Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect.
Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C(o)t-1 DNA). Contrary to expectation, we demonstrated that C(o)t-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C(o)t-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C(o)t-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 C(o)t-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 C(o)t-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 C(o)t-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.
Newkirk
Heather L
HL
Laboratories of Genomic Disorders, Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine, KS, USA.
Knoll
Joan H M
JH
Rogan
Peter K
PK
D004247:DNA / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D023281:Genomics / Q000379:methods; D008863:Microspheres; D009693:Nucleic Acid Hybridization / Q000379:methods; D015341:Nucleic Acid Probes / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results
D015341:Nucleic Acid Probes; D004247:DNA