Biotechnology - Microarray

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Biotechnology - Microarray

  1. 1. Genomic librarycDNA libraryMicroarrayBiotechnology
  2. 2. Genomic LibraryA collection of genesA complete set of recombinant plasmids cloneseach carrying copies of particular segment of thegenome (comprehensive)
  3. 3. Shotgun ApproachUses restriction enzyme to randomly cut upgenomic DNA and place it in plasmidsThis process of generating a genomic library cancut up genes because restriction enzymes do notrespect gene boundariesAnimation: http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html
  4. 4. cDNA LibrarycDNA = complementary DNAReverse transcribed from mRNAAnimation: Making a cDNAhttp://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swfQ: Why can’t eukaryotic DNA be cloned directlyinto prokaryotic cells?
  5. 5. Refer to Fig 20.5http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/cdna.gif
  6. 6. http://www.chem.agilent.com/cag/feature/03-04/feature_graphics/rice_microarray.jpgMicroarrayhttp://talon.niagara.edu/~391s08/chapman/Images/cag_42_microarray_dna_print.jpg Slide can hold up to 100 000 pieces of known DNA. The sequence and position of each DNA is known. Information is kept in a database. DNA sequence can be cDNA (genes) oroligonucleotides (i.e. in GeneChip technology)
  7. 7. Manufacturing a microarrayAnimation: printing a microarray by robothttp://upload.wikimedia.org/wikipedia/commons/0/0a/Microarray_printing.ogg
  8. 8. MicroarrayBenefit: can examine thousands of mRNA (cDNA)strands at the same timeUsed to compare a test sample (treated) to thecontrol (wild type, normal, untreated)
  9. 9. Animation: Microarray http://www.sumanasinc.com/webcontent/animations/content/dnachips.html http://www.youtube.com/watch?v=VNsThMNjKhM (1:31) http://www.dnalc.org/resources/3d/26-microarray.html(0:52) http://www.youtube.com/watch?v=9U-9mlOzoZ8 (DNAmicroarray hybridization close up, no sound, 0:55) http://www.dnai.org/d/index.htmlGo to “Genes and Medicine”  “Genetic Profiling” “Technique”  “Microarray” animation “Patrick Brown” on the development of the microarray
  10. 10. Animation: GeneChip Technologyhttp://highered.mcgraw-hill.com/olc/dl/120078/micro50.swf (includesmaking a gene chip array)http://www.dnai.org/d/index.htmlGo to “Genes and Medicine”  “Genetic Profiling”“Technique”  “GeneChips” for click-throughanimation“Stephen Fodor” on the development of GeneChiptechnology
  11. 11. MicroarrayProcedure1. Extract mRNA2. Reverse transcribe intocDNA incorporatingfluorescent probes green is control /normal / wild typesample red is test sample3. Hybridize to microarray4. Laser scannerquantitates data
  12. 12. http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/images/Affymetrixarray.jpg
  13. 13. http://www.agilent.com/about/newsroom/lsca/imagelibrary/images/cag_43_microarray_dna.jpghttp://talon.niagara.edu/~391s08/chapman/Images/cag_42_microarray_dna_print.jpg
  14. 14. http://www.pharmapolis.net/images/stories/microarray%20profile.jpg
  15. 15. Scanned Microarray
  16. 16. Microarray Green: mRNA producedin control sample (wildtype, normal, untreated) Red: mRNA produced intest sample (treated) Yellow: both samplesproduce the sameamount of mRNA
  17. 17. MicroarrayWhich colour dots arethe ones that willinterest theresearcher? Why?Hint: Relate colours togene expression
  18. 18. Gene Expression Green: signifies downregulation of a gene intesting conditions Red: signifies upregulation of a gene intesting conditions Yellow: no changes ingene expression whentesting conditions arechanged
  19. 19. Activity: MicroarrayThe BackgroundmRNA was isolated from seedlings exposed to lightand darkness during germination. The mRNA willbe reverse transcribed into cDNA and hybridizedonto complementary nucleotides on a microarray todetermine the different genes that are expressed inthe two conditions.

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