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Preparation of fluorescent in situ hybridisation probes without the need for optimisation of fragmentation

Author:
McCoy, Patrick J., Costello, Anthony J., Corcoran, Niall M., Hovens, Christopher M., Clarkson, Michael J.
Source:
MethodsX 2019 v.6 pp. 22-34
ISSN:
2215-0161
Subject:
DNA fragmentation, chromosomes, equipment, fluorescence in situ hybridization, genomics, hybridization, plasmids, restriction endonucleases, sonication
Abstract:
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.
Agid:
6253012