Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes.
Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40-200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.
Fiegler
Heike
H
The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. heike@sanger.ac.uk
Redon
Richard
R
Carter
Nigel P
NP
D023281:Genomics / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
D009841:Oligonucleotides