Damir Sudar

Bio: Damir Sudar is an academic researcher from Oregon Health & Science University. The author has contributed to research in topics: Comparative genomic hybridization & Fluorescence in situ hybridization. The author has an hindex of 21, co-authored 52 publications receiving 7754 citations. Previous affiliations of Damir Sudar include University of California, San Francisco & Lawrence Livermore National Laboratory.

Comparative Genomic Hybridization for Molecular Cytogenetic Analysis of Solid Tumors

Abstract: Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays

Abstract: Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization3(CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies2,3,4,5. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

The Library of Integrated Network-Based Cellular Signatures NIH Program: System-Level Cataloging of Human Cells Response to Perturbations

Abstract: The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Computer image analysis of comparative genomic hybridization

Abstract: We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented.

CellProfiler: image analysis software for identifying and quantifying cell phenotypes

Abstract: Biologists can now prepare and image thousands of samples per day using automation, enabling chemical screens and functional genomics (for example, using RNA interference). Here we describe the first free, open-source system designed for flexible, high-throughput cell image analysis, CellProfiler. CellProfiler can address a variety of biological questions quantitatively, including standard assays (for example, cell count, size, per-cell protein levels) and complex morphological assays (for example, cell/organelle shape or subcellular patterns of DNA or protein staining).

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

Abstract: We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

Cell cycle control and cancer

Abstract: Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.

Distinct Sets of Genetic Alterations in Melanoma

Abstract: Background Exposure to ultraviolet light is a major causative factor in melanoma, although the relationship between risk and exposure is complex. We hypothesized that the clinical heterogeneity is explained by genetically distinct types of melanoma with different susceptibility to ultraviolet light. Methods We compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and N-RAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. Results We found significant differences in the frequencies of regional changes in the number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Samples could be correctly classified into the four groups with 70 percent accuracy on the basis of the changes in the number of copies of...