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Dna microarray (dna chips)

May 24, 2015




2. 2 Introduction Principle Steps involved in DNA microarray Technique used for Preparing DNA microarray Photolithography Types of DNA Chips cDNA - based chips Oligonucleotide - based chips Applications Advantages and disadvantages Conclusion Reference CONTENTS 3. 3 Introduction: Definition: DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached in an organized grid fashion. Each spot of DNA, called a probe, represents a single gene. There are several synonyms of DNA microarrays such as DNA chips, gene chips, DNA arrays, gene arrays and biochips. [Ref: Presscot (Book for Microbiology),] History: Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known DNA sequence. 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. 4. 4 a) A DNA chip can be manufactured to contain hundreds of thousands of synthetic single-stranded DNA sequences. b) Unknown DNA from a patient is separated into single strands, enzymatically cut and labeled with a fluorescent dye. 5. 5 c) The unknown DNA is inserted into the chip and allowed to hybridize with the DNA on the chip. d) The tagged DNA will bind only to the complementary DNA on the chip. The bound DNA will be detected by its fluorescent dye and analyzed by a computer. The red light is a gene expressed in normal cells; green is a mutated gene expressed in tumor cells; and yellow, in both cells. Fig: DNA Chip Technology 6. 6 Principl e The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization. The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. 7. 7 i. DNA Chip ii. Target sample (Fluorescently labelled) ii. Enzymes iii. Fluorescent dyes iv. Probes v. Scanner Requirement s: There are certain requirements for designing a DNA microarray system and they are: 8. 8 Design of a DNA Microarray System 9. 9 1. Sample preparation 2. Purification 10. 10 3. Reverse Transcription 4. Labelling 11. 11 5.Hibridization 6. Scanning 7.Normalization and analysis 12. 12 Technique used for the Preparation of DNA chip Fig: Photolithography 13. 13 There are 2 types of DNA Chips/Microarrays: Types of DNA chips 1. cDNA based microarray 2. Oligonucleotide based microaaray 14. 14 This type of chips are prepared by using cDNA, it is called cDNA chips or cDNA microarray or probe DNA. The cDNAs are amplified by using PCR. Then these immobilized on a solid support made up of nylon filtre of glass slide (1 x 3 inches). The probe DNA are loaded into a a spotting spin by capillary action. Small volume of this DNA preparation is spotted on solid surface making physical contact between these two. DNA is delivered mechanically or in a robotic manner. When one DNA spotting is done, the pin is washed and loaded with fresh DNA to Start the second cycle. cDNA based chips: 15. 15 Oligonucleotide-based chips Fig: Construction of a DNA chip with attached oligonucleotide sequences (two cycles are shown) 16. 16 Comparative Microarray hybridization: Fig: Steps of comparative microarray hybridization. [Ref:] 17. 17 Table 1. Steps in the design and implementation of a DNA microarray 1) Probe (cDNA/oligo with known identity) 2) Chip fabrication (Putting probes on the chip) 3) Target (fluorecently labeled sample) 4) Assay 5) Readout 6) Informatics Small oligonucleoti des, cDNAs, chromosome . Photolithogr a--phy, pipette, piezoelectric . RNA, (mRNA) cDNA. Hybridizatio n. Fluorescenc e, probeless (conductanc e, MS, electrophor esis). Robotics control, Image processing, DBMS,bioin formatics. 18. 18 The DNA chips are used in many areas as given below: Gene expression profiling Discovery of drugs Diagnostics and genetic engineering Alternative splicing detection Proteomics Functional genomics DNA sequencing Toxicological research (Toxicogenomics) Applications 19. 19 Provides data for thousands of genes. One experiment instead of many. Fast and easy to obtain results. Huge step closer to discovering cures for diseases and cancer. Different parts of DNA can be used to study gene expresion. ADVANTAGES [Ref:,] 20. 20 Disadvantages: The biggest disadvantage of DNA chips is that they are expensive to create. The production of too many results at a time requires long time for analysis, which is quite complex in nature. The DNA chips do not have very long shelf life, which proves to be another major disadvantage of the technology. [Ref:,] 21. 21 DNA Microarrays are one of the most effective invention ever developed. A DNA Microarray is a test that allows for the comparison of thousands of genes at once. Microarray technology uses chips with attached DNA sequences as probes for gene expression. Any DNA in the sample that is complementary to a probe sequence will become bound to the chip. Microarray technology is most powerful when it used on species with a sequenced genome. The microarray chip can hold sequences from every gene in the entire genome and the expression of every gene can be studied simultaneously. Gene expression data can provide information on the function of previously uncharacterized genes. Conclusion 22. 22 Source: Lehninger, 2005, DNA Chips, Principle of Biochemistry, W.H. Freeman and Company, Newyork, forth Edition, Pg. 326- 328. Presscot M. Lansing, 2002, DNA Chips, Microbiology, Athenaeum Press Ltd, Gateshead, Tyne and Wear, Britain, Fifth Edition, 353 354. Dubey C. R, 2008, DNA Chips, A textbook for Biotechnology, S. Chand and Company Ltd., New Delhi, 13th Edition, Pg. 194 197. Tortora J. Gerard, DNA Chips, Book for Microbiology, Pearson Education, Inc., San Francisco, CA, tenth edition, Pg. 292 293. Net Source: Reference 23. 23 THANK YOU

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