aCGH is performed essentially in the same way as metaphase CGH. Test and reference DNA are differentially labeled, preannealed with Cotl DNA and then hybridized to the microarray. After washing, the slide is scanned and the intensity of each fluorescent DNA for each clone on the array measured. The fluorescence ratio is then plotted along the length of each chromosome using the mapped position of the clones (see Fig. 1). Again, gains are represented by increased ratios and losses by decreased ratios. With aCGH, analysis resolution is determined by the insert size and the density along the genome of the clones spotted onto the slide.

Clone DNA was originally extracted from large scale cultures for direct spotting onto slides (6,7). Carrying out such large scale cultures quickly becomes a costly and time consuming process when expanded to the number of clones required to construct an array with a resolution of one clone every 1 Mb of the human genome (approx 3000 clones). To overcome this problem, several approaches have been applied to amplify the clone DNA enzymatically for spotting, thus removing the requirement for large-scale DNA preparations. These have included methods such as linker adapter polymerase chain reaction (PCR) (8), rolling circle PCR using bacteriophage Phi29 (9), or degenerate oligonucleotide primed PCR (DOP-PCR) (10) using an amine modified version of the standard DOP-PCR primer 6MW (11,12). Escherichia coli genomic DNA however is a common contaminant of DNA preparations of large insert clones. The degree of contamination has been estimated by real-time PCR to be between 6 and 26% dependent on the method used for purification (13). This contaminating E. coli DNA will reduce the capacity of each probe spotted on the array to hybridize with the DNA of interest and may contribute to increased nonspecific background signal. In order to overcome this

Fig. 1. Principle of array-comparative genomic hybridization.

Position along Chromosome Sequence

Fig. 1. Principle of array-comparative genomic hybridization.

problem, we designed three new DOP-PCR primers (DOP1, -2, and -3) that were chosen to be efficient in amplifying human genomic DNA but inefficient in amplifying the contaminating E. coli DNA (14). The use of these three new DOP-PCR primers, particularly in combination, revealed a significant increase in sensitivity and reproducibility in genomic hybridizations compared to arrays constructed with the standard DOP-PCR primer 6MW (14).

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