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.