Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography
- Cite this article as:
- Craig, J., Kraus, J. & Cremer, T. Hum Genet (1997) 100: 472. doi:10.1007/s004390050536
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.