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AFFYMETRIX chips. What are DNA chips/arrays?. Small silica (glass) chips covered with thousands of nucleotides of know sequence Underlying principle: Base pairing or hybridization Medium for matching known and unknown DNA samples Two types of arrays-Macro and Micro

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what are dna chips arrays
What are DNA chips/arrays?
  • Small silica (glass) chips covered with thousands of nucleotides of know sequence
  • Underlying principle: Base pairing or hybridization
  • Medium for matching known and unknown DNA samples
  • Two types of arrays-Macro and Micro

Macro-Sample spot sizes >= 300 microns

Micro-Sample spot sizes < 200 microns in diameter

micro arrays
Micro Arrays
  • Fabricated by high-speed robotics; generally on glass, but sometimes on nylon substrates
  • 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
micro arrays contd
Micro Arrays (contd.)
  • Two major variants, based on the property of the arrayed DNA sequence with known identity :

1) probe CDNA

2) AFFYMETRIX DNA chips

slide6
Micro Array: Another picture(intensity and color of each spot encode information on a specific gene from tested sample)
affymetrix
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
affymetrix chip how is it made
AFFYMETRIX chip: how is it made?
  • Very few synthesis steps
  • Produces arrays with hundreds of thousands of probes packed at very high density
  • Very high density helps obtain high-quality genomic data using small sample volumes
  • Number of synthesis steps determined by length of probes, not number
construction
CONSTRUCTION
  • 5-inch square quartz wafer used
  • Wafer washed to ensure uniform hydroxylation across its surface
  • Quartz; good substrate for linker molecules, which are used to position the probes on the arrays
  • Wafer placed in bath of silane, which reacts with hydroxyl groups of quartz, and forms a matrix of covalently linked molecules
construction contd
Construction (contd.)
  • Distance between silane molecules determines the probes’ packing density ( the arrays can hold over 500,000 probe locations)
  • Each of these probe locations harbors millions of identical DNA molecules.
  • Probe synthesis occurs in parallel: addition of an A,C,G or T nucleotide to multiple growing chains happens simultaneously
probe synthesis
Probe synthesis
  • Photolithographic masks, carrying 18-20 square micron windows are 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.
  • CRITICAL: Alignment of mask with wafer before each synthesis step
nucleotide attachment
Nucleotide attachment
  • Solution containing single type of deoxynucleotide (with removable protection group) is flushed over wafer’s surface
  • Nucleotide attaches to activated linkers, initiating synthesis
  • Although a very efficient process, some activated linkers fail to attach the new nucleotide:

- Truncation used to prevent formation of probes with missing nucleotides

nucleotide attachment contd
Nucleotide attachment (contd.)

- Also, side chains of nucleotides are protected, to prevent the formation of branched oligonucleotides.

  • Another mask placed over wafer, for next round of deprotection and coupling
  • Algorithms used, to minimize mask usage
  • Process repeated, till probes reach their full length (typically 25 nucleotides)
beyond synthesis
Beyond synthesis
  • After synthesis is complete, the wafers are deprotected, diced and the resulting arrays are packaged in flowcell cartridges.
  • Depending on the number of probe locations per array, a single wafer can yield 49-400 arrays.
  • Quality control tests
  • Prepared chips well-suited to ambitious goals, like discovery of biological mechanisms and disease therapy
applications of affymetrix chips
APPLICATIONS of Affymetrix chips

TWO MAIN CLASSES:

  • Gene expression/RNA analysis
  • DNA analysis
gene expression
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.
melanoma cells research
Melanoma cells research
  • Researchers from Whitehead Institute and MIT identified a gene in melanoma cells that appears to play a crucial role in metastasis, the spread of cancer throughout the body.
  • Used data from Human Genome Project, DNA arrays
  • Screened the expression of about 10,000 genes
  • Found that a gene called rhoC plays a central role in metastasis
dana farber cancer institute
Dana-Farber Cancer Institute
  • Scientists from the institute have discovered a rare and lethal form of blood cancer that affects infants.
  • Data on next slide
  • MLL does not match with ALL or AML.
slide21

Colors representing gene activity (red is high, blue is low) show that MLL has a distinct pattern from both ALL and AML. Each vertical column of squares represents a tumor sample. Each horizontal row of squares represents the activity of one gene.

dna analysis
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.
references
References
  • http://www.gene-chips.com/GeneChips.html#What
  • http://www.gene-chips.com/sample1.html
  • http://www.polymerassemblytech.com/pages/4/
  • http://www.ruderfinn.com/press_release.asp?release_id=79&bhcp=1
  • http://www.affymetrix.com
  • http://www.dartmouth.edu/~cbbc/courses/bio4/bio4-1999/papers/JeffIsaacs.html
  • http://dnabridges.com/hottop/2000/2000NOV11.htm
  • http://www.genomenewsnetwork.org/articles/02_02/Whole_genome_leukemia_image1.shtml
  • http://pharmabiotech.ch/articles/20000701.htm
  • http://web.mit.edu/newsoffice/nr/2000/metastasis.html