1 / 16

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.

mahola
Télécharger la présentation

Microarrays

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related