Genomic LibraryA collection of genesA complete set of recombinant plasmids cloneseach carrying copies of particular segment of thegenome (comprehensive)
Shotgun ApproachUses restriction enzyme to randomly cut upgenomic DNA and place it in plasmidsThis process of generating a genomic library cancut up genes because restriction enzymes do notrespect gene boundariesAnimation: http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html
cDNA LibrarycDNA = complementary DNAReverse transcribed from mRNAAnimation: Making a cDNAhttp://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swfQ: Why can’t eukaryotic DNA be cloned directlyinto prokaryotic cells?
Refer to Fig 20.5http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/cdna.gif
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)
Manufacturing a microarrayAnimation: printing a microarray by robothttp://upload.wikimedia.org/wikipedia/commons/0/0a/Microarray_printing.ogg
MicroarrayBenefit: can examine thousands of mRNA (cDNA)strands at the same timeUsed to compare a test sample (treated) to thecontrol (wild type, normal, untreated)
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
Animation: GeneChip Technologyhttp://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
MicroarrayProcedure1. Extract mRNA2. Reverse transcribe intocDNA incorporatingfluorescent probes green is control /normal / wild typesample red is test sample3. Hybridize to microarray4. Laser scannerquantitates data
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
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.