Automated Screening for Genomic Imbalances using Matrix-Based Comparative Genomic Hybridization

Abstract

SUMMARY:Genome-wide screening for chromosomal imbalances using comparative genomic hybridization (CGH) revealed a wealth of data on previously unrecognized tumor-specific genomic alterations. CGH to microarrays of DNA, an approach termed matrix-CGH, allows detection of genomic imbalances at a much higher resolution. We show that matrix CGH is also feasible from small tissue samples requiring universal amplification of genomic DNA. Because widespread application of matrix-CGH experiments using large numbers of DNA targets demands a high degree of automation, we have developed a protocol for a fully automated procedure. The use of specialized instrumentation for the generation of DNA chips, their hybridization, scanning, and evaluation required numerous alterations and modifications of the initial protocol. We here present the elaboration and testing of automated matrix-CGH. A chip consisting of 188 different genomic DNA fragments, cloned in bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors and immobilized in replicas of 10, was used to assess the performance of the automated protocol in determining the gene dosage variations in tumor cell lines COLO320-HSR, HL60, and NGP. Although ratios of matrix-CGH were highly concordant with results of chromosomal CGH (85%), the dynamic range of the matrix-CGH ratios was highly superior. Investigation of the two amplicons on 8q24 in COLO320-HSR and HL60, containing the MYC gene, revealed a homogeneous amplicon in COLO320-HSR but a heterogeneous amplification pattern in HL60 cells. Although control clones for normalization of the signal ratios can be predicted in cases with defined chromosomal aberrations, in primary tumors such data are often not available, requiring alternative normalization algorithms. Testing such algorithms in a primary high-grade B-cell lymphoma, we show the feasibility of this approach. With the matrix-CGH protocol presented here, robust and reliable detection of genomic gains and losses is accomplished in an automated fashion, which provides the basis for widespread application in tumor and clinical genetics. (Lab Invest 2002, 82:47-60).

A tiling resolution DNA microarray with complete coverage of the human genome

“…A detailed analysis of our COLO320 profile identified new microamplifications on chromosome arms 13q, 15q, 16p and 22q ( Supplementary Fig. 1 online), which were not detected by the two previous high-resolution CGH studies 7,23 . For example, we identified a 300-kb microamplification at 13q12.2 containing only three genes (according to University of California Santa Cruz Genome Browser, April 2003 Freeze): caudal type homeobox transcription factor 2 (CDX2), insulin promoter factor 1 (IPF1) and GS homeobox 1 (GSH1; Fig.…”

“…22), which has been characterized in two previous array CGH studies 7,23 . We confirmed the amplification at 8q24 in the MYC region identified by these studies.…”

Construction of a natural panel of 11p11.2 deletions and further delineation of the critical region involved in Potocki–Shaffer syndrome

“…25,27 The subject and reference DNA were cohybridized to one microarray and then oppositely labeled and cohybridized to a second microarray as previously described. 25,28 Shortly after the labeling, probes were purified with Microcon (Millipore, Billerica, MA, USA), and B500 ng of subjects' DNA, combined with an equal amount of opposite-sex control DNA, was coprecipitated with 50 mg of Cot1-DNA (Invitrogen) and hydrated with 15.5 ml ULTRAhyb (Ambion, Austin, TX, USA). The labeled genomic DNAs were denatured at 721C for 5 min, preannealed immediately after at 371C for 1 h, placed onto a microarray, and covered with a 22 Â 22 mm 2 coverslip.…”

Delineation of mechanisms and regions of dosage imbalance in complex rearrangements of 1p36 leads to a putative gene for regulation of cranial suture closure

“…19 We used a dye-reversal strategy on two separate microarrays in which study subject and reference DNAs were labeled with cyanine 3 (Cy3) and cyanine 5 (Cy5), respectively, cohybridized to one microarray and then oppositely labeled and cohybridized to a second microarray. 20,21 Images were acquired using a GenePix 4000B (Axon Instruments) dual-laser scanner in combination with GenePix Pro 4.0 imaging software. Two simultaneous scans of each array were obtained at wavelengths of 635 and 532 nm, and data analysis was performed as described previously 20 to identify gains or losses within this particular region of 1p36.…”