title: Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH.
abstract: The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.
authors.affiliation: Agilent Laboratories, Agilent Technologies, Santa Clara, Calif., USA. alice_yamada@agilent.com
authors.forename: N A
authors.initials: NA
authors.lastname: Yamada
authors.forename: L S
authors.initials: LS
authors.lastname: Rector
authors.forename: P
authors.initials: P
authors.lastname: Tsang
authors.forename: E
authors.initials: E
authors.lastname: Carr
authors.forename: A
authors.initials: A
authors.lastname: Scheffer
authors.forename: M C
authors.initials: MC
authors.lastname: Sederberg
authors.forename: M E
authors.initials: ME
authors.lastname: Aston
authors.forename: R A
authors.initials: RA
authors.lastname: Ach
authors.forename: A
authors.initials: A
authors.lastname: Tsalenko
authors.forename: N
authors.initials: N
authors.lastname: Sampas
authors.forename: B
authors.initials: B
authors.lastname: Peter
authors.forename: L
authors.initials: L
authors.lastname: Bruhn
authors.forename: A R
authors.initials: AR
authors.lastname: Brothman
mesh_terms: D058674:Chromosome Duplication / Q000235:genetics*; D002877:Chromosomes, Human / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008677:Metaphase / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D056916:Segmental Duplications, Genomic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D057345:Sequence Inversion