Jamie Mashek

What we will be discussing… What is DNA microarray? The purpose of using DNA

microarray. The plate. Steps to perform a microarray. Benefits. Problems.

What is DNA Microarray?

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

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 of mitosis.

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

To observe mutations in DNA.

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 needle applies the cDNA to the plate, or by a method similar to making silicon chips for computers. The latter is called a Gene Chip.

Let’s perform a microarray!1) Collect Samples.

2) Isolate mRNA.

3) Create Labelled DNA.

4) Hybridization.

5) Microarray Scanner.

6) Analyze Data.

STEP 1: Collect Samples.

This can be from a variety of organisms. We’ll use two samples – cancerous human skin tissue & healthy human skin tissue

STEP 2: Isolate mRNA.

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

After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing 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.

STEP 3: Create Labelled DNA. Add a labelling mix to the

RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.

We will add cyanine 3 (fluoresces green) to the healthy cells and cyanine 5 (fluoresces red) to the cancerous cells.

The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA

STEP 4: Hybridization.

Apply the cDNA we have just created to a microarray plate.

When comparing two samples, apply both samples to the same plate.

The ssDNA will bind to the cDNA already present on the plate.

STEP 5: LASERS!

STEP 5: Microarray Scanner. The scanner has a laser, a

computer, and a camera.

The laser causes the hybrid bonds to fluoresce.

The camera records the images produced when the laser scans the plate.

The computer allows us to immediately view our results and it also stores our data.

STEP 6: Analyze the Data. GREEN – the healthy sample

hybridized more than the diseased sample.

RED – the diseased/cancerous sample hybridized more than the nondiseased sample.

YELLOW - both samples hybridized equally to the target DNA.

BLACK - areas where neither sample hybridized to the target DNA.

By comparing the differences in gene expression between the two samples, we can understand more about the genomics of a disease.

Benefits.

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

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

Fast - Thousands of genes can be analyzed at once.

Problems.

Oligonucleotide libraries – redundancy and contamination.

DNA Microarray only detects whether a gene is turned on or off.

Massive amounts of data.

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

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 between therapeutic responses to drugs and the genetic profiles of the patients.

Toxicogenomics – microarray technology allows us to research the impact of toxins on cells. Some toxins can change the genetic profiles of cells, which can be passed on to cell progeny.

Sources.

DNA Microarray Technology. National Human Genome Research Institute, 17 Dec. 2009. 19 Feb. 2010 <http://www.genome.gov/10000533>

Microarrays: Chipping Away at the Mysteries of Science and Medicine. National Center for Biotechnology Information, 27 July 2007. 19 Feb. 2010. <http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html>

Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA Microarrays. Nature Genetics Supplement. 21. (1999): 33-37. <http://www.ctu.edu.vn/~dvxe/Bioinformatic/PDF%20Files/Volume21/ng0199supp_33.pdf>

Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in the Use of DNA Microarray Data 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 to Finish – For Obtaining Data From DNA Microarrays II. Nature Genetics Supplement. 32. (2002): 482-489. <http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/ng1030.pdf>