McCoy Patrick J, Costello Anthony J, Corcoran Niall M, Hovens Christopher M, Clarkson Michael J
Departments of Surgery and Urology, University of Melbourne, Royal Melbourne Hospital, Parkville, Australia.
Australian Prostate Cancer Research Centre Epworth, Victoria, Australia.
MethodsX. 2018 Nov 27;6:22-34. doi: 10.1016/j.mex.2018.11.015. eCollection 2019.
DNA-fluorescence in situ hybridisation (DNA-FISH) allows visualisation of chromosome organisation and rearrangement. FISH probes are pools of short fluorescently labelled DNA fragments that are often produced from template plasmids that contain large genomic inserts. For effective sample penetration and target hybridisation it is critical that probe fragments are between 200 and 500bp. Production of these short probes requires significant optimisation and can be confounded access to expensive sonication equipment or inherent sequence features that influence enzymatic fragmentation or amplification. Here we demonstrate that effective FISH probes can be prepared without the need for optimisation of fragmentation using a cocktail of two the 4bp recognition sequence restriction enzymes CviQI and AluI.
DNA荧光原位杂交（DNA-FISH）可实现染色体组织和重排的可视化。FISH探针是短荧光标记DNA片段的集合，通常由含有大基因组插入片段的模板质粒产生。为了实现有效的样本穿透和靶标杂交，探针片段长度在200至500bp之间至关重要。制备这些短探针需要进行大量优化，且可能因难以获得昂贵的超声破碎设备或影响酶切片段化或扩增的固有序列特征而受到干扰。在此，我们证明，使用两种4bp识别序列限制酶CviQI和AluI的混合物，无需对片段化进行优化即可制备有效的FISH探针。
