ARRAY COMPARATIVE GENOMIC HYBRIDIZATION:
A NEW GENOMIC APPROACH FOR HIGH-RESOLUTION
ANALYSIS OF COPY NUMBER CHANGES
Dimova Iv
*Corresponding Author: Dr. Ivanka Dimova, Department of Medical Genetics, Medical University Sofia, 2 Zdrave str, 1431 Sofia, Bulgaria; Tel.Fax: +359-2-952-03-57; E-mail: idimova73@yahoo.com
page: 11
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ARRAY COMPARATIVE GENOMIC HYBRIDIZATION PERFORMANCE
Several different array platforms have been used for CGH measurements in mammalian genomes. The initial step is selection and preparation of the clones. Various approaches have used large insert genomic clones such as bacterial artificial chromosomes (BACs) [4], as well as cDNA clones and oligonucleotides for array spots [3,7].
The BAC clones are thought to be superior to cDNAs and oligonucleotides as aCGH probes because their large genomic size (~150 Kb) confers high and consistent binding specifity [8]. The use of BACs for aCGH provide sufficiently intense signals so that accurate measurements can be obtained over a wide range in copy number from below single-copy level to at least two orders of magnitude higher (for amplifications of at least 1,000-fold) [9]. Thus, the precision of measurement is such that single copy changes affecting individual clones on the array can be readily detected [4] and aberration boundaries can be located to within a fraction of the length of a BAC [10]. Arrays described in early reports of aCGH used whole BACs isolated from large bacterial cultures, and the DNA was often sonicated to reduce the molecular mass [9,11, 12]. Since growth and processing of large bacterial cultures is not practical for large analysis, methods have been devised that use representations of BACs prepared by ligation-mediated polymerase chain reaction (PCR) [4,13], degenerate oligonucleotide primed PCR (DOP-PCR) [5] or a modified DOP-PCR protocol [14]. If the complexity and sequence balance of the source clones is maintained, the BAC representation is expected to yield sufficiently intense signals to perform as well as whole BACs.
Arrays that contain spots made from cDNAs [15] or oligonucleotides were initially produced to measure gene expression. The cDNA arrays can detect amplified sequences using several micrograms of genomic DNA isolated from cell lines and frozen tissues [3,7]. The large changes in copy number provide enough signal so that boundaries of the amplicons can be determined with the highest resolution because the amplification status of individual array elements could be determined.
Analyses of genomes by aCGH have employed arrays focused on: i) a particular region of the genome [7]; ii) selected regions known to be frequently aberrant in tumors [16]; and iii) genome-wide arrays [17,18]. In all cases, the enhanced resolution possible with aCGH compared with chromosome CGH, has demonstrated copy number aberrations not evident using chromosome CGH.
The PCR products are arrayed in triplicate (custom arrays) or duplicate (1 Mb-resolution whole genome array) onto amine-binding slides. The procedure of aCGH includes the following steps: digestion of genomic DNA; labeling of genomic DNA by random priming; purification of labeled products; precipitation in the presence of blocking DNA; hybridization to microarrays; image and data analysis [3-5].
For data analysis, global intensities of fluorescence signals for test (T) and reference (N) DNA are normalized with the entire set of spots on the array, the T:N ratios are calculated, the median values of replicate spots are calculated, and these values are used to define the selection threshold for individual spots. The profiles are represented with log 2 values of the ratios on the Y-axis and with the values of Mb position of the clones along the chromosome on the X-axis (Fig. 1) [3-5].
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