DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.
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.
Fiegler
Heike
H
Wellcome Trust Sanger Institute/Cancer Research UK Genomic Microarray Group, Hinxton, Cambridge, CB10 1SA, United Kingdom.
Carr
Philippa
P
Douglas
Eleanor J
EJ
Burford
Deborah C
DC
Hunt
Sarah
S
Scott
Carol E
CE
Smith
James
J
Vetrie
David
D
Gorman
Patricia
P
Tomlinson
Ian P M
IP
Carter
Nigel P
NP
D000818:Animals; D002292:Carcinoma, Renal Cell / Q000235:genetics; D002460:Cell Line; D002869:Chromosome Aberrations; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004269:DNA, Bacterial; D004247:DNA