Jamie Mashek DNA Microarray
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! • Collect Samples. • Isolate mRNA. • Create Labelled DNA. • Hybridization. • Microarray Scanner. • 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 (oligodT) 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: 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>