Representational Oligonucleotide Microarray Analysis Research Articles

High-throughput identification of genetic markers using representational oligonucleotide microarray analysis

We describe a novel approach for high-throughput development of genetic markers using representational oligonucleotide microarray analysis. We test the performance of the method in sugar beet (Beta vulgaris L.) as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridized on microarrays containing in total 146,554 custom made oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences and expressed sequence tags (ESTs). Oligonucleotides showing a signal with one parental line only, were selected as potential marker candidates and placed onto an array, designed for genotyping of 184 F(2) individuals from the mapping population. Utilizing known co-dominant anchor markers we obtained 511 new dominant markers (392 derived from BAC end sequences, and 119 from ESTs) distributed over all nine sugar beet linkage groups and calculated genetic maps. Further improvements for large-scale application of the approach are discussed and its feasibility for the cost-effective and flexible generation of genetic markers is presented.

Progress in the detection of human genome structural variations

The emerging of high-throughput and high-resolution genomic technologies led to the detection of submicroscopic variants ranging from 1 kb to 3 Mb in the human genome. These variants include copy number variations (CNVs), inversions, insertions, deletions and other complex rearrangements of DNA sequences. This paper briefly reviews the commonly used technologies to discover both genomic structural variants and their potential influences. Particularly, we highlight the array-based, PCR-based and sequencing-based assays, including array-based comparative genomic hybridization (aCGH), representational oligonucleotide microarray analysis (ROMA), multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), paired-end mapping (PEM), and next-generation DNA sequencing technologies. Furthermore, we discuss the limitations and challenges of current assays and give advices on how to make the database of genomic variations more reliable.

Protumorigenic Role of HAPLN1 and Its IgV Domain in Malignant Pleural Mesothelioma

Tumor extracellular matrix (ECM) plays a crucial role in cancer progression mediating and transforming host-tumor interactions. Targeting the ECM is becoming an increasingly promising therapeutic approach in cancer treatment. We find that one of the ECM proteins, HAPLN1, is overexpressed in the majority of mesotheliomas. This study was designed to characterize the protumorigenic role of HAPLN1 in mesothelioma. Overexpression of HAPLN1 was assessed and validated on a large set of normal/mesothelioma specimens on the RNA and protein levels. We also analyzed DNA copy number alterations in the HAPLN1 genomic locus using the array-based comparative genomic hybridization representational oligonucleotide microarray analysis tool. Tumorigenic activities of the HAPLN1 domains were evaluated in vitro on mesothelioma cells transfected with HAPLN1-expressing constructs. We found that HAPLN1 is 23-fold overexpressed in stage I mesothelioma and confirmed it for 76% samples (n = 53) on RNA and 97% (n = 40) on protein levels. The majority of lung cancers showed no differential expression of HAPLN1. Analysis of DNA copy number alterations identified recurrent gain in the 5q14.3 HAPLN1 locus in approximately 27% of tumors. Noteworthy, high expression of HAPLN1 negatively correlated with time to progression (P = 0.05, log-rank test) and overall survival (P = 0.006). Proliferation, motility, invasion, and soft-agar colony formation assays on mesothelioma cells overexpressing full-length HAPLN1 or its functional domains strongly supported the protumorigenic role of HAPLN1 and its SP-IgV domain. Overexpression of HAPLN1 and its SP-IgV domain increases tumorigenic properties of mesothelioma. Thus, targeting the SP-IgV domain may be one of the therapeutic approaches in cancer treatment.

Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA)

AbstractWe examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

High resolution representational oligonucleotide microarray analysis (ROMA) suggests that TOPO2 and HER2 co-amplification is uncommon in human breast cancer.

Abstract Abstract #2023 
 Background
 Clinical studies relating TOPO2A expression or amplification by FISH have yielded inconsistent results regarding prediction of benefit from various drug therapies, particularly in the setting of HER2 amplification by FISH. Because HER2 and TOPO2A are relatively small genes that are close to each other, and because the relevant commercially-available FISH probes hybridize to DNA sequences beyond the genes of interest, there is significant potential for false positive results. We therefore conducted an exploratory study comparing HER2 and TOPO2A amplification by ROMA and FISH.
 Methods:
 Forty-two archived formalin-fixed paraffin-embedded primary breast cancer specimens were pre-selected to contain 36 HER2 amplified and 6 HER2 non-amplified cases by FISH. These specimens were evaluated for HER2 and TOPO2A amplification by FISH at Memorial Sloan-Kettering Cancer Center and by ROMA at Cold Spring Harbor Laboratory in a double-blinded experiment. Two HER2 amplified by FISH specimens proved inevaluable for technical reasons. The results for the remaining 40 evaluable specimens were then compared.
 Results:
 Of the 40 evaluable specimens, the 6 cases selected as HER2 non-amplified by FISH were HER2 non-amplified by ROMA and TOPO2A non-amplified by both FISH and ROMA. Thirty-two specimens were HER2 amplified by both FISH and ROMA. Two specimens with HER2 FISH results of 2.2 and 2.4 did not demonstrate focal amplification by ROMA. Twenty-five (74%) of the 34 specimens with HER2 amplification by FISH were TOPO2A non-amplified by both FISH and ROMA (Table 1). However, only 2 (22%) of the 9 specimens with HER2 and TOPO2A co-amplification by FISH demonstrated TOPO2A amplification by ROMA.
 
 Conclusions:
 Our results suggest that by ROMA, the determination of HER2 status (amplified and non-amplified) and TOPO2A non-amplification by FISH is accurate. The absence of TOPO2A amplification in HER2 non-amplified breast cancer is consistent with published reports. We also confirmed the published frequency of HER2 and TOPO2A co-amplification by FISH. However, we found that co-amplification of HER2 and TOPO2A by ROMA is uncommon, which suggests that commercially-available FISH probes over-estimate the frequency of TOPO2A amplification in HER2 amplified cases. Studies evaluating the clinical implications of these findings are underway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2023.

Mechanisms of FUS1/TUSC2 deficiency in mesothelioma and its tumorigenic transcriptional effects

BackgroundFUS1/TUSC2 is a novel tumor suppressor located in the critical 3p21.3 chromosomal region frequently deleted in multiple cancers. We previously showed that Tusc2-deficient mice display a complex immuno-inflammatory phenotype with a predisposition to cancer. The goal of this study was to analyze possible involvement of TUSC2 in malignant pleural mesothelioma (MPM) - an aggressive inflammatory cancer associated with exposure to asbestos.MethodsTUSC2 insufficiency in clinical specimens of MPM was assessed via RT-PCR (mRNA level), Representational Oligonucleotide Microarray Analysis (DNA level), and immunohistochemical evaluation (protein level). A possible link between TUSC2 expression and exposure to asbestos was studied using asbestos-treated mesothelial cells and ROS (reactive oxygen species) scavengers. Transcripional effects of TUSC2 in MPM were assessed through expression array analysis of TUSC2-transfected MPM cells.ResultsExpression of TUSC2 was downregulated in ~84% of MM specimens while loss of TUSC2-containing 3p21.3 region observed in ~36% of MPMs including stage 1 tumors. Exposure to asbestos led to a transcriptional suppression of TUSC2, which we found to be ROS-dependent. Expression array studies showed that TUSC2 activates transcription of multiple genes with tumor suppressor properties and down-regulates pro-tumorigenic genes, thus supporting its role as a tumor suppressor. In agreement with our knockout model, TUSC2 up-regulated IL-15 and also modulated more than 40 other genes (~20% of total TUSC2-affected genes) associated with immune system. Among these genes, we identified CD24 and CD274, key immunoreceptors that regulate immunogenic T and B cells and play important roles in systemic autoimmune diseases. Finally, clinical significance of TUSC2 transcriptional effects was validated on the expression array data produced previously on clinical specimens of MPM. In this analysis, 42 TUSC2 targets proved to be concordantly modulated in MM serving as disease discriminators.ConclusionOur data support immuno-therapeutic potential of TUSC2, define its targets, and underscore its importance as a transcriptional stimulator of anti-tumorigenic pathways.

Frequent genomic copy number gain and overexpression of GATA-6 in pancreatic carcinoma

Multiple genetic alterations are well recognized as contributing to pancreatic carcinogenesis, although the finding of recurrent copy number changes indicates additional targets remain to be found. The objective of this study was to identify novel targets of genetic alteration that contribute to pancreatic cancer development or progression. We used Representational Oligonucleotide Microarray Analysis (ROMA) to identify copy number changes in pancreatic cancer xenografts, and validated these findings using FISH, quantitative PCR, Western blotting and immunohistochemical labeling. With this approach, we identified a 0.36-Mb amplification at 18q11.2 containing two known genes, GATA-6 and cTAGE1. Using a cutoff value of 3.0 fold compared to haploid controls, copy number gain or amplification was confirmed in 4 of 42 (9.5%) pancreatic carcinomas analyzed. Combined genetic and transcriptional analyses showed consistent overexpression of GATA-6 in all carcinomas with 18q11.2 gain, as well as in the majority of pancreatic cancers examined (17 of 30 cancers, 56.7%) that did not have gain of this region. By contrast, overexpression of cTAGE1 was rare in these same cancers suggesting GATA-6 is the true target of this copy number increase. GATA-6 mRNA overexpression corresponded to robust nuclear protein expression in cancer cell lines and resected tissues consistent with its role as a transcription factor. Intense nuclear labeling was significantly increased in PanIN-3 lesions and infiltrating carcinomas compared to normal duct epithelium (p

Copy-number variants in patients with a strong family history of pancreatic cancer

Copy-number variants such as germ-line deletions and amplifications are associated with inherited genetic disorders including familial cancer. The gene or genes responsible for the majority of familial clustering of pancreatic cancer have not been identified. We used representational oligonucleotide microarray analysis (ROMA) to characterize germ-line copy number variants in 60 cancer patients from 57 familial pancreatic cancer kindreds. Fifty-seven of the 60 patients had pancreatic cancer and three had non-pancreatic cancers (breast, ovary, ovary). A familial pancreatic cancer kindred was defined as a kindred in which at least two first-degree relatives have been diagnosed with pancreatic cancer. Copy-number variants identified in 607 individuals without pancreatic cancer were excluded from further analysis. A total of 56 unique genomic regions with copy-number variants not present in controls were identified, including 31 amplifications and 25 deletions. Two deleted regions were observed in two different patients, and one in three patients. The germ-line amplifications had a mean size of 662Kb, a median size of 379Kb (range 8.2Kb to 2.5Mb) and included 425 known genes. Examples of genes included in the germ-line amplifications include the MAFK, JunD and BIRC6 genes. The germ-line deletions had a mean size of 375Kb, a median size 151Kb (range 0.4Kb to 2.3Mb) and included 81 known genes. In multivariate analysis controlling for region size, deletions were 90% less likely to involve a gene than were duplications (p

PD6-2-8: High-resolution ROMA analysis of chromosomal rearrangements in mesothelioma patients with poor and good survival

Representational oligonucleotide microarray analysis (ROMA) is a recently developed microarray-based high-resolution technology that helps linking chromosomal losses and gains with specific genetic events. ROMA identifies the boundaries of deletions, duplications, and amplifications with the accuracy of roughly 50-100 kb throughout the whole human genome. Mesothelioma is an aggressive asbestos-related cancer with short (median 11-22 months) survival and poorly investigated genetic components.

Copy Number Variations in del22q11.2 Syndrome

RATIONALE: Del22q11.2 syndrome is usually due to a deletion involving the Tbx gene on chromosome 22, but not all patients have the typical deletion, and not all patients with the deletion have full expression of the disease. A whole-genome approach may be better to understand and diagnose the disorder. We evaluated the use of 500,000 SNP mapping arrays and representational oligonucleotide microarray analysis (ROMA) in the delineation of the 22q11.2 deletion and identification of additional copy number variations (CNVs) elsewhere in the del22 syndrome genomes.METHODS: We evaluated CNVs using very-high-density ROMA in five patients with del22 syndrome. Identification of CNVs and pathologic deletions was by a new normalization and segmentation procedure (which we refer to as vivaROMA) that expanded on existing methods to improve comparisons between different arrays.RESULTS: The 22q11.2 deletion is easily identified, and our results agreed with the results of clinical flouresence in situ hybridization studies in all five patients. In addition, both patients and controls had numerous other CNVs elsewhere in the genome, consistent with the findings of other groups investigating CNVs in normal genomes. Some deletions elsewhere in del22 syndrome genomes involve coding regions that could affect immune system function.CONCLUSIONS: Genetic diversity outside of the 22q11.2 region may affect the phenotype in del22q11.2 syndrome. Use of whole genome approaches to the study of this disorder could enable the identification of new genes and hypotheses about the origins of immune system dysfunction. RATIONALE: Del22q11.2 syndrome is usually due to a deletion involving the Tbx gene on chromosome 22, but not all patients have the typical deletion, and not all patients with the deletion have full expression of the disease. A whole-genome approach may be better to understand and diagnose the disorder. We evaluated the use of 500,000 SNP mapping arrays and representational oligonucleotide microarray analysis (ROMA) in the delineation of the 22q11.2 deletion and identification of additional copy number variations (CNVs) elsewhere in the del22 syndrome genomes. METHODS: We evaluated CNVs using very-high-density ROMA in five patients with del22 syndrome. Identification of CNVs and pathologic deletions was by a new normalization and segmentation procedure (which we refer to as vivaROMA) that expanded on existing methods to improve comparisons between different arrays. RESULTS: The 22q11.2 deletion is easily identified, and our results agreed with the results of clinical flouresence in situ hybridization studies in all five patients. In addition, both patients and controls had numerous other CNVs elsewhere in the genome, consistent with the findings of other groups investigating CNVs in normal genomes. Some deletions elsewhere in del22 syndrome genomes involve coding regions that could affect immune system function. CONCLUSIONS: Genetic diversity outside of the 22q11.2 region may affect the phenotype in del22q11.2 syndrome. Use of whole genome approaches to the study of this disorder could enable the identification of new genes and hypotheses about the origins of immune system dysfunction.

Representational oligonucleotide microarray analysis (ROMA) and comparison of binning and change-point methods of analysis: application to detection of del22q11.2 (DiGeorge) Syndrome

DiGeorge (del22q11.2) syndrome is estimated to occur in 1:4,000 births, is the most common contiguous-gene deletion syndrome in humans, and is caused by autosomal dominant deletions in the 22q11.2 DiGeorge syndrome critical region (DGCR). Multiple microarray methods have been developed recently for analyzing such copy number changes, but data analysis and accurate deletion detection remains challenging. Clinical use of these microarray methods would have many advantages, particularly when the possibility of a chromosomal disorder cannot be determined simply on the basis of history and physical examination data alone. We investigated the use of the microarray technique, representational oligonucleotide microarray analysis (ROMA), in the detection of del22q11.2 syndrome. Genomic DNA was isolated from three well-characterized cell lines with 22q11.2 DGCR deletions and from the blood of a patient suspected of having del22q11.2 syndrome, and analyzed using both the binning and change-point model algorithms. Though the 22q11.2 deletion was easily identified with either method, change-point models provide clearer identification of deleted regions, with the potential for fewer false-positive results. For circumstances in which a clear, a priori, copy-number change hypothesis is not present, such as in many clinical samples, change-point methods of analysis may be easier to interpret.

Novel patterns of genome rearrangement and their association with survival in breast cancer

Representational Oligonucleotide Microarray Analysis (ROMA) detects genomic amplifications and deletions with boundaries defined at a resolution of approximately 50 kb. We have used this technique to examine 243 breast tumors from two separate studies for which detailed clinical data were available. The very high resolution of this technology has enabled us to identify three characteristic patterns of genomic copy number variation in diploid tumors and to measure correlations with patient survival. One of these patterns is characterized by multiple closely spaced amplicons, or "firestorms," limited to single chromosome arms. These multiple amplifications are highly correlated with aggressive disease and poor survival even when the rest of the genome is relatively quiet. Analysis of a selected subset of clinical material suggests that a simple genomic calculation, based on the number and proximity of genomic alterations, correlates with life-table estimates of the probability of overall survival in patients with primary breast cancer. Based on this sample, we generate the working hypothesis that copy number profiling might provide information useful in making clinical decisions, especially regarding the use or not of systemic therapies (hormonal therapy, chemotherapy), in the management of operable primary breast cancer with ostensibly good prognosis, for example, small, node-negative, hormone-receptor-positive diploid cases.

Mouse genomic representational oligonucleotide microarray analysis: detection of copy number variations in normal and tumor specimens.

Genomic amplifications and deletions, the consequence of somatic variation, are a hallmark of human cancer. Such variation has also been observed between "normal" individuals, as well as in individuals with congenital disorders. Thus, copy number measurement is likely to be an important tool for the analysis of genetic variation, genetic disease, and cancer. We developed representational oligonucleotide microarray analysis, a high-resolution comparative genomic hybridization methodology, with this aim in mind, and reported its use in the study of humans. Here we report the development of a representational oligonucleotide microarray analysis microarray for the genomic analysis of the mouse, an important model system for many genetic diseases and cancer. This microarray was designed based on the sequence assembly MM3, and contains approximately 84,000 probes randomly distributed throughout the mouse genome. We demonstrate the use of this array to identify copy number changes in mouse cancers, as well to determine copy number variation between inbred strains of mice. Because restriction endonuclease digestion of genomic DNA is an integral component of our method, differences due to polymorphisms at the restriction enzyme cleavage sites are also observed between strains, and these can be useful to follow the inheritance of loci between crosses of different strains.

PROBER: oligonucleotide FISH probe design software

PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. http://prober.cshl.edu

Design considerations for array CGH to oligonucleotide arrays

Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

High-resolution representational oligonucleotide microarray analysis and fluorescence in situ hybridization analysis of aneuploid and diploid breast tumors

High-resolution representational oligonucleotide microarray analysis and fluorescence in situ hybridization analysis of aneuploid and diploid breast tumors

Application of ROMA (representational oligonucleotide microarray analysis) to patients with cytogenetic rearrangements

PurposeTo demonstrate the accuracy and sensitivity of Representational Oligonucleotide Microarray Analysis (ROMA) to describe copy number changes in patients with chromosomal abnormalities. MethodsROMA was performed using BglII digested DNA from two cases with cytogenetically detected deletions and one case with an unbalanced terminal rearrangement detected only by subtelomeric FISH. Hybridization was to an 85,000-probe oligonucleotide microarray, providing an average resolution of 35 kb. FISH was used to confirm some of the ROMA findings. ResultsBy ROMA, a del(13)(q14.3q21.2) was shown to be noncontiguous, with deletions extending from 53.08 to 61.40 Mb and from 72.88 to 74.83 Mb. The 10-Mb deletion contained only six known genes. FISH confirmed the noncontiguous nature of the deletion, as well as a small amplification in 6q that was also found in the patient's mother. A del(4)(q12q21.2) was found by ROMA to be 23 Mb in length, from 58.8 to 81.9 Mb on chromosome 4, in agreement with the cytogenetically assigned breakpoints. ROMA showed that an unbalanced “subtelomeric” rearrangement involved a 6-Mb deletion of 22q and an 8-Mb duplication of 16q. ConclusionsROMA can define cytogenetic aberrations with extraordinary precision. Unexpected findings included the interrupted nature of the deletion in 13q and the large size of the imbalances in the “subtelomeric” rearrangement. Together with the information from the human genome sequence and proteomics, the ability to define rearrangements with “ultra-high” resolution will improve the ability to provide accurate prognosis both prenatally and postnatally to parents of offspring with chromosomal aberrations.

High-Resolution ROMA CGH and FISH Analysis of Aneuploid and Diploid Breast Tumors

Combining representational oligonucleotide microarray analysis (ROMA) of tumor DNA with fluorescence in situ hybridization (FISH) of individual tumor cells provides the opportunity to detect and validate a wide range of amplifications, deletions, and rearrangements directly in frozen tumor samples. We have used these combined techniques to examine 101 aneuploid and diploid breast tumors for which long-term follow-up and detailed clinical information were available. We have determined that ROMA provides accurate and sensitive detection of duplications, amplifications, and deletions and yields defined boundaries for these events with a resolution of <50 kbp in most cases. We find that diploid tumors exhibit fewer rearrangements on average than aneuploids, but rearrangements occur at the same locations in both types. Diploid tumors reflect at least three consistent patterns of rearrangement. The reproducibility and frequency of these events, especially in very early stage tumors, provide insight into the earliest chromosomal events in breast cancer. We have also identified correlations between certain sets of rearrangement events and clinically relevant parameters such as long-term survival. These correlations may enable novel prognostic indicators for breast and other cancers as more samples are analyzed.

Generation and Analysis of Genetically Defined Liver Carcinomas Derived from Bipotential Liver Progenitors

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

Apply innovative technologies to explore cancer genome

Molecular genetic alterations characterize the development of human cancer. Recent advances in molecular genetic technology and the success of the human genome project have empowered investigators with new tools in dissecting the cancer genome for discovery of new cancer-associated genes. The purpose of this review is to highlight the emerging molecular genetic methodologies and summarize their principles, applications, and potential technical challenges. The critical issue in sample preparation and a strategy that combines different molecular techniques to facilitate the identification of novel cancer-associated genes will be discussed. Digital karyotyping and array-based techniques including array comparative genomic hybridization and representational oligonucleotide microarray analysis have been recently developed to study the genomic landscape in human cancer. These innovations provide tools to quantitatively measure DNA copy number changes in cancer and to map those changes directly onto the human genome. Digital karyotyping is based on counting the sequence tags that are distributed in the human genome and thus, it provides a digital readout to precisely outline the amplified and deleted chromosomal regions. Array-based technologies, on the other hand, compare the content of cancer and reference genomes followed by localizing the amplified or deleted signals in chromosomal regions using an array hybridization technique. In addition, a high-throughput mutational analysis platform has been available for a large-scale mutational analysis by using an automated capillary sequencing device and sophisticated bioinformatic tools. A number of examples have demonstrated the promise of these new molecular genetic approaches in identifying several potential new oncogenes and tumor suppressors. As compared with conventional cytogenetics methods, digital karyotyping, array comparative genomic hybridization, and representational oligonucleotide microarray analysis provide an unprecedented mapping resolution that allows a precise localization of the amplified and deleted chromosomal regions. These technologies can be combined with gene expression profiling and high-throughput mutational analysis to facilitate the search for new cancer-associated genes. It is expected that applying these new technologies will lead to discovery of a host of novel oncogenes and tumor suppressors, which will have a significant impact in our understanding of tumorigenesis and in the clinical management of cancer patients.