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DNA microarrays for comparative genomic hybridization based on DOP‐PCR amplification of BAC and PAC clones
Author(s) -
Fiegler Heike,
Carr Philippa,
Douglas Eleanor J.,
Burford Deborah C.,
Hunt Sarah,
Smith James,
Vetrie David,
Gorman Patricia,
Tomlinson Ian P.M.,
Carter Nigel P.
Publication year - 2003
Publication title -
genes, chromosomes and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.754
H-Index - 119
eISSN - 1098-2264
pISSN - 1045-2257
DOI - 10.1002/gcc.10155
Subject(s) - dna microarray , biology , insert (composites) , microbiology and biotechnology , primer (cosmetics) , multiple displacement amplification , genomic dna , dna , polymerase chain reaction , comparative genomic hybridization , genome , chromosome , genetics , dna extraction , gene , chemistry , gene expression , mechanical engineering , organic chemistry , engineering
Abstract We have designed DOP‐PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP‐PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome‐specific clones spotted on microarrays by use of DNA amplified from flow‐sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1‐Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet. © 2003 Wiley‐Liss, Inc.