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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.

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AFFYMETRIX chips

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  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.

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