1 / 18

AFFYMETRIX chips

AFFYMETRIX chips. What are DNA chips/arrays?. Orderly arrangement of samples Medium for matching known and unknown DNA samples Two types of arrays-Macro and Micro Macro-Sample spot sizes of 300 microns or more Micro-Sample spot sizes of less than 200 microns in diameter.

weldon
Télécharger la présentation

AFFYMETRIX chips

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. AFFYMETRIX chips

  2. What are DNA chips/arrays? • Orderly arrangement of samples • Medium for matching known and unknown DNA samples • Two types of arrays-Macro and Micro Macro-Sample spot sizes of 300 microns or more Micro-Sample spot sizes of less than 200 microns in diameter

  3. Micro Arrays • Fabricated by high-speed robotics • Known probes used to determine complementary binding • Enable massive parallel gene expression and gene studies • Dramatic increase in throughput • Two major applications-sequence identification and determination of gene expression level

  4. Micro Arrays (contd.) • Two major variants: 1) probe CDNA 2) AFFYMETRIX DNA chips

  5. What does one of them look like??

  6. AFFYMETRIX • Pioneer in making tools for the genomic revolution • Applies semiconductor technology to life science • Manufactures AFFYMETRIX chips using a combination of photolithography and combinatorial chemistry

  7. AFFYMETRIX chip: how is it made? • Very few synthesis steps • Produces arrays with hundreds of thousands of probes packed at very high density • The very high density enables researches to obtain high-quality genomic data using small sample volumes • Number of synthesis steps determined by length of probes, not number

  8. Diagrammatic Representation

  9. CONSTRUCTION • 5-inch square quartz wafer used • Wafer washed to ensure uniform hydroxylation across its surface • Quartz provides a good substrate for linker molecules, which are used to position the probes on the arrays. • Wafer placed in bath of silane, reacts with hydroxyl groups of quartz, and forms a matrix of covalently linked molecules

  10. Construction (contd.) • Distance between silane molecules determines the probes’ packing density • Each of these probe locations harbors millions of identical DNA molecules. • Probe synthesis occurs in parallel: addition of A,C,G or T nucleotide to multiple growing chains happens simultaneously.

  11. Probe synthesis • Photolithographic masks, carrying 18-20 square micron windows: placed over coated wafer • Windows distributed over mask depending on desired sequence of each probe • First step of synthesis: UV light is shown over the mask, rendering the exposed linkers unprotected and available for nucleotide coupling. • CRITCAL: Alignment of mask with wafer before each synthesis step

  12. Nucleotide attachment • Solution containing a single type of deoxynucleotide with a removable protection group is flushed over wafer’s surface • Nucleotide attaches to activated linkers, initiating synthesis • Side chains of nucleotides protected to prevent the formation of branched oligonucleotides

  13. Synthesis and beyond • Another mask placed over wafer, for next round of deprotection and coupling • Process repeated, till probes reach their full length (typically 25 nucleotides) • After synthesis is complete, the wafers are deprotected, diced and the resulting arrays are packaged. • Depending on the number of probe features per array, a single wafer can yield 49-400 arrays. • Quality control tests

  14. APPLICATIONS TWO MAIN CLASSES: • Gene expression/RNA analysis • DNA analysis

  15. Gene Expression • AFFYMETRIX developed a chip called Cytochrome P450, that could rapidly detect variation in gene activity affected by several therapeutics, including many beta-blockers and anti-depressants. • AFFYMETRIX teamed up with a bacterial genomics company to develop an inexpensive chip to test drinking water for the presence of many different strains of pathogenic molecules.

  16. Gene Expression (contd.) • Researchers from Whitehead Institute and MIT were able to identify a gene in melanoma cells that appears to play a crucial role in the spread of cancer throughout the body. • Scientists claimed that they were able to distinguish between two types of adult leukemia that can be confused with one another - each requiring a different course of treatment.

  17. DNA analysis • AFFYMETRIX developed a chip to quickly scan a DNA sample for the presence of any of the mutant versions of the HIV virus. • A high-density DNA probe array, developed by AFFYMETRIX has important applications for the identification of Mycobacterium tuberculosis species and the ability to detect of rifampicin resistance in a single platform.

More Related