Microarray CGH


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A micro-array is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.

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  • Less than 200 micron. The numbers of spotsmay vary from less than 100 to many 100 000. The molecules are attached to a solid support that can be made from glass,silicon or a polymer
  • "array" as "to place in an orderly arrangement".
  • In the 1980's, bacterial colonies were spotted on nylon membranes with different genomic inserts or cDNAsThe 1990's resulted in the emergence of ink-jet spotting methods which allowed the in situ synthesis of 60-mer oligo spots on glass slides.Affymetrix further developed DNA microarrays which were based on high-density 25-mer oligos from human cDNA sequences.
  • Some variations among normal individualsCan cause defects in human developmentContributors to cancerCan effect function and gene expression
  • Currently, over 50,000 cDNA probes can be spotted onto a 25X75mm slide by robotic printing. It is thus possible to array the entire human genome compliment using this high-density array approach (e.g. Affymetrix Arrays).The central process in all biochip or microarray experiments is a bindingevent, the hybridisation. One binding partner (either receptor or ligand,probe or target) is immobilised within a small but well-defined area on a flatsolid support of glass or polymer; referred to as the “spot” or “feature” ofthe microarray. There are also examples of prestructured slides, structuredfor example by microcavities (nanotiter plates) or by chemical structuringas well as by electronic features, such as microelectrodes. 103-106 of features per array, 105-106 of probes per featureall probes within a feature contain the same sequenceeach feature has a different sequence
  • . Dna sequences are build up using light directed chemical synthesis.first a substrate with a ntd is fixed ontu the chip at specific locations.ntd has a protecting group x that blocks polymerisation.this protector group is photoliable. And is released on exposure to uvlight.without the protector polymerization and chain build up occur.a filter is added to the chip so that some of the ntds are exposed to light.thesedeprotected groups are then free to add the next nucleotide to the chain.by altering the position of the filter, fodor can build a gene chip with an array of different sequences about 20 ntdslong.fodder added a cdna probe to the gene chip he can simultaneously add tens of thousands of different seq s at the same time.when a cdna probe is added on chip thousands of different seqs can b analysed at the same time.since this entire process is done with a computer the matctingseqs can b quickly pinpointed .and then be matched to available dnaseq in gene database.Affymetrix gene chipsPhotolithography technology from computer industry allows building many 25-mers
  • Developed by Pat Brown’s lab at StanfordPCR products of full-length genes (>100nt)
  • 60 mers.
  • The RNA from 2 or more specimens are compared.  The RNA from each specimen is reverse transcribed using RT-PCR to cDNA (complementary cDNA). The cDNA is labeled with two different fluorescent dyes which are colored differently (red and green), with Cy3 and Cy5.
  • The cDNA probes are hybridized to the DNA microarray. The DNA array is then washed.
  • Fluorescent hybridization signals are scanned and detected using laser confocal devices. Data is the filtered and processed.
  • Data analysis is conducted using DNA Microarray bioinformatic tools and/or image processing software.The signal intesities of the spots are correlated with the concentrations of target mRNA samples. Data mining is also conducted using statistical programs and algorithms to determine if the gene of interest is up-regulated, down-regulated, or unchanged.Data is then organized using a database. Gene Annotation can be then performed on the data, with GO (gene ontology) analysis, clustering analysis (which groups similar genes and pathways together), and analysis of pathways and networks of genes.Results are interpreted as log2 ratios (test intensity/reference intensity
  • In case of a deletion in the test DNA, less test DNA will bind to the corresponding spots and the red label of the reference DNA will prevail; gains in the test genome can be identified by a dominance of the green label of the test DNA. Spots, representing sequences with the same copy number in the test genome relative to the reference genome appear yellow. For BAC arrays, an excess of repetitive Cot DNA (blue spheres) has to be added in order to suppress otherwise unspecifically binding repetitive sequences. Using our array CGH platform, we have been able to detect heterozygous deletion as small as 80kb.
  • This evolving technology is, however, somewhat hampered by the large DNA input requirement—a minimum of 150,000 copies of a human genome, or 0.5 μg, are generally needed per sample to process one CGH array.When compared with conventional karyotyping, array CGH provides a higher resolution, a higher dynamic range and better possibilities for automation. In addition, it allows for direct linking of copy number alterations to known genomic sequences. Examples of substrates used for hybridization are bacterial artificial chromosomes (BACs) , cDNAs , oligonucleotides and exon-specific PCR products .
  • Microarray CGH

    1. 1. A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern. A microarray works by exploiting the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated. By using an array containing many DNA samples, scientists can determine, in a single experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. With the aid of a computer, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell.
    2. 2. The basic premise of the DNA microarray is that RNA samples or targets are hybridized to known cDNAs/oligo probes on the arrays. Microarrays were originally designed to measure gene expression levels of a few genes. Recently, high density microarrays have been developed which have allowed the global analysis of gene expression or the transcriptome. This global analysis allows one to determine the cellular function of genes, the nature and regulation of biochemical pathways, and the regulatory mechanisms at play during certain signalling conditions or diseases
    3. 3. DNA Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, the size of two side-by-side pinky fingers, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. It is important that the gene sequences in a microarray are attached to their support in an orderly or fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides.
    4. 4. • Timeline of Recent DNA Microarray Developments • 1991: Photolithographic printing (Affymetrix) • 1994: First cDNA collections are deve;oped at Stranford • 1995: Quantitative monitoring of gene expression patterns with a complementary DNA microarray. • 1996: Commercialization of arrays (Affymetrix) • 1997: Genome- wide expression monitoring in S. cerevisiae (yeast) • 2000: Portraits/ Signatures of cancer. • 2003: Introduction into clinical practices • 2004: Whole human genome on one microarray
    5. 5. Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Array comparative genomic hybridization (also CMA, Chromosomal Microarray Analysis, Microarray-based comparative genomic hybridization, array CGH, aCGH, or Virtual Karyotype) is a technique to detect genomic copy number variations at a higher resolution level than chromosome-based comparative genomic hybridization (CGH). Dedicated tools are needed to analyse the results of such experiments, which include appropriate visualisation, and to take into consideration the physical relation in
    6. 6. • Comparative genomic hybridization (CGH) microarray is an emerging tool in bioclinical research that allows to identify genomic alterations with a higher resolution than the conventional CGH • To study aberrations of the genome, investigators competitively hybridize fluorescein-labeled normal and pathological samples to an array containing clones designed to cover certain areas of the genome. • Once hybridization has been performed, the signal intensities of the dyes are quantified. Thus, this technique provides a means to quantitatively measure DNA copy-number changes and to map them directly onto a genomic sequence. In oncology, where carcinogenesis is associated with complex chromosomic alterations, CGH arrays can be used for detailed analysis of genomic changes in copy number (in terms of gains or loss of genetic information) in the tumor sample.
    7. 7. • Manufacturing of Microarrays For microarray production, two different approaches are used: • 1. Synthesis on the chip; and • 2. bulk synthesis with subsequent deposition on the chip. The first method is applicable to generate chemical libraries, for example, of short oligonucleotides or peptides; the second method may also be adapted to long polynucleotides or proteins, or any of the many receptors.
    8. 8. . . . . . . . . . . . . . . Spotting technique.
    9. 9. Nimblegen Maskless Array Synthesis These "virtual masks" reflect the desired pattern of UV light with individually addressable aluminum mirrors controlled by the computer. The DMD controls the pattern of UV light projected on the microscope slide in the reaction chamber, which is coupled to the DNA synthesizer. The UV light selectively cleaves a UV-labile protecting group at the precise location where the next nucleotide will be coupled. The patterns are coordinated with the DNA synthesis chemistry in a parallel, combinatorial manner such that 385,000 to 2.1 million unique probe features are synthesized in a single array.
    11. 11. mRNA tRNA rRNA ELUTION BUFFER Bead containing poly T chain TT TTT T T WASHING SOLUTION mRNA tRNA mRNA rRNA
    12. 12. G A C G U A A A mRNA C T G C A T T T cDNA G A C Normal cells. G T A A A mRNA G A C G U A A A cDNA C T G C A T T T G A C G T A A A Cancer cells.
    13. 13. Array CGH Reference DNA (cy5) Array containing probes corresponding to genomic DNA Mix and hybridize to array Test DNA (cy3) Scan and analyze image
    14. 14. Gene 1 Gene 2 Gene 3
    15. 15. In this schematic: GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. Each spot on an array is associated with a particular gene. Each color in an array represents either healthy (control) or diseased (sample) tissue. Depending on the type of array used, the location and intensity of a color will tell us whether the gene, or mutation, is present in either the control and/or sample DNA. It will also provide an estimate of the expression level of the gene(s) in the sample and control DNA.
    16. 16. DNA microarrays have been used to examine the gene expression changes under diseases such as cancer. Tumour profiling using DNA microarrays allows the analysis of the development and the progression of such complex diseases. Using DNA microarrays, one can examine targets for drug discovery and potential diagnostic and prognostic biomarkers for many complex diseases. DNA microarrays are used commonly to detect viruses and other pathogens from blood samples and thus can be used as a pathogen detection method. DNA microarrays have been more recently used to identify inheritable markers, and therefore have been used as a genotyping tool. SNP chips based on DNA microarray technology have allowed the high throughput profiling of single nucleotide polymorphisms using a chip or array approach. This has allowed polymorphisms to be more quickly assayed and also their relavence to disease to
    17. 17. DNA microarrays are better than other profiling methods (such as SAGE, SH, PCR methods) in that they are: Easier to use Are high-throughput (can analyze thousands of genes or markers at a time) Generate large amounts of data in little time Do not require large-scale sequencing - Allow the quantitation of thousands of genes from many samples
    18. 18. • High-throughput – Identify: candidate genes, patterns • Compare two different populations – wild type vs. evolved – normal tissue vs. cancerous tissue • Studing specific chromosomal regions.
    19. 19. •Parental diagnosis •Disease diagnosis like cancer •Gene expression •Chromosomal abberations. •Determines gain and loss of chromosomal regions
    20. 20. • Do not necessarily reflect true levels of proteins - protein levels are regulated by translation initiation & degradation as well • Generally, do not “prove” new biology - simply suggest genes involved in a process, a hypothesis that will require traditional experimental verification • Expensive! $20-$100K to make your own / buy enough to get publishable data
    21. 21. Identification of disease genes by whole genome CGH arrays Lisenka E.L.M. Vissers, Joris A. Veltman*, Ad Geurts van Kessel and Han G. Brunner Department of Human Genetics <http://www.nimblegen.com/> <http://www.affymetrix.com/> <http://smd.stanford.edu/resources/resinfshtml> Validation of Sequence-Optimized 70-base Oligonucleotides for Use on DNA Microarrays (http://www.westburg.nl/download/arrayposter.pdf). By John Ten Bosch, Chris Seidel, Sajeev Batra, Hugh Lam, Nico Tuason, Sepp Saljoughi, and Robert Saul. Assessment of the specificity and Sensitivity of the oligonucleotides (50 mer ) microarrays. By Dr. Susanne Schröder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul 1 WG Biotech AG, Microarray Development1 and Department of Bioinformatics2. 50 nucleotide long probes on microarrays enable high signal intensity and high specificity. By Dr. Susanne Schröder1, Dr. Jaqueline Weber2, and Dr. Hubert Paul1 MWG Biotech AG,microarray Development1and Department of Bioinformatics 2, Anzinger Str. 7, 85560 Ebersberg, Germany. Optimization of Oligonucleotide - based DNA microarrays. By Angela Relogio, Christian Aschwager, Alexandra Ritcher, Wilhelm Ansorge and Juan Valcarel.
    22. 22. An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH by: Joke Allemeersch, Steven Van Vooren, Femke Hannes, Bart De Moor, Joris Vermeesch, Yves Moreau Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays Michael D. Kane, Timothy A. Jatkoe, Craig R. Stumpf, Jia Lu1, Jeffrey D. Thomas and Steven J. Madore* DNA Microarrays: Background, Interactive Databases, and Hands-on Data Analysis A. Malcolm Campbell1 and Laurie J. Heyer2 Microarray CGH Ben Beheshti, Paul C. Park, Ilan Braude, and Jeremy A. Squire http/www.wikipedia.com Search engines: