Published online 14 December 2005
Methods Online |
Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect
Laboratories of Genomic Disorders, Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine KS, USA 1Human Molecular Genetics, Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine KS, USA
*To whom correspondence should be addressed. Tel: +1 816 983 6511; Fax: +1 816 983 6515; Email: progan{at}cmh.edu
Received October 21, 2005. Revised November 23, 2005. Accepted November 23, 2005.
Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (Cot-1 DNA). Contrary to expectation, we demonstrated that Cot-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, Cot-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, Cot-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using Cot-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of Cot-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for Cot-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.