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Microarrays

Microarrays. w ith 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. . Common Types of Microarrays.

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Microarrays

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

  2. Formal Definition A microarray is a hybridization-based technique that allows simultaneous analysis of thousands of samples of biological material on a solid substrate.

  3. Common Types of Microarrays • Additional types of assays: • Chemical compound microarrays • Carbohydrate microarrays • Phenotype micro arrays • Many more specific types under development

  4. 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.

  5. 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 (cDNAor 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. *RNA microarrays are very similar to DNA microarrays, but involve RNA probes hybridizing to target cRNA (also known as anti-sense RNA)

  6. 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

  7. 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

  8. General Process Overview

  9. 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

  10. 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

  11. 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)

  12. In situ Synthesized Arrays Nucleotides Protecting groups

  13. 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

  14. Applications • Gene discovery • Drug discovery • Individualized treatments • Toxicological research • Gene expression studies • Disease diagnosis • Pathogen analysis • Rapid genotyping

  15. Microarray Pros and Cons

  16. 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.

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