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DNA MICROARRAY

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What we will be discussing

What is DNA microarray?

The purpose of using DNA microarray.

The plate.

Steps to perform a microarray.

Benefits.

Problems.

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What is DNA Microarray?

Scientists used to be able to perform genetic analyses of a fewgenes at once. DNA microarray allows us to analyze thousands ofgenes in one experiment!

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Purposes.

So why do we use DNA microarray? To measure changes in gene expression levels two samples gene expression

can be compared from different samples, such as from cells of different stages ofmitosis.

To observe genomic gains and losses. Microarray Comparative GenomicHybridization (CGH)

To observe mutations in DNA.

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The Plate.

Usually made commercially.

Made of glass, silicon, or nylon.

Each plate contains thousands of spots, and each spot contains a

probe for a different gene. A probe can be a cDNA fragment or a synthetic oligonucleotide,

such as BAC (bacterial artificial chromosome set).

Probes can either be attached by robotic means, where a needleapplies the cDNA to the plate, or by a method similar to making

silicon chips for computers. The latter is called a Gene Chip.

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Lets perform a microarray!

1) Collect Samples.

2) Isolate mRNA.

3) Create Labelled DNA.

4) Hybridization.

5) Microarray Scanner.

6) Analyze Data.

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STEP 1: Collect Samples.

This can be from a variety of organisms. Well use twosamples cancerous human skin tissue & healthy human

skin tissue

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STEP 2: Isolate mRNA.

Extract the RNA from the samples. Using either a column, or asolvent such as phenol-chloroform.

After isolating the RNA, we need to isolate the mRNA from therRNA and tRNA. mRNA has a poly-A tail, so we can use a columncontaining beads with poly-T tails to bind the mRNA.

Rinse with buffer to release the mRNA from the beads. The buffer

disrupts the pH, disrupting the hybrid bonds.

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STEP 3: Create Labelled DNA.

Add a labelling mix to the RNA.The labelling mix contains poly-T(oligo dT) primers, reversetranscriptase (to make cDNA),and fluorescently dyednucleotides.

We will add cyanine 3 (fluorescesgreen) to the healthy cells andcyanine 5 (fluoresces red) to thecancerous cells.

The primer and RT bind to themRNA first, then add thefluorescently dyed nucleotides,creating a complementary strandof DNA

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STEP 4: Hybridization.

Apply the cDNA we havejust created to a microarrayplate.

When comparing twosamples, apply bothsamples to the same plate.

The ssDNA will bind to thecDNA already present onthe plate.

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STEP 5: LASERS!

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STEP 5: Microarray Scanner.

The scanner has a laser, a computer,and a camera.

The laser causes the hybrid bonds tofluoresce.

The camera records the imagesproduced when the laser scans theplate.

The computer allows us toimmediately view our results and italso stores our data.

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STEP 6: Analyze the Data.

GREEN the healthy sample hybridized morethan the diseased sample.

RED the diseased/cancerous samplehybridized more than the nondiseasedsample.

YELLOW - both samples hybridized equally tothe target DNA.

BLACK - areas where neither samplehybridized to the target DNA.

By comparing the differences in geneexpression between the two samples, we canunderstand more about the genomics of adisease.

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Benefits.

Relatively affordable (for some people!), about $60,000 for anarrayer and scanner setup.

The plates are convenient to work with because they are small.

Fast - Thousands of genes can be analyzed at once.

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Problems.

Oligonucleotide libraries redundancy andcontamination.

DNA Microarray only detects whether a geneis turned on or off.

Massive amounts of data.

http://www.stuffintheair.com/very-big-problem.html

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The Future of DNA Microarray.

Gene discovery.

Disease diagnosis: classify the types of cancer on the basis of the

patterns of gene activity in the tumor cells.

Pharmacogenomics = is the study of correlations betweentherapeutic responses to drugs and the genetic profiles of thepatients.

Toxicogenomics microarray technology allows us to research theimpact of toxins on cells. Some toxins can change the geneticprofiles of cells, which can be passed on to cell progeny.

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Sources.

DNA Microarray Technology. National Human Genome Research Institute, 17Dec. 2009. 19 Feb.2010

Microarrays: Chipping Away at the Mysteries of Science and Medicine. National Center forBiotechnology Information, 27 July 2007. 19 Feb. 2010.

Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA Microarrays.Nature Genetics Supplement. 21. (1999): 33-37.

Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in theUse of DNA MicroarrayData for Diagnostic and Prognostic Classification. Journal of the National Cancer Institute. 95.(2003): 14-18. http://jnci.oxfordjournals.org/cgi/content/full/95/1/14

Holloway, A.J., Van Laar, R.K., Tothill, R.W., & Bowtell, D.D.L. Options Available From Start toFinish ForObtaining Data From DNA Microarrays II. Nature Genetics Supplement. 32. (2002):482-489.