DNA microarray                Isfahan University of Medical Science, School of Pharmacy                          Departmen...
DNA                    MICROARRAY                       (An overview)By:A.N. Emami Razavi
DNA microarray                          O u t lin e s         In t r o d u c t io n         A p p lic a t io n s       ...
IntroductionDNA microarray
DNA microarray                     What is a Microarray?         Microarray” has become a general term, there are        ...
DNA microarray         A DNA microarray (also commonly known as gene or          genome chip, DNA chip, or gene array) is...
DNA microarray         The affixed DNA segments are known as probes          (although some sources will use different   ...
DNA microarray         DNA microarrays can be used to detect DNA (e.g., in          comparative genomic hybridization); i...
DNA microarray         Since there can be tens of thousands of distinct          probes on an array, each microarray expe...
DNA microarray         These early gene arrays were made by spotting          cDNAs onto filter paper with a pin-spotting...
DNA microarray                        Microarrays are Popular         PubMed search "microarray"= 23,559 papers (february...
ApplicationsDNA microarray
DNA microarray         Gene expression profiling               In different cells/tissues               During the cour...
DNA microarray         Comparative genomic hybridization               Assessing genome content in different cells or cl...
DNA microarray      Areas being studied with microarrays         Differential gene expression between two (or more) sampl...
DNA microarray      mRNA levels compared in many different contexts         Different tissues, same organism (brain v. li...
Types of   DNAMicroarrayDNA microarray
DNA microarray                             Types of Microarrays         Spotted DNA arrays (“cDNA arrays”)              ...
DNA microarray                        Spotted DNA arrays         In spotted microarrays, the probes are oligonucleotides,...
DNA microarray                                    Building a cDNA chip                                        PCR amplific...
DNA microarray                                     Make Chip    Microarray “spotters” are high-precision robots    with me...
DNA microarray         This technique is used by research scientists around the world          to produce "in-house" prin...
DNA microarray                     Oligonucleotide arrays         In oligonucleotide microarrays, the probes are short se...
DNA microarray         Sequences may be longer (60-mer probes such as the Agilent          design) or shorter (25-mer pro...
DNA microarrayJune 26, 2012         25         Total slides : 51
DNA microarray                Spotted Vs. Oligonucleotide array      Spotted Arrays                             Affy Gene ...
DNA Microarray   Process DNA microarray
DNA microarray                   Overview of the process                             Questio                             n...
DNA microarray                     Gene Expression Patterns         Genes are expressed when they are copied into mRNA or...
DNA microarrayJune 26, 2012         30         Total slides : 51
DNA microarrayJune 26, 2012         31         Total slides : 51
DNA microarrayJune 26, 2012         32         Total slides : 51
DNA microarrayJune 26, 2012         33         Total slides : 51
DNA microarrayJune 26, 2012         34         Total slides : 51
DNA microarrayJune 26, 2012         35         Total slides : 51
DNA microarray                        Experiment process      1.        Collect tissue      2.        Isolate RNA      3. ...
DNA microarray                EquipmentsJune 26, 2012           37         Total slides : 51
DNA microarray                Collect tissueJune 26, 2012            38         Total slides : 51
DNA microarray                Isolate RNAJune 26, 2012           39         Total slides : 51
DNA microarray                Isolate mRNAJune 26, 2012            40         Total slides : 51
DNA microarray                Make labeled DNA copyJune 26, 2012                41         Total slides : 51
DNA microarray                Apply DNAJune 26, 2012           42         Total slides : 51
DNA microarray                Scan microarrayJune 26, 2012             43         Total slides : 51
DNA microarray                Analyze dataJune 26, 2012           44         Total slides : 51
DNA microarray      Quantification                Cy5 at 635                     Cy3 at 532                             Ov...
DNA microarray            Expression profiling with DNA microarrays             cDNA “control” Data acquistion cDNA “tream...
DNA microarray                Image Analysis & Data Visualization                                                         ...
DNA microarray                Sample DataJune 26, 2012           48         Total slides : 51
DNA microarray                Data storage and retrieval                        Filtering                     Normalizatio...
DNA microarray                       Analysis of Microarray Data         Clustering               Idea: Groups of genes ...
Thank you  Questions ?
DNA microarray                Two-color vs. one-color detection         Two-Color microarrays are typically hybridized wi...
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DNA microarray

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DNA microarray:
A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.

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  • Fluorophore:A fluorophore , in analogy to a chromophore , is a component of a molecule which causes a molecule to be fluorescent . It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, eg. in immunofluorescence and immunohistochemistry . Fluorescein isothiocyanate , a reactive derivative of fluorescein , has been one of the most common fluorophores chemically attached to other, non-fluorescent molecules to create new and fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine , coumarin and cyanine . A newer generation of fluorophores such as the Alexa Fluors and the DyLight Fluors are generally more photostable, brighter, and less pH -sensitive than other standard dyes of comparable excitation and emission. ---------------------------------
  • Spatially فضائی،فاصله ای = Affixed = متصل شده
  • Drug development or preclinical development is defined in many pharmaceutical companies as the process of taking a new chemical lead through the stages necessary to allow it to be tested in human clinical trials , although a broader definition would encompass the entire process of drug discovery and clinical testing of novel drug candidates. Pharmacogenomics is the branch of pharmacology which deals with the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity . By doing so, pharmacogenomics aims to develop rational means to optimise drug therapy, with respect to the patients' genotype , to ensure maximum efficacy with minimal adverse effects . Such approaches promise the advent of " personalized medicine ", in which drugs and drug combinations are optimised for each individual's unique genetic makeup. Pharmacogenomics is the whole genome application of pharmacogenetics , which examines the single gene interactions with drugs.
  • Assessing= ارزیابی ، تشخیص Occupancy = اشغال ، تصرف ------------------------------- SNP array From Wikipedia, the free encyclopedia Jump to: navigation , search In molecular biology and bioinformatics , a SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA , is the most frequent type of variation in the genome. For example, there are an estimated 5-10 million SNPs in the human genome . As SNPs are highly conserved throughout evolution and within a population , the map of SNPs serves as an excellent genotypic marker for research. [ edit ] Principles The basic principles of SNP array are the same as the DNA microarray . These are the convergence of DNA hybridization , fluorescence microscopy , and solid surface DNA capture. The three mandatory components of the SNP arrays are: The array that contains immobilized nucleic acid sequences or target; One or more labeled probes; A detection system that records and interprets the hybridization signal. To achieve relative concentration independence and minimal cross-hybridization, raw sequences and SNPs of multiple databases are scanned to design the probes. Each SNP on the array is interrogated with different probes. Depending on the purpose of experiments, the amount of SNPs present on an array is considered. [ edit ] Applications An SNP array is a useful tool to study the whole genome . The most important application of SNP array is in determining disease susceptibility and consequently, in pharmacogenomics by measuring the efficacy of drug therapies specifically for the individual. As each individual has many single nucleotide polymorphisms that together create a unique DNA sequence, SNP-based genetic linkage analysis could be performed to map disease loci, and hence determine disease susceptibility genes for an individual. The combination of SNP maps and high density SNP array allows the use of SNPs as the markers for Mendelian diseases with complex traits efficiently. For example, whole-genome genetic linkage analysis shows significant linkage for many diseases such as rheumatoid arthritis , prostate cancer , and neonatal diabetes . As a result, drugs can be personally designed to efficiently act on a group of individuals who share a common allele - or even a single individual. In addition, SNP array can be used for studying the Loss of heterozygosity (LOH). LOH is a form of allelic imbalance that can result from the complete loss of an allele or from an increase in copy number of one allele relative to the other. While other chip -based methods (e.g. Comparative genomic hybridization can detect only genomic gains or deletions), SNP array has the additional advantage of detecting copy number neutral LOH due to uniparental disomy (UPD). In UPD, one allele or whole chromosome from one parent are missing leading to reduplication of the other parental allele (uni-parental = from one parent, disomy = duplicated). In a disease setting this occurrence may be pathologic when the wildtype allelle (e.g. from the mother) is missing and instead two copies of the heterozygous allelle (e.g. from the father) are present. Using high density SNP array to detect LOH allows identification of pattern of allelic imbalance with potential prognostic and diagnostic utilities. This usage of SNP array has a huge potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers. Recent studies based on the SNP array technology have shown that not only solid tumors (e.g. gastric cancer , liver cancer etc) but also hematologic malignancies ( ALL , MDS , CML etc) have a high rate of LOH due to genomic deletions or UPD and genomic gains. The results of these studies may help to gain insights into mechanisms of these diseases and to create targeted drugs. -------------------------------- Chromatin immunoprecipitation From Wikipedia, the free encyclopedia Jump to: navigation , search Chromatin immunoprecipitation ( ChIP ) assay , is a method used for experiments in molecular biology . The purpose of this assay is to determine whether proteins including (but not limited to) transcription factors bind to a particular region on the endogenous chromatin of living cells or tissues. The in vivo nature of this method is in contrast to other approaches traditionally employed to answer the same questions (e.g. SELEX ). The principle underpinning this assay is that DNA -bound proteins (including transcription factors) in living cells can be cross-linked to the chromatin where they are situated. This is usually accomplished by a gentle formaldehyde fixation, although it is sometimes advantageous to use the reversible crosslinker DTBP instead. Following fixation, the cells are lysed and the DNA is broken into pieces 0.2-1 kb in length by sonication . Once the proteins are immobilized on the chromatin and the chromatin is fragmented, whole protein-DNA complexes can be immunoprecipitated using an antibody specific for the protein in question. The DNA from the isolated protein/DNA fraction can then be purified. The identity of the DNA fragments isolated in complex with the protein of interest can then be determined by PCR using primers specific for the DNA regions that the protein in question is hypothesized to bind. Alternatively, when one wants to find where the protein binds across the whole genome , a DNA microarray can be used ( ChIP-on-chip or ChIP-chip ) allowing for the characterization of the cistrome . [ edit ] Major disadvantages and solutions The major disadvantage is the requirement for highly specific antibodies for each protein to be tested. This can be overcome by the construction of proteins fused to either epitopes (like HA or c-myc) recognized by antibodies widely available, or amino acid sequences recognized by enzymes that add biotin to some residues. Biotin has the great advantage of binding with extremely high affinity and specificity to the proteins avidin , streptavidin and NeutrAvidin .
  • Customizable= قابل هماهنگ کردن
  • Consolidate This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that is customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator.
  • Photolithography From Wikipedia, the free encyclopedia Jump to: navigation , search For earlier uses of photolithography in printing, see Lithography . Photolithography (also optical lithography ) is a process used in microfabrication to selectively remove parts of a thin film (or the bulk of a substrate). It uses light to transfer a geometric pattern from a photomask to a light-sensitive chemical ( photoresist , or simply "resist") on the substrate. A series of chemical treatments then engraves the exposure pattern into the material underneath the photoresist. In a complex integrated circuit (for example, modern CMOS ), a wafer will go through the photolithographic cycle up to 50 times. Photolithography resembles the conventional lithography used in printing , and shares some fundamental principles with photography . It is used because it affords exact control over the shape and size of the objects it creates, and because it can create patterns over an entire surface simultaneously. Its main disadvantages are that it requires a flat substrate to start with, it is not very effective at creating shapes that are not flat, and it can require extremely clean operating conditions. Basic procedure This article needs additional citations for verification . Please help improve this article by adding reliable references . Unsourced material may be challenged and removed. (July 2007) The wafertrack portion of an aligner that uses 365 nm ultraviolet light. A single iteration of photolithography combines several steps in sequence. Modern cleanrooms use automated, robotic systems to coordinate the process. The procedure described here omits some advanced treatments, such as thinning agents or edge-bead removal. [1] [ edit ] Preparation The wafer is initially heated to a temperature sufficient to drive off any moisture that may be present on the wafer surface. Wafers that have been in storage must be chemically cleaned to remove contamination. A liquid or gaseous "adhesion promoter", such as hexamethyldisilazane (HMDS), is applied to promote adhesion of the photoresist to the wafer. [ edit ] Photoresist application The wafer is covered with photoresist ("PR") by spin coating . A viscous, liquid solution of photoresist is dispensed onto the wafer, and the wafer is spun rapidly to produce a uniformly thick layer. The spin coating typically runs at 1200 to 4800 rpm for 30 to 60 seconds, and produces a layer between 2.5 and 0.5 micrometres thick. The photoresist-coated wafer is then "soft-baked" or "prebaked" to drive off excess solvent, typically at 90 to 100 ° C for 5 to 30 minutes. [ citation needed ] Sometimes a nitrogen atmosphere is used. [ edit ] Exposure and developing After prebaking, the photoresist is exposed to a pattern of intense light. Optical lithography typically uses ultraviolet light (see below). Positive photoresist, the most common type, becomes less chemically robust when exposed; negative photoresist becomes more robust. This chemical change allows some of the photoresist to be removed by a special solution, called "developer" by analogy with photographic developer . A post-exposure bake is performed before developing, typically to help reduce standing wave phenomena caused by the destructive and constructive interference patterns of the incident light. The develop chemistry is delivered on a spinner, much like photoresist. Developers originally often contained sodium hydroxide (NaOH). However, sodium is considered an extremely undesirable contaminant in MOSFET fabrication because it degrades the insulating properties of gate oxides. Metal-ion-free developers such as tetramethylammonium hydroxide (TMAH) are now used. The resulting wafer is then "hard-baked", typically at 120 to 180 °C [ citation needed ] for 20 to 30 minutes. The hard bake solidifies the remaining photoresist, to make a more durable protecting layer in future ion implantation , wet chemical etching , or plasma etching . [ edit ] Etching Main article: Etching ( microfabrication ) In the etching step, a liquid ("wet") or plasma ("dry") chemical agent removes the uppermost layer of the substrate in the areas that are not protected by photoresist. In semiconductor fabrication , dry etching techniques are generally used, as they can be made anisotropic, in order to avoid significant undercutting of the photoresist pattern. This is essential when the width of the features to be defined is similar to or less than the thickness of the material being etched (ie when the aspect ratio approaches unity). Wet etch processes are generally isotropic in nature, which is often indispensable for microelectromechanical systems ( MEMS ), where suspended structures must be "released" from the underlying layer. The development of low-defectivity anisotropic dry-etch process has enabled the ever-smaller features defined photolithographically in the resist to be transferresd to the substrate material. [ edit ] Photoresist removal After a photoresist is no longer needed, it must be removed from the substrate. This usually requires a liquid "resist stripper", which chemically alters the resist so that it no longer adheres to the substrate. Alternatively, photoresist may be removed by a plasma containing oxygen , which oxidizes it. This process is called ashing , and resembles dry etching. [ edit ] Exposure ("printing") systems Karl Süss manual contact aligner for small volume processing. Exposure systems typically produce an image on the wafer using a photomask. The light shines through the photomask, which blocks it in some areas and lets it pass in others. ( Maskless lithography projects a precise beam directly onto the wafer without using a mask, but it is not widely used in commercial processes.) Exposure systems may be classified by the optics that transfer the image from the mask to the wafer. [ edit ] Contact and proximity Main article: Contact lithography A contact printer, the simplest exposure system, puts a photomask in direct contact with the wafer and exposes it to a uniform light. A proximity printer puts a small gap between the photomask and wafer. In both cases, the mask covers the entire wafer, and simultaneously patterns every die. Contact printing is liable to damage both the mask and the wafer, and this was the primary reason it was abandoned for high volume production. Both contact and proximity lithography require the light intensity to be uniform across an entire wafer, and the mask to align precisely to features already on the wafer. As modern processes use increasingly large wafers, these conditions become increasingly difficult. Research and prototyping processes often use contact lithography, because it uses inexpensive hardware and can achieve high optical resolution . The resolution is approximately the square root of the product of the wavelength and the gap distance. Hence, contact printing offers the best resolution, because its gap distance is approximately zero (neglecting the thickness of the photoresist itself). In addition, nanoimprint lithography may revive interest in this familiar technique, especially since the cost of ownership is expected to be low. [ edit ] Projection See also: Stepper Very-large-scale integration lithography uses projection systems. Unlike contact or proximity masks, which cover an entire wafer, projection masks (also called "reticles") show only one die. Projection exposure systems (steppers) project the mask onto the wafer many times to create the complete pattern. [ edit ] Photomasks Main article: Photomask The image for the mask originates from a computerized data file. This data file is converted to a series of polygons and written onto a square fused quartz substrate covered with a layer of chrome using a photolithographic process. A beam of electrons is used to expose the pattern defined in the data file and travels over the surface of the substrate in either a vector or raster scan manner. Where the photoresist on the mask is exposed, the chrome can be etched away, leaving a clear path for the light in the stepper/scanner systems to travel through. [ edit ] Resolution in projection systems The filtered fluorescent lighting in photolithography cleanrooms contains no ultraviolet or blue light in order to avoid exposing photoresists. The spectrum of light emitted by such fixtures gives virtually all such spaces a bright yellow color. The ability to project a clear image of a small feature onto the wafer is limited by the wavelength of the light that is used, and the ability of the reduction lens system to capture enough diffraction orders from the illuminated mask. Current state-of-the-art photolithography tools use deep ultraviolet (DUV) light with wavelengths of 248 and 193 nm , which allow minimum feature sizes down to 50 nm. The minimum feature size that a projection system can print is given approximately by: where is the is a coefficient that encapsulates process-related factors, and typically equals 0.5 is the wavelength of light used is the numerical aperture of the lens as seen from the wafer According to this equation, minimum feature sizes can be decreased by decreasing the wavelength, and increasing the numerical aperture, i.e. making lenses larger and bringing them closer to the wafer. However, this design method runs into a competing constraint. In modern systems, the depth of focus is also a concern: The depth of focus restricts the thickness of the photoresist and the depth of the topography on the wafer. Chemical mechanical polishing is often used to flatten topography before high-resolution lithographic steps. [ edit ] Light sources Historically, photolithography has used ultraviolet light from gas-discharge lamps using mercury , sometimes in combination with noble gases such as xenon . These lamps produce light across a broad spectrum with several strong peaks in the ultraviolet range. This spectrum is filtered to select a single spectral line , usually the "g-line" (436 nm) or "i-line" (365 nm). More recently, lithography has moved to "deep ultraviolet", produced by excimer lasers . (In lithography, wavelengths below 300 nm are called "deep UV".) Krypton fluoride produces a 248-nm spectral line, and a 193-nm line. Optical lithography can be extended to feature sizes below 50 nm using 193 nm and liquid immersion techniques. Also termed immersion lithography , this enables the use of optics with numerical apertures exceeding 1.0. The liquid used is typically ultra-pure, deionised water, which provides for a refractive index above that of the usual air gap between the lens and the wafer surface. This is continually circulated to eliminate thermally-induced distortions. Water will only allow NA' s of up to ~1.4, but materials with higher refractive indices will allow the effective NA to be increased further. Tools using 157 nm wavelength DUV in a manner similar to current exposure systems have been developed. These were once targeted to succeed 193 nm at the 65 nm feature size node but have now all but been eliminated by the introduction of immersion lithography. This was due to persistent technical problems with the 157 nm technology and economic considerations that provided strong incentives for the continued use of 193 nm technology. High-index immersion lithography is the newest extension of 193 nm lithography to be considered. In 2006, features less than 30 nm were demonstrated by IBM using this technique [2] . [ edit ] Experimental methods See also: Nanolithography Photolithography has been defeating predictions of its demise for many years. For instance, it was predicted that features smaller than 1 micrometre could not be printed optically. Modern techniques already print features several times smaller than the wavelength of light used - an amazing optical feat. Current research is exploring new tricks in the ultraviolet regime, as well as alternatives to conventional UV, such as electron beam lithography , X-ray lithography , extreme ultraviolet lithography , and immersion lithography .
  • Acquistion= بدست آوری
  • Retrieval بازیافت =
  • Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Uses They have many uses as fluorescent dyes. Depending on the structure, they cover the spectrum from IR to UV. They were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelengths which will form an image on the film. They are used in CD-R and DVD-R media. The ones used are mostly green or light blue in color, and are chemically unstable. This makes cyanine discs unsuitable for archival CD and DVD use, as they can fade and become unreadable in a few years. [edit] Cy3 and Cy5 Cy3 and Cy5 are reactive water-soluble fluorescent dyes of the cyanine dye family. Cy3 is red and Cy5 is far-red. They are usually synthesized with reactive groups on either one or both of the nitrogen side chains so that they can be chemically crosslinked to either nucleic acids or protein molecules. Labeling is done for visualization and quantification purposes. They are used in a wide variety of biological applications including comparative genomic hybridization and in gene chips, which are used in transcriptomics. They are also used to label proteins for various studies including proteomics
  • DNA microarray

    1. 1. DNA microarray Isfahan University of Medical Science, School of Pharmacy Department of Clinical BiochemistryJune 26, 2012 1 Total slides : 51
    2. 2. DNA MICROARRAY (An overview)By:A.N. Emami Razavi
    3. 3. DNA microarray O u t lin e s  In t r o d u c t io n  A p p lic a t io n s  T y p e s o f D N A m ic r o a r r a y  M ic r o a r r e y p r o c e s s  M ic r o a r r a y a n a ly s isJune 26, 2012 3 Total slides : 51
    4. 4. IntroductionDNA microarray
    5. 5. DNA microarray What is a Microarray?  Microarray” has become a general term, there are many types now  DNA microarrays  Protein microarrays  Transfection microarrays  Antibody microarray  Tissue microarray  Chemical compound microarray …  We’ll be discussing DNA microarraysJune 26, 2012 5 Total slides : 51
    6. 6. DNA microarray  A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high- stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.June 26, 2012 6 Total slides : 51
    7. 7. DNA microarray  The affixed DNA segments are known as probes (although some sources will use different nomenclature such as reporters), thousands of which can be placed in known locations on a single DNA microarray. Microarray technology evolved from Southern blotting, whereby fragmented DNA is attached to a substrate and then probed with a known gene or fragment.June 26, 2012 7 Total slides : 51
    8. 8. DNA microarray  DNA microarrays can be used to detect DNA (e.g., in comparative genomic hybridization); it also permits detection of RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins, which is referred to as "expression analysis" or expression profiling.June 26, 2012 8 Total slides : 51
    9. 9. DNA microarray  Since there can be tens of thousands of distinct probes on an array, each microarray experiment can potentially accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations. The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon.June 26, 2012 9 Total slides : 51
    10. 10. DNA microarray  These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays for gene expression profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997.June 26, 2012 10 Total slides : 51
    11. 11. DNA microarray Microarrays are Popular  PubMed search "microarray"= 23,559 papers (february 17,2008)  // // = 28,027 papers (march,9,2008) 5000 4500 4406  2005 = 4406 4000 3500 3509  2004 = 3509 3000  2003 = 2421 2500 2421 2000  2002 = 1557 1500 1557  2001 = 834 1000 834 500  2000 = 294 0 294 2000 2001 2002 2003 2004 2005June 26, 2012 11 Total slides : 51
    12. 12. ApplicationsDNA microarray
    13. 13. DNA microarray  Gene expression profiling  In different cells/tissues  During the course of development  Under different environmental or chemical stimuli  In disease state versus healthy  Molecular diagnosis:  Molecular classification of disease  Drug development  Identification of new targets  Pharmacogenomics  Individualized medicineJune 26, 2012 13 Total slides : 51
    14. 14. DNA microarray  Comparative genomic hybridization  Assessing genome content in different cells or closely related organisms.  SNP detection arrays  Identifying single nucleotide polymorphism among alleles within or between populations.  Chromatin immunoprecipitation (ChIP) studies  Determining protein binding site occupancy throughout the genome, employing ChIP-on-chip technology.June 26, 2012 14 Total slides : 51
    15. 15. DNA microarray Areas being studied with microarrays  Differential gene expression between two (or more) sample types  Similar gene expression across treatments  Tumor sub-class identification using gene expression profiles  Classification of malignancies into known classes  Identification of “marker” genes that characterize different tumor classes  Identification of genes associated with clinical outcomes (e.g. survival)June 26, 2012 15 Total slides : 51
    16. 16. DNA microarray mRNA levels compared in many different contexts  Different tissues, same organism (brain v. liver)  Same tissue, same organism (tumor v. non-tumor)  Same tissue, different organisms (wt v. mutant)  Time course experiments (development)June 26, 2012 16 Total slides : 51
    17. 17. Types of DNAMicroarrayDNA microarray
    18. 18. DNA microarray Types of Microarrays  Spotted DNA arrays (“cDNA arrays”)  Developed by Pat Brown (Stanford)  PCR products (or long oligos) from known genes (~100 nt) spotted on glass, plastic, or nylon support  Customizable and off the shelf  Gene Chips  Oligonucleotide arrays (Affymetrix)  Large number of 20-25mers/gene  Enabled by photolithography from the computer industry  Off the shelf  Ink-jet microarrays (Agilent)  25-60mers “printed” directly on glass  Four cartridges: A, C, G, and T  Flexible, rapid, but expensiveJune 26, 2012 18 Total slides : 51
    19. 19. DNA microarray Spotted DNA arrays  In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs. There probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive cDNA derived from experimental or clinical samples.June 26, 2012 19 Total slides : 51
    20. 20. DNA microarray Building a cDNA chip PCR amplification of Consolidate target DNA into plates (cDNA or portion of genomic DNA) Arrayed Library (96 or 384-well plates of bacterial glycerol stocks) Spot as microarray on glass slidesJune 26, 2012 20 Total slides : 51
    21. 21. DNA microarray Make Chip Microarray “spotters” are high-precision robots with metal pins that dip into DNA solution & Robot spotter tap down on glass slide (pins work like a fountain pen) Glass slideJune 26, 2012 21 Total slides : 51
    22. 22. DNA microarray  This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that is customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator.June 26, 2012 22 Total slides : 51
    23. 23. DNA microarray Oligonucleotide arrays  In oligonucleotide microarrays, the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames. Although oligonucleotide probes are often used in "spotted" microarrays, the term "oligonucleotide array" most often refers to a specific technique of manufacturing. Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene by synthesizing this sequence directly onto the array surface instead of depositing intact sequences.June 26, 2012 23 Total slides : 51
    24. 24. DNA microarray  Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer probes produced by Affymetrix) depending on the desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture.  One technique used to produce oligonucleotide arrays include photolithographic synthesis (Agilent and Affymetrix) on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array.June 26, 2012 24 Total slides : 51
    25. 25. DNA microarrayJune 26, 2012 25 Total slides : 51
    26. 26. DNA microarray Spotted Vs. Oligonucleotide array Spotted Arrays Affy Gene Chips  Relative cheap to make (~$10  Expensive ($500 or more) slide)  Limited types avail, no chance  Flexible - spot anything you of specialized chips want  Fewer repeated experiments  Cheap so can repeat experiments many times usually  Highly variable spot  More uniform DNA feaures deposition  Usually have to make your  Can buy off the shelf ownJune 26, 2012 26 Total slides : 51
    27. 27. DNA Microarray Process DNA microarray
    28. 28. DNA microarray Overview of the process Questio n Data Sample Analysis Preparatio n Microarra y Microarray DetectionJune 26, 2012 28 Hybridizatio Total slides : 51
    29. 29. DNA microarray Gene Expression Patterns  Genes are expressed when they are copied into mRNA or RNA (transcription)  Differential gene expression: which genes are expressed in which cells or tissues at a given point in time or in the life of the organism.  Total RNA can be isolated from cells or tissues under different experimental conditions and the relative amounts of transcribed RNA can be measured  The change in expression pattern in response to an experimental condition, environmental change, drug treatment, etc. sheds light into the dynamic functioning of a cellJune 26, 2012 29 Total slides : 51
    30. 30. DNA microarrayJune 26, 2012 30 Total slides : 51
    31. 31. DNA microarrayJune 26, 2012 31 Total slides : 51
    32. 32. DNA microarrayJune 26, 2012 32 Total slides : 51
    33. 33. DNA microarrayJune 26, 2012 33 Total slides : 51
    34. 34. DNA microarrayJune 26, 2012 34 Total slides : 51
    35. 35. DNA microarrayJune 26, 2012 35 Total slides : 51
    36. 36. DNA microarray Experiment process 1. Collect tissue 2. Isolate RNA 3. Isolate mRNA 4. Make labeled DNA copy 5. Apply DNA 6. Scan microarray 7. Analyze dataJune 26, 2012 36 Total slides : 51
    37. 37. DNA microarray EquipmentsJune 26, 2012 37 Total slides : 51
    38. 38. DNA microarray Collect tissueJune 26, 2012 38 Total slides : 51
    39. 39. DNA microarray Isolate RNAJune 26, 2012 39 Total slides : 51
    40. 40. DNA microarray Isolate mRNAJune 26, 2012 40 Total slides : 51
    41. 41. DNA microarray Make labeled DNA copyJune 26, 2012 41 Total slides : 51
    42. 42. DNA microarray Apply DNAJune 26, 2012 42 Total slides : 51
    43. 43. DNA microarray Scan microarrayJune 26, 2012 43 Total slides : 51
    44. 44. DNA microarray Analyze dataJune 26, 2012 44 Total slides : 51
    45. 45. DNA microarray Quantification Cy5 at 635 Cy3 at 532 Overlay imagesJune 26, 2012 45 Total slides : 51
    46. 46. DNA microarray Expression profiling with DNA microarrays cDNA “control” Data acquistion cDNA “treament” Cy3 labeled Cy5 labeled 1 1 6 2 3 6 2 5 5 5 5 3 5 4 2 5 6 2 3 5 6 6 6 5 Relative Relative intensity Output HYBRIDIZATION intensity Output 1 Spot #1 Spot #4 1 4 2 5 55 2 Spot #2 5 Spot #5 2 2 5 5 5 5 3 6 3 6 Spot #3 6 6 Spot #6 3 46 6 6 Total slides : 51June 26, 2012
    47. 47. DNA microarray Image Analysis & Data Visualization Cy5 Cy5 log2 Cy3 Cy3 Cy5 Cy3 200 10000 50.00 5.64 4800 4800 1.00 0.00 9000 300 0.03 -4.91 Experiments Underexpressed 8 4 2 Genes fold 2 4 Overexpressed 8June 26, 2012 47 Total slides : 51
    48. 48. DNA microarray Sample DataJune 26, 2012 48 Total slides : 51
    49. 49. DNA microarray Data storage and retrieval Filtering Normalization AnalysisJune 26, 2012 49 Total slides : 51
    50. 50. DNA microarray Analysis of Microarray Data  Clustering  Idea: Groups of genes that share similar function have similar expression patterns  Hierarchical clustering  k-means  Bayesian approaches  Projection techniques  Principal Component Analysis  Independent Component Analysis  Classification  Idea: A cell can be in one of several states  (Diseased vs. Healthy, Cancer X vs. Cancer Y vs. Normal)  Can we train an algorithm to use the gene expression patterns to determine which state a cell is in?  Support Vector Machines  Decision Trees  Neural Networks  K-Nearest NeighborsJune 26, 2012 50 Total slides : 51
    51. 51. Thank you Questions ?
    52. 52. DNA microarray Two-color vs. one-color detection  Two-Color microarrays are typically hybridized with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different fluorophores. Fluorescent dyes commonly used for cDNA labelling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labelled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up- regulated and down-regulated genes.June 26, 2012 52 Total slides : 51
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