ABSTRACT
PCR (DOP PCR) using an amine-modified version of the standard DOP PCR primer 6MW (8).
We have also chosen a DOP PCR-based approach for the construction of a large insert clone DNA microarray. DOP PCR uses a mixture of primers, whereby each of the primers consists of a defined 5′ sequence and six defined bases at the 3′ end flanking random hexanucleotide sequence (9, 10). Thus, DOP PCR allows a general amplification of any target DNA. However, the reaction relies on the frequency with which bases in the target sequence match the six 3′ bases of the primer. If the matches to the primer are infrequent in a particular target sequence, the DOP PCR product will be a poor representation of the target (9, 10). In order to increase the level of representation, we have designed three DOP PCR primers that would be efficient in amplifying human genomic DNA, but inefficient in the amplification of Escherichia coli DNA, a known contaminant of DNA preparations from clone cultures (11). Amplification of the contaminating E. coli DNA together with the clone insert will reduce the capacity of the spotted product to hybridize with the DNA of interest thus reducing the sensitivity of the array. We found that the use of these three DOP PCR primers in combination resulted in a significant increase in signal to background ratio, sensitivity and reproducibility. Following this strategy, we have constructed a large insert clone DNA microarray consisting of 3523 Golden Path sequencing clones spaced at approximately 1 Mb intervals across the human genome. Each of the clones was amplified in three separate PCR reactions using the three different DOP PCR primers. This was followed by a secondary PCR reaction with a 5′ amine-modified primer designed such that the 3′ end matched the 5′ end of the DOP PCR primers to enable covalent attachment of the products to specially coated glass slides (12). In order to interpret and report copy number changes correctly across the genome it is essential to map the exact position of each clone along the chromosomes. Clone information is available through various genome browsers but often not easily accessible for large clone collections. We have therefore generated a specific view of the human genome within the Ensembl genome browser (Cytoview, www.ensembl.org/homo_sapiens/cytoview) that displays the 1 Mb clone set in relation to the Golden Path sequencing clones. In particular, Cytoview facilitates the downloading of clone lists (from specific regions of interest to whole chromosomes and the whole genome) together with their corresponding map position. Additional information, such as location by fluorescent in situ hybridization (FISH), BAC-end sequence data, genes or expressed sequence tags (ESTs) for any region of interest can be viewed within the context of the 1 Mb clone set. Ensembl also provides an automatic update of all this information with every new assembly of the human genome (12).
