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Statistics for Microarrays

Statistics for Microarrays. Biological background: Molecular Laboratory Techniques. Class web site: http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/. Molecular Laboratory Techniques. Hybridizing DNA Copying DNA Cutting DNA Probing DNA. Hybridization.

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Statistics for Microarrays

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  1. Statistics for Microarrays Biological background: Molecular Laboratory Techniques Class web site: http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/

  2. Molecular Laboratory Techniques • Hybridizing DNA • Copying DNA • Cutting DNA • Probing DNA

  3. Hybridization • Hybridization exploits a potent feature of the DNA duplex – the sequence complementarity of the two strands • Strands can be separated (denatured) by heating • Remarkably, DNA can reassemble with perfect fidelity from separated strands

  4. Polymerase Chain Reaction (PCR) • PCR is used to amplify (copy) specific DNA sequences in a complex mixture when the ends of the sequence are known • Source DNA is denatured into single strands • Two synthetic oligonucleotides complementary to the 3’ ends of the segment of interest are added in great excess to the denatured DNA, then the temperature is lowered • The genomic DNA remains denatured since the complementary strands are at too low a concentration to encounter each other during the period of incubation • The specific oligonucleotides hybridize with complementary sequences in the genomic DNA

  5. PCR, ctd • The hybridized oligos then serve as primers for DNA synthesis, which begins upon addition of a supply of nucleotides and a temperature resistant polymerase such as Taq polymerase, from Thermus aquaticus (a bacterium that lives in hot springs) • Taq polymerase extends the primers at temperatures up to 72˚C • When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes • Repeated cycles (25—30) of synthesis (cooling) and melting (heating) quickly provide many DNA copies

  6. (RT)

  7. Types of Viruses A virus is a nucleic acid in a protein coat. Reverse transcriptase makes a complementary DNA copy from RNA.

  8. Reverse transcription Clone cDNA strands, complementary to the mRNA G U A A U C C U C mRNA Reverse transcriptase T T A G G A G cDNA C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G

  9. RT-PCR

  10. Restriction Enzymes Cut DNA

  11. Probing DNA • One way to study a specific DNA fragment within a genome is to probe for the sequence of the fragment • A probe is a labeled (usually radioactive or fluorescent) single-stranded oligonucleotide, synthesized to be complementary to the sequence of interest – probe sequence is known • Attach single-stranded DNA to a membrane (or other solid support) and incubate with the probe so that it hybridizes • Visualize the probe (e.g. by X-ray for radioactive probes)

  12. Measuring Gene Expression Idea: measure the amount ofmRNAto see whichgenesare beingexpressedin (used by) the cell. Measuringproteinmight be more direct, but is currently harder.

  13. Principal Uses of Microarrays • Genome-scale gene expression analysis • Differential gene expression between two (or more) sample types • Responses to environmental factors • Disease processes (e.g. cancer) • Effects of drugs • Identification of genes associated with clinical outcomes (e.g. survival) • Detection of sequence variation • Genetic typing • Detection of somatic mutations (e.g. in oncogenes) • Direct sequencing

  14. Major technologies • cDNA probes (> 200 nt), usually produced by PCR, attached to either nylon or glass supports • Oligonucleotides (25-80 nt) attached to glass support • Oligonucleotides (25-30 nt) synthesized in situ on silica wafers (Affymetrix) • Probes attached to tagged beads

  15. Brief outline of steps for producing a cDNA microarray • Probes are cDNA fragments, usually amplified by PCR • Probes are deposited on a solid support, either positively charged nylon or glass slide • Samples (normally poly(A)+ RNA) are labelled using fluorescent dyes • At least two samples are hybridized to chip • Fluorescence at different wavelengths measured by a scanner

  16. Pins collect cDNA from wells 384 well plate -- Contains cDNA probes Print-tip group 1 cDNA clones Spotted in duplicate Print-tip group 6 Glass Slide Array of bound cDNA probes 4x4 blocks = 16 print-tip groups

  17. Building the chip Ngai Lab arrayer , UC Berkeley Print-tip head

  18. cDNA microarrays PRINT cDNA from one gene on each spot SAMPLES cDNA labelled red/green Compare gene expression in two samples e.g. treatment/ control or normal/ tumor tissue

  19. HYBRIDIZE Add equal amounts of labelled cDNA samples to microarray. SCAN Laser Detector

  20. Yeast genome on a chip

  21. Web animation of a cDNA microarray experiment http://www.bio.davidson.edu/courses/genomics/chip/chip.html

  22. Probe selection Non-redundant set of probes Includes genes of interest to project Corresponds to physically available clones Chip layout Grouping of probes by function Correspondence between wells in microtiter plates and spots on the chip cDNA Microarray Design

  23. Nylon arrays Up to about 1000 probes per filter Use radiolabeled cDNA target Can use phosphorimager or X-ray film Glass arrays Up to about 40,000 probes per slide, or 10,000 per 2cm2 area (limited by arrayer’s capabilities) Use fluorescent targets Require specialized scanner cDNA arrays on nylon and glass

  24. Choice of coupling method Physical (charge), non-specific chemical, specific chemical (modified PCR primer) Choice of printing method Mechanical pins: flat tip, split tip, pin & ring Piezoelectric deposition (“ink-jet”) Robot design Precision of movement in 3 axes Speed and throughput Number of pins, numbers of spots per pin load Glass chip manufacturing

  25. Laser scanners Excellent spatial resolution Good sensitivity, but can bleach fluorochromes Still rather slow CCD scanners Spatial resolution can be a problem Sensitivity easily adjustable (exposure time) Faster and cheaper than lasers In all cases, raw data are images showing fluorescence on surface of chip Scanning the arrays

  26. Probes are oligos synthesized in situ using a photolithographic approach There are at least 5 oligos per cDNA, plus an equal number of negative controls The apparatus requires a fluidics station for hybridization and a special scanner Only a single fluorochrome is used per hybridization Expensive, but getting cheaper Affymetrix GeneChips

  27. Affymetrix chip production

  28. Clontech, Incyte, Research Genetics - filter-based arrays with up to about 8000 clones Incyte / Synteni – 10,000 probe chips, not distributed (have to send them target RNA) Affymetrix - oligo-based chips with 12,000 genes of known function (16 oligos/gene) and 4x10’000 genes from ESTs Commercial chips

  29. Synthesis of probes on microbeads Hybridization in solution Identification of beads by fluorescent bar coding by embedding transponders Readout using micro-flow cells or optic fiber arrays Production of “universal” arrays Array uses a unique combination of oligos, and probes containing the proper complements Alternative technologies

  30. cDNA microarray experiments mRNA levels compared in many different contexts • Different tissues, same organism (brain v. liver) • Same tissue, same organism (ttt v. ctl, tumor v. non-tumor) • Same tissue, different organisms (wt v. ko, tg, or mutant) • Time course experiments (effect of ttt, development) • Other special designs (e.g. to detect spatial patterns)

  31. Mutation detection Oligos (Affymetrix type) representing all known alleles PCR followed by primer extension, with detection of alleles by MALDI-TOF mass spectroscopy (Sequenom) Gene loss and amplification Measure gene dosage in genomic DNA by hybridization to genomic probes Arrays for Genetic Analysis

  32. Many groups have made their raw data available, but in many formats Some groups have created searchable databases Several initiatives to create “unified” databases EBI: ArrayExpress NCBI: Gene Expression Omnibus Some companies are beginning to sell microarray expression data (e.g. Incyte) Microarray data on the Web

  33. Biological question Differentially expressed genes Sample class prediction etc. Experimental design Microarray experiment 16-bit TIFF files Image analysis (Rfg, Rbg), (Gfg, Gbg) Normalization R, G Estimation Testing Clustering Discrimination Biological verification and interpretation

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