DNA Microarray

1
DNA Microarray

• Jamie Mashek

2
What we will be discussing

• What is DNA microarray?
• The purpose of using DNA microarray.
• The plate.
• Steps to perform a microarray.
• Benefits.
• Problems.

3
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!

4
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.

5
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.

6
Lets perform a microarray!

• Collect Samples.
• Isolate mRNA.
• Create Labelled DNA.
• Hybridization.
• Microarray Scanner.
• Analyze Data.

7
STEP 1 Collect Samples.

• This can be from a variety of organisms. Well
  use two samples cancerous human skin tissue
  healthy human skin tissue

8
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.

9
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

10
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.

11
STEP 5 LASERS!
12
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.

13
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.

14
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.

15
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
16
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.

17
Sources.

• DNA Microarray Technology. National Human Genome
  Research Institute, 17 Dec. 2009. 19 Feb. 2010
  lthttp//www.genome.gov/10000533gt
• Microarrays Chipping Away at the Mysteries of
  Science and Medicine. National Center for
  Biotechnology Information, 27 July 2007. 19
  Feb. 2010. lthttp//www.ncbi.nlm.nih.gov/About/pri
  mer/microarrays.htmlgt
• Brown, P.O. Botstein, D. Exploring the New
  World of the Genome with DNA Microarrays. Nature
  Genetics Supplement. 21. (1999) 33-37.
  lthttp//www.ctu.edu.vn/dvxe/Bioinformatic/PDF20F
  iles/Volume21/ng0199supp_33.pdfgt
• 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. lthttp//web.cs.mun.ca/harold/Courses/Old
  /CS6754.W04/Diary/ng1030.pdfgt