7.91/7.36/BE490 Lecture #7 May4,2004 DNA Microarrays& Clustering Chris burge
7.91 / 7.36 / BE.490 Lecture #7 May 4, 2004 DNA Microarrays & Clustering Chris Burge
DNA Microarrays Clustering ° Why the hype? ° Microarray platforms CDNA VS oligo technologies Sample applications Analysis of microarray data clustering of co-expressed genes some classic microarray papers
DNA Microarrays & Clustering • Why the hype? • Microarray platforms - cDNA vs oligo technologies • Sample applications • Analysis of microarray data - clustering of co-expressed genes - some classic microarray papers
Stanford U. Dept. of Biochemistry Web site tanford University.: DEPARTMENT OF Biochemistry http://cmgm.stanford.edu/biochem/
Stanford U. Dept. of Biochemistry Web Site http://cmgm.stanford.edu/biochem/
Why microarrays? Changes in gene expression are important in many biological contexts Development Cancer other Diseases Environmental Adaptation DNA microarrays provide a high throughput way to study these changes
Why Microarrays? • Changes in gene expression are important in many biological contexts: – Development – Cancer – Other Diseases – Environmental Adaptation • DNA microarrays provide a high throughput way to study these changes
What's new? progression to chip technology Hybrid detection radioactive labeling fluorescent labeling Solid support for sample fixation Southern blots northern blots etc Main advantage of microarrays is scale Probes are attached to solid support Efficient robotics Bioinformatic analysis Parallel measurement of thousands of genes at a time
What’s new? … progression to chip technology • Hybrid detection – radioactive labeling – fluorescent labeling • Solid support for sample fixation – Southern blots, Northern blots, etc. • Main advantage of microarrays is scale – Probes are attached to solid support – Efficient robotics – Bioinformatic analysis • Parallel measurement of thousands of genes at a time
Array platforms CDNA arrays(spotted arrays Probes are PCR products from cDNA libraries or clone collections May be printed on glass slides(e.g, P. Brown lab, Stanford), OR May be printed on nylon membranes(e.g, Millennium Spots are 100-300 um in size and about the same distance apart 30,000 CDNAs can be fit onto the surface of a microscope slide Oligonucleotide arrays 20-25 mers synthesized onto silicon wafers in situ or printed onto glass slides by: photolithography(Affymetrix)or ink-jet printing(Rosetta/Agilent Presynthesized oligos can also be printed onto glass slides Other technologies (e.g, bead arrays attached to optical fibers
Array Platforms • cDNA arrays (spotted arrays) – Probes are PCR products from cDNA libraries or clone collections – May be printed on glass slides (e.g., P. Brown lab, Stanford), OR – May be printed on nylon membranes (e.g., Millennium) – Spots are 100-300 µm in size and about the same distance apart – ~30,000 cDNAs can be fit onto the surface of a microscope slide • Oligonucleotide arrays – 20-25 mers synthesized onto silicon wafers in situ or printed onto glass slides by: by: photolithography (Affymetrix) or ink-jet printing (Rosetta/Agilent) – Presynthesized oligos can also be printed onto glass slides • Other technologies (e.g., bead arrays attached to optical fibers)
CDNA Arrays I-Overview Please see Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. " Expression Profiling using CDNA Microarrays. "Nat Genet. 21, no. 1 Suppl (January 1999 ): 10-4
cDNA Arrays I - Overview Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. "Expression Profiling using cDNA Microarrays." Nat Genet. 21, no. 1 Suppl (January 1999): 10-4. Please See
CDNA Arrays Printing 1. Templates for genes of interest obtained and amplified by pcr 2. After purification and quality control aliquots of -5 nl printed on coated glass microscope slide using high speed robot Please see Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. " Expression Profiling using CDNA Microarrays. "Nat Genet. 21, no. 1 Suppl (January 1999): 10-4
cDNA Arrays II - Printing 1. Templates for genes of interest obtained and amplified by PCR 2. After purification and quality control, aliquots of ~5 nl printed on coated glass microscope slide using high speed robot Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. "Expression Profiling using cDNA Microarrays." Nat Genet. 21, no. 1 Suppl (January 1999): 10-4. Please See
CDNA Arrays -Labeling Hybing 1. Total RNA from test and reference samples is fluorescently labeled with Cy5/Cy3 dye using a single round of reverse transcription 2. Pooled fluorescent targets are hybridized to the clones on the array Please see Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. " Expression Profiling using CDNA Microarrays. "Nat Genet. 21, no. 1 Suppl (January 1999): 10-4
1. Total RNA from test and reference samples is fluorescently labeled with Cy5/Cy3 dye using a single round of reverse transcription 2. Pooled fluorescent targets are hybridized to the clones on the array cDNA Arrays III - Labeling, Hybing Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. "Expression Profiling using cDNA Microarrays." Nat Genet. 21, no. 1 Suppl (January 1999): 10-4. Please See
CDNA ArraysⅣ- Scanning Laser excitation of hybridized targets-emission spectra measured using a scanning confocal laser microscope 2. Monochrome images(from scanner) are imported into software in which images are pseudo colored and merged 3. Data analyzed as normalized ratio ( Cy3/Cy5)-gene expression increase or decrease relative to reference sample Please see Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. " Expression Profiling using CDNA Microarrays. "Nat Genet. 21, no. 1 Suppl (January 1999 ) 10-4
1. Laser excitation of hybridized targets - emission spectra measured using a scanning confocal laser microscope 2. Monochrome images (from scanner) are imported into software in which images are pseudocolored and merged 3. Data analyzed as normalized ratio (Cy3/Cy5) - gene expression increase or decrease relative to reference sample cDNA Arrays IV - Scanning Duggan, DJ, M Bittner, Y Chen, P Meltzer, and JM Trent. "Expression Profiling using cDNA Microarrays." Nat Genet. 21, no. 1 Suppl (January 1999): 10-4. Please See
