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Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography
journal contribution
posted on 1997-08-01, 00:00 authored by Jeffrey CraigJeffrey Craig, J Kraus, T CremerThe vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA.
History
Journal
Human geneticsVolume
100Issue
3-4Pagination
472 - 476Publisher
SpringerLocation
Berlin, GermanyPublisher DOI
ISSN
0340-6717Language
engPublication classification
C1.1 Refereed article in a scholarly journalCopyright notice
1997, Springer-Verlag Berlin HeidelbergUsage metrics
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Keywords
Chromatography, AffinityDNAHumansIn Situ Hybridization, FluorescenceMalePolymerase Chain ReactionRepetitive Sequences, Nucleic AcidHuman ChromosomeMagnetic ParticleScience & TechnologyLife Sciences & BiomedicineGenetics & HeredityCOMPARATIVE GENOMIC HYBRIDIZATIONINSITU HYBRIDIZATIONCYTOGENETIC ANALYSISCHROMOSOMESGENERATIONSELECTIONLIBRARIESGenetics
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