaphase chromosomes using fluorescence microscopy.  DNA for probe use is labeled with fluorescent(direct method)or non fluorescent molecules which are then detected by fluorescent antibodies (indirect method). The probes bind to a specific region or regions on the target chromosome. The chromosomes are then stained using a contrasting color, and the cells are viewed using a fluorescence microscope.  General FISH protocol Slide preparation flat nuclei spread chromosomes Labeled Isolation and probe rification of abeling the DNA probe Nick translation 0 Cover with coverslip PCR 75° C Denaturing Examination under fluorescent microscope Detection hybridization 37°C,0N Various antibodies/washing steps  Fluorescence In-Situ Hybr dization F工aHy taoTIe+ ! A TC T C  荧光原位杂交的特点: 1)快速;2)信号强;3)特异性高;4)多色。 FISH有上述优点的原因 使用了荧光标记与检测系统取代放射性标记与检测系统 标记探针的物质:(1)生物素 biotin/地高辛( digoxigenin) 半抗原报告分子(间接标记) (2)荧光物质(直接标记)  FLUoresceNt labeled HAPTENE labeled dUTP duTP AMCA-6-dUTP Biotin (BIo)-11-dUTP Cascadeblue-4-dUTP Digoxygenin(DIG)-11 Fluorescein-12-dUTP duTP Rhodamine-6-dUTP Dinitrophenyl (DNP TexasRed-6-dUTP 11-dUTP Cy3-6-dUTP Cy5-dUTP  染色体复染的物质: Propidium lodide(PD)(红色) DAPI(兰色) quinacrine(绿色) chromomycin A3(绿色)