Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography.
The 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.
Craig
J M
JM
Institute of Anthropology and Human Genetics, University of Munich, Germany.
Kraus
J
J
Cremer
T
T
D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
D004247:DNA