with an emphasis on DNA microarrays BE 4332 Final Project Natalie Derise

Formal Definition A microarray is a hybridization-based technique that allows simultaneous analysis of thousands of samples of biological material on a solid substrate. http://faculty.ucr.edu/~tgirke/HTML_Presentations/Manuals/Microarray/arrayBasics.pdf

Common Types of Microarrays Type of array General function Protein Protein expression and interactions profiling Tissue Compare histologic sections from unique tissues or tumors; tissues from multiple patients on same slide Cellular Reverse transfection and PMHC; study cell responses Antibody Detects antigens and protein expression; many diagnostic applications DNA Measure gene activity and genotyping 1) http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=14562005 2) http://en.wikipedia.org/wiki/Microarray 3)http://medicine.yale.edu/pathology/research/tissueservices/tissuemicroarrayfacility/what.aspx 4) http://www.abcam.com/index.html?pageconfig=resource&rid=15165 Additional types of assays: Chemical compound microarrays Carbohydrate microarrays Phenotype micro arrays Many more specific types under development

DNA Microarrays A DNA microarray consists of pre-designed synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix. DNA microarrays rely on the hybridization between cDNA that is reverse transcribed from a biological sample to the pre-designed probes on the array. http://bitesizebio.com/articles/introduction-to-dna-microarrays/

Common Terms Array: refers to the glass, plastic, or silicon slide that the DNA probes will be spotted or built on. Target DNA/RNA*: the nucleic acid (cDNA or cRNA) sample that is being identified and/ or measured. Probe: short sections of oligonucleotides that are attached to the array and hybridize with the target cDNA or cRNA Hybridization: the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. Video of DNA hybridization: http://www.youtube.com/watch?v=0qoqzErrae4 http://ghr.nlm.nih.gov/glossary=dnahybridization *RNA microarrays are very similar to DNA microarrays, but involve RNA probes hybridizing to target cRNA (also known as anti-sense RNA)

General Process Overview Using PCR or other techniques, synthesize probes specific to sequence(s) of interest and attach them to array; another option is to purchase premade arrays specific to gene(s) of interest Isolate mRNA from cells of interest Transform this mRNA into cDNA by reverse transcriptase using fluorescently labeled nucleotides in order to create labeled cDNA for RNA microarrays reverse transcriptase is omitted and the mRNA is labeled The labeled cDNA is washed over the microarray and will bind to any matching probes **videos of the general process can be found in the notes on the last slide http://www.docstoc.com/docs/86089378/DNA-Microarray-%28PowerPoint%29

General Process Overview Excess cDNA is washed away, leaving only hybridized sequences. Microarray is excited with laser The labeled, hybridized cDNA will fluoresce at different intensities Microarray is scanned and quantified The brighter/ more concentrated the color, the more DNA is present The picture shown here is a two color array, which will be further explained on slide 13. http://www.unictbiolmol-lab.it/figure_didattica/arrays.pdf Image from: http://people.uwec.edu/piercech/tox/techniques.htm

General Process Overview Note: Here is another picture of the microarray process. The process shown here is a one color microarray. Stains can be used in place of fluorescent molecules, as shown in the above process.

Attaching the Probe to the Array DNA fragments are chemically tethered to glass, plastic, nylon, or silicon biochips (also known as DNA chips) This is done in different ways according to which type of microarray is being used. The two common types are: Spotted arrays In situ synthesized arrays

Spotted Arrays The nucleotide sequences of interest are generated by PCR and “spotted” onto the array surface by a robot. Sequences are ~1 kb in length Relatively low costs and easy to synthesize for smaller gene sets http://www.youtube.com/watch?v=3ZXq_aDfSB8 video: shows spotting process Modified from ChE 4420 notes

In situ Synthesized Arrays Also known as photolithographic arrays Sequences of interest are synthesized directly on the array surface Sequences are ~30-70 bp long  more specific binding and more probes per slide Much more expensive than spotted The individual masks must be designed and manufactured. This is the most tedious step, and is what makes the process pricey. To combat this cost, Digital Light Processing (DLP) has been developed; this process uses a set of movable micromirrors to apply light to certain areas on the array. This computerized process bypasses the need for the masks. http://www.youtube.com/watch?v=MuN54ecfHPw (view up to 30 second mark) Video: shows photolithography process http://cmr.asm.org/content/22/4/611/F3.expansion.html and ChE 4420 notes

In situ Synthesized Arrays Nucleotides Protecting groups *microfluidics are often used in this process *The mask blocks the light over certain nucleotides. The light removes the protecting groups which prevent the attachment of the new nucleotide. With the protecting groups removed, the attached, unprotected nucleotides are free to bind with the next nucleotides that are washed over the array. Repeating this process with different variations of masks, will eventually lead to building different specific nucleotide strands ~30-70 bp long on the microarray.

Types of Signal Detection Two color Control and experimental samples labeled with two different fluorophores Hybridized on same array Allows for direct comparisons Cheaper (also commonly used with spotted) Can compare ratio of two genes to determine if their expression levels are related Sensitive to error, more complex design One color Each sample labeled with same fluorophore Expression is compared between multiple arrays Usually used with in situ; also requires twice as many arrays  $$ Less prone to error Simpler design http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1000543 Images: http://www.people.vcu.edu/~mreimers/OGMDA/image.html http://www.mathworks.com/matlabcentral/fx_files/2573/2/ScreenShotSmall.gif

Applications Gene discovery Gene expression studies Drug discovery Image: http://www.bumc.bu.edu/microarray/ --Shows gene expression in different cells; helps determine which genes are working together as well where they are being expressed most frequently. http://www.premierbiosoft.com/tech_notes/microarray.html http://www.unictbiolmol-lab.it/figure_didattica/arrays.pdf Gene discovery Drug discovery Individualized treatments Toxicological research Gene expression studies Disease diagnosis Pathogen analysis Rapid genotyping

Microarray Pros and Cons Many pre-made probes available in market Prior knowledge to gene sequence is not required Large cDNA works well with hybridization Spotting technology is readily available Process is fast and flexible Can be used for all organisms Study multiple genes at once Cons Contamination potential extremely high Cross-hybridization with similar, repetitive gene families can create false positives Not extremely quantitative In situ can become very expensive Many possible errors during scanning due to background noise http://www.unictbiolmol-lab.it/figure_didattica/arrays.pdf

Complete process overview: In Summary: Complete process overview: http://www.youtube.com/watch?v=3jX_08zdYCE Microarray technology is a very powerful tool used for many applications and if often paired with PCR. This field is continuing to grow, leading to cheaper prices as well as increasingly specialized applications. Video: summary of microarrays Image: http://commons.wikimedia.org/wiki/File:Affymetrix_5.0_microarray.jpg --GeneChip: A microarray that can be used to assay half a million or so variable positions in the human genome. ADDITIONAL VIDEOS: http://www.youtube.com/watch?v=SNbt--d14P4 (theoretical working of microarray) http://www.youtube.com/watch?v=UgL1Pq2sk3M (microarray process video) http://www.youtube.com/watch?v=9U-9mlOzoZ8 (generic microarray video)