同时显示杂交信号和荧光R带的FISH技术  

A FISH TECHNIQUE FOR SIMULTANEOUS DETECTION OF FLUORESCENT R BANDS AND IN SITU HYBRIDIZATION SIGNALS

作　　者：赵非非[1,2] 高春生[1,2] 刘国仰 杨焕明[1,2] 程在玉[1,2] 

机构地区：[1]中国医学科学院基础医学研究所 [2]中国协和医科大学基础医学院医学遗传室

出　　处：《中华医学遗传学杂志》1998年第2期108-110,共3页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金

摘　　要：目的建立直接将ＤＮＡ探针定位于显示Ｒ带染色体上的简便方法。方法正常人外周血淋巴细胞培养６７小时，同时加入Ｈｏｅｃｈｓｔ３３２５８和ＢＵｄＲ继续培养５～６小时，常规方法制片。标本浸于２×ＳＳＣ中，在距其１０ｃＭ的２０Ｗ紫外灯下７５℃照射２０分钟。此标本以生物素标记的ｃｏｓｍｉｄ和ＹＡＣ克隆以及ｐＢａｍＸ７进行原位杂交，Ａｖｉｄｉｎ－ＦＩＴＣ和Ａｎｔｉａｖｉｄｉｎ进行信号的检测与放大，ＰＩ复染。于Ｏ１ｙｍｐｕｓＢＸ６０型荧光显微镜下，通过ＷＩＢ滤光镜同时观察显示Ｒ带的染色体和杂交信号。结果在显微镜下，黄绿色杂交信号直接定位于红色并显示Ｒ带的染色体上。Objective To develop a simple method for detecting DNA probes directly on R banded chromosomes. Methods After sixty seven hours culture,human peripheral blood lymphocytes were synchronized for 5 to 6 hours by adding Hoechst 33258 and BUdR,and then arrested by standard cytogenetic procedures.The slides were mounted with 2×SSC and exposed with a 20W UV light which was about 10cM above the slides for 20 min at 75℃.The biotinylated probes,such as the cosmid and YAC clones on 5p specific region and pBamX7,were hybridized on to the slides.After washing,the slides were treated with avidin FITC and amplified with additional layer of biotinylated anti avidin and avidin FITC,and counterstained with propidium iodide in an antifade solution.Fluorescent signals and R bands were observed simultaneously under Olympus BX 60 fluorescence microscope equipped with a WIB filter. Results The chromosomal location of the greenish yellow signals could be directly identified on the R banded chromosome background.Conclusion This method can serve as a rapid and precise system for chromosomal localization of DNA markers.

关 键 词：荧光原位杂交 荧光R带 染色体定位 医学遗传学 

分 类 号：R394[医药卫生—医学遗传学] Q343.2[医药卫生—基础医学]