MICROARRAY

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MICROARRAY

  1. 1. 1. INTRODUCTION 2. HISTORY 3. PRINCIPLE 4. DNA MICROARRAY TECHNOLOGY 5. PRINCIPLES OF DNA MICROARRAY TECHNOLOGY 6. TYPES OF DNA MICROARRAY  GLASS cDNA MICROARRAYS  IN SITU OLIGONUCLEOTIDE ARRAY FORMAT 7. APPLICATIONS OF MICROARRAY TECHNOLOGY
  2. 2. The large-scale genome sequencing effort and the ability to immobilize thousands of DNA fragments on coated glass slide or membrane, have led to the development of microarray technology. A microarray is a pattern of ssDNA probes which are immobilized on a surface called a chip or a slide.  Microarrays use hybridization to detect a specific DNA or RNA in a sample.  DNA microarray uses a million different probes, fixed on a solid surface.
  3. 3.  An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules.  An array experiment makes use of common assay systems such as microplates or standard blotting membranes. Fig-01 Robotic arm with spotting slides
  4. 4.    Microarray technology evolved from Southern blotting. The concept of microarrays was first proposed in the late 1980s by Augenlicht and his colleagues. They spotted 4000 cDNA sequences on nitrocellulose membrane and used radioactive labeling to analyze differences in gene expression patterns among different types of colon tumors in various stages of malignancy.
  5. 5.  The core principle behind microarrays is hybridization between two DNA strands.  Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases. Fig-02 Array hybridization
  6. 6.    DNA microarray technology may be defined as a high-throughput and versatile technology used for parallel gene expression analysis for thousands of genes of known and unknown functions. Used for detection of polymorphisms and mutations in genomic DNA A DNA microarray is a collection of microscopic DNA spots on solid surface. Each spot contains picomoles of a specific DNA sequence, known as probes or reporters.
  7. 7.  Each identified sequenced gene on the glass, silicon chips or nylon membrane corresponds to a fragment of genomic DNA, cDNAs, PCR products or chemically synthesized oligonucleotides of up to 70mers and represents a single gene.  Probe-target hybridization is usually detected and quantified by detection of fluorophore, silver, or chemiluminescence labeled targets to determine relative abundance of nucleic acid sequences in the target.
  8. 8.   The principle of DNA microarray technology is based on the fact that complementary sequences of DNA can be used to hybridise, immobilised DNA molecules. There are four major steps in performing a typical microarray experiment. Sample preparation and labeling Hybridisation Washing Image acquisition and Data analysis
  9. 9.  Isolate a total RNA containing mRNA that ideally represents a quantitative copy of genes expressed at the time of sample collection.  Preparation of cDNA from mRNA using a reversetranscriptase enzyme.  Short primer is required to initiate cDNA synthesis.  Each cDNA (Sample and Control) is labelled with fluorescent cyanine dyes (i.e. Cy3 and Cy5). Fig-03 Sample labeling
  10. 10.    Here, the labelled cDNA (Sample and Control) are mixed together. Purification After purification, the mixed labelled cDNA is competitively hybridised against denatured PCR product or cDNA molecules spotted on a glass slide. Fig-04 Array Hybridisation
  11. 11.     Slide is dried and scanned to determine how much labelled cDNA (probe) is bound to each target spot. Hybridized target produces emissions. Microarray software often uses green spots on the microarray to represent upregulated genes. Red to represent those genes that are downregulated and yellow to present in equal abundance Fig-05 Gene chip showing different type of color spots
  12. 12. 1) Glass cDNA microarrays which involves the micro spotting of pre-fabricated cDNA fragments on a glass slide. 2) High-density oligonucleotide microarrays often referred to as a "chip" which involves in situ oligonucleotide synthesis.
  13. 13.    Glass cDNA microarrays was the first type of DNA microarray technology developed. It was pioneered by Patrick Brown and his colleagues at Stanford University. Produced by using a robotic device which deposits (spots) a nanoliter of DNA onto a coated microscopic glass slide (50-150 µm in diameter) . Fig-06 Contact printer with robotic pins
  14. 14. Selection of the material to spot onto the microscope glass surface. Preparation and purification of DNA sequences representing the gene of interest. Spotting DNA solution onto chemically modified glass slides via a contact printing or inkjet printing. FIG-07 Spotting of slides
  15. 15.    Advantages of Glass cDNA microarrays include their relative affordability with a lower cost. Its accessibility requiring no specific equipment for use such that hybridisation does not need specialised equipment. Data capture can be carried out using equipment that is very often already available in the laboratory.
  16. 16.   Glass cDNA microarray have a few disadvantages such as intensive labour requirement for synthesizing, purifying, and storing DNA solutions before microarray fabrication. They may hybridise to spots designed to detect transcript from a different gene.
  17. 17.    Oligonucleotides are synthesized on the chip. Presently, the commercial versions of Affymetrix Gene Chips hold up to 500,000 probes/sites in a 1.28-cm2 chip area. Due to such very high information content (genes) they are finding widespread use in the hybridisation-based detection and analysis of mutations and polymorphisms, such as single nucleotide polymorphisms.
  18. 18.     Light is directed through a photolithographic mask to specific areas of array surface. Activation of areas for chemical coupling. Attachment of A nucleotide containing photolabile protecting group X (MeNPOC). Next light is Directed to a different region of the array surface through a new mask. Addition of 2nd building block T containing a photolabile protecting group X. This process is repeated until the desired product is obtained. Fig-08 Photolithography process
  19. 19.  Advantages offered by the in situ oligonucleotide array format include speed, specificity and reproducibility.
  20. 20.  In situ oligonucleotide array formats tend to have expensive specialised equipments e.g. to carry out the hybridisation, staining of label, washing, and quantitation process.  Short-sequences used on the array have decreased sensitivity/binding compared with glass cDNA microarrays.
  21. 21. MICROARRA Y AS A GENE EXPRESSION PROFILING TOOL MICROARRA Y AS A COMPARATIV E GENOMICS TOOL DISEASE DIAGNOSIS DRUG DISCOVERY TOXICOLOGIC AL RESEARCH
  22. 22.  The principle aim of using microarray technology as a gene expression profiling tool is to answer some of the fundamental questions in biology such as "when, where, and to what magnitude genes of interest are expressed.  Microarray analysis measure changes in the multigene patterns of expression to better understand about regulatory mechanisms and broader bioactivity functions of genes.
  23. 23.  Microarray technology have widespread use in comparative gene mutation analysis to analyse genomic alterations such as sequence and single nucleotide polymorphisms.  In microbiology microarray gene mutation analysis is directed to characterisation of genetic differences among microbial isolates, particularly closely related species.
  24. 24.  Different types of cancer have been classified on the basis of the organs in which the tumors develop.  Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells.
  25. 25.  Microarray technology has extensive application in Pharmacogenomics.  Comparative analysis of the genes from a diseased and a normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes.
  26. 26.    Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. The microarray permits researchers to examine thousands of different genes in the same experiment and thus to obtain a good understanding of the relative levels of expression between different genes in an organism.
  27. 27.  Microarray is a recently developed functional genomics technology that has powerful applications in a wide array of biological medical sciences, agriculture, biotechnology and environmental studies. Since many universities research institutions and industries have established microarray based core facilities and services, microarrays have become a readily accessible, widely used technology for investigating biological systems.
  28. 28. THANK YOU

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