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Genomic librarycDNA libraryMicroarray
Biotechnology
Genomic Library
A collection of genes A complete set of recombinant plasmids
clones each carrying copies of particular segment of the genome (comprehensive)
Shotgun Approach
Uses restriction enzyme to randomly cut up genomic DNA and place it in plasmids
This process of generating a genomic library can cut up genes because restriction enzymes do not respect gene boundaries
Animation: http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html
cDNA LibrarycDNA = complementary DNAReverse transcribed from mRNAAnimation: Making a cDNA
http://highered.mcgraw-hill.com/olc/dl/120078/bio_h.swf
Q: Why can’t eukaryotic DNA be cloned directly into 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.jpg
Microarray
http://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) or
oligonucleotides (i.e. in GeneChip technology)
Manufacturing a microarray
Animation: 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 the control (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
(DNA microarray hybridization close up, no sound, 0:55)
http://www.dnai.org/d/index.html Go 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 (includes making a gene chip array)
http://www.dnai.org/d/index.html Go to “Genes and Medicine” “Genetic
Profiling”“Technique” “GeneChips” for click-
through animation“Stephen Fodor” on the development of
GeneChip technology
Microarray Procedure
1. Extract mRNA2. Reverse transcribe into
cDNA incorporating fluorescent probes green is control /
normal / wild type sample
red is test sample3. Hybridize to
microarray4. Laser scanner
quantitates data
http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/images/Affymetrixarray.jpg
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
http://www.pharmapolis.net/images/stories/microarray%20profile.jpg
Scanned Microarray
Microarray
Green: mRNA produced in control sample (wild type, normal, untreated)
Red: mRNA produced in test sample (treated)
Yellow: both samples produce the same amount of mRNA
Microarray
Which colour dots are the ones that will interest the researcher? Why?
Hint: Relate colours to gene expression
Gene Expression
Green: signifies down regulation of a gene in testing conditions
Red: signifies up regulation of a gene in testing conditions
Yellow: no changes in gene expression when testing conditions are changed
Activity: Microarray
The BackgroundmRNA was isolated from seedlings exposed to light and darkness during germination. The mRNA will be reverse transcribed into cDNA and hybridized onto complementary nucleotides on a microarray to determine the different genes that are expressed in the two conditions.