简并寡核苷酸引物PCR在荧光原位杂交检测染色体微缺失综合征中的应用  

Detection of microdeletion syndromes by fluorescence in situ hybridization based on degenerate oli- gonucleotide primed. PCR

作　　者：张占辉[1] 王小竹[1] 孟岩[2] 闫明[1] 刘卉[1] 韩玲[1] 黄昱[1] 黄尚志[2] 吴白燕[1] 

机构地区：[1]北京大学医学部基础医学院医学遗传学系,100191 [2]中国医学科学院基础医学研究所医学遗传学系北京协和医学院基础医学院,北京100730

出　　处：《国际遗传学杂志》2013年第5期185-190,195,共7页International Journal of Genetics

基　　金：国家自然科学基金（81270426）

摘　　要：目的优化荧光原位杂交（fluorescence in situ hybridization，FISH）所需探针的制备工作，应用FISH检测染色体7q11．2、15q11．2以及22q11．2缺失。方法采用简并寡核苷酸引物聚合酶链式反应（degenerate oligonucleotide primedPCR，DOP—PCR）的方法，扩增人工细菌染色体（bacterial artificial chromosome，BAC）DNA，通过切口平移对扩增的DNA进行间接标记，使用制备好的探针检测染色体微缺失。结果DOP—PCR方法得到的BAC—DNA扩增产物，经标记后用于FISH，可以观察到清晰且背景低的信号；FISH结果确认了1例染色体7q11．2微缺失嵌合体、1例辣色体15q11．2缺失。结论经过优化的实验方法能够快速制备探针，节约抽提BAC—DNA所需的时间，可以应用于FISH以检测微缺失综合征。Objective To optimize the work of probe preparation used for fluorescence in situ hybridization （FISH） and aplly FISH analysis for the detection of deletions 7ql 1.2, 15ql 1.2, and 2 2 q 1 1.2. Methods The DNA of bacterial artificial chromosome （ BAC ） was amplified by degenerate oligonucleotide primed PCR （DOP-PCR）. Indirect labeling of the amplified products was performed, by nick translation. Subsequently, the prepared FISH probes were used for detection of chromosome mierode- letion. Results By means of DOP-PCR, we got sufficient amounts of FISH probes which provided clear hybridization signals and low backgroud. FISH analysis showed one case with of 15 q 1 1.2 deletion and another case with a mosaic 7 q 1 1.2 deletion. Conclusion The optimized method developed by us en- ables generation of probes to run faster without spending much time on isolating BAC-DNA. The probes could be applied in FISH analysis for the detection of microdeletion syndromes.

关 键 词：简并寡核苷酸引物聚合酶链反应 荧光原位杂交 微缺失综合征 嵌合体 

分 类 号：R596.1[医药卫生—内科学]