Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH.
We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labelling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries.
Dugan
Lawrence C
LC
Biology and Biotechnology Research Program, L-448 Lawrence Livermore National Laboratory, Livermore, CA 94550, USA. dugan3@llnl.gov
Pattee
Melissa S
MS
Williams
Jennifer
J
Eklund
Mike
M
Sorensen
Karen
K
Bedford
Joel S
JS
Christian
Allen T
AT
D000818:Animals; D002871:Chromosome Banding; D020223:Chromosome Painting; D033481:Chromosomes, Mammalian / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid
D015342:DNA Probes