Biotechnology - Microarray
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Transcript of Biotechnology - Microarray
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Genomic librarycDNA libraryMicroarray
Biotechnology
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Genomic Library
A collection of genes A complete set of recombinant plasmids
clones each carrying copies of particular segment of the genome (comprehensive)
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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
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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?
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Refer to Fig 20.5http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/cdna.gif
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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)
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Manufacturing a microarray
Animation: printing a microarray by robothttp://upload.wikimedia.org/wikipedia/commons/0/0a/Microarray_printing.ogg
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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)
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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
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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
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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
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http://www.columbia.edu/~bo8/undergraduate_research/projects/sahil_mehta_project/images/Affymetrixarray.jpg
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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
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http://www.pharmapolis.net/images/stories/microarray%20profile.jpg
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Scanned Microarray
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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
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Microarray
Which colour dots are the ones that will interest the researcher? Why?
Hint: Relate colours to gene expression
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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
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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.