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DNA Microarrays

Benefits and DrawbacksWaasil Kareem

Biochemistry 118Prof. Douglas Brutlag

01 June 2004

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What is a DNA Microarray?

_ A basic premise of drug research is that differencesin gene expressions (which ones are active) willprovide clues about where to look for drugs. DNAmicroarrays are a fundamental tool for doing suchdifferential gene expression analysis

_ Microarray is a sliver of glass or silicon roboticallystudded with two dimensional orderly arrangementof thousands of DNA spots. It provides a mediumfor matching known and unknown DNA samplesbased on base-pairing rules (hybridization) andautomating the process of identifying the unknowns

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Need for Microarrays_ Despite the recent flood of new biological data from the

human genome sequencing, scientists are struggling toanswer many basic questions

_ There is a need to accelerate figuring out which genesare active, not by tackling one gene one experiment at atime, but implementing a massively parallel process

_ Microarrays allow researchers information on thousandsof genes simultaneously - a dramatic increase inthorough output: months to days

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How does a DNA Microarray work?Microarray works by exploiting the property of a given mRNA moleculeto bind specifically to, or hybridize to, the DNA template from which itoriginated

In a single experiment, using an array with many DNA samples fromknown genes, we can determine the expression levels of hundreds orthousands of genes within a cell by measuring the amount of mRNAbound to each site on the array

The amount of mRNA, fluorescently labeled, bound to the spots on themicroarray is precisely measured using a computer, generating aprofile of gene expression in the cell

DNA MicroArray MicroArray Analyzer Profile

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Hybridization is the KeySource: National Human Genome Research Institute (NHGRI)

Analyzing the location and intensity of fluorescent signals, one can determine the level of activity of each gene

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Types of DNA Microarrays_ cDNA Microarrays

– each spot contains a cDNA clone from a known gene (PCR product)

– better matching results because of longer length cDNA

– initially developed at Stanford University

_ Oligo(nucleotide) Microarrays– made by prefabricated or synthesizing single stranded oligo from

known database

– Since oligos are usually short, the density of these arrays is muchhigher

– Traditionally called GeneChips by commercial suppliers, such asAffymetrix, Agilent

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Benefits of DNA Microarrays

_ Gene Discovery

_ Gene Expression

_ Disease Diagnosis

_ Drug Discovery and toxicology

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Drawbacks of MicroArrays_ Lack of standardization

_ Inadequate computer based tools

_ Statistical problems

_ Insufficient quality

_ Insufficient sensitivity

_ Lack of reproducibility

_ Variability of outcome

_ Fidelity of gene expression data

_ Effects of probe length

_ Cost

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Lack of Standardization_ Data collected from different microarray platforms can’t

be accurately compared

_ Absence of a unified “language” for exchange ofmicroarray data between different groups

_ Microarray Gene Expression Data Society, MGED, hasdeveloped guidelines for the publication of DNAmicroarray data called MIAME, minimal information

_ Microarray Markup Language, developed by MGED, isan attempt to provide a standard platform for submittingand analyzing the microarray expression data generatedby different laboratories around the world

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Inadequate Computer Tools_ Interpreting microarray experiments is very taxing – too

much, and too little information

_ Lack of turnkey bioinformatics models and tools inhibitquick and efficient analysis of the large data sets createdwithin each experiments

_ Statistical problems ranging from image analysis topattern discovery and classification exist.

_ Efforts are being made to improve standards ofexperimental design, data presentation and analysis –advance bioinformatics

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Insufficient Quality, Sensitivityand Reproducibility

_ Ways to characterize the quality of microarrays to ensurefull functionality are lacking

_ Poor sensitivity and background noise forcesamplification which may not give the researcher a truepicture

_ Array to array coefficients of variation is high loweringthe reproducibility of microarray results

_ Hybridization process still involves a great deal ofmanual handling. Microfluidics combined withmicroarrays has the promise of enhancing ease of useand reproducibility of results

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Fidelity of Gene Expression Data

_ Some researchers have shown that for complexdiseases a considerable discrepancy exists between thedifferentially expressed genes identified on oligo arraysarrays and those identified on cDNA microarrays

_ Separation of genes causally involved in a disease frominnocent bystander genes whose expression levels havebeen secondarily altered by primary changes elsewhereis a challenge

_ In addition, different bioinformatic protocols applied tothe same microarray data yield quite different gene setsmaking clinical decisions difficult

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Cost_ Initial cost of designing and fabricating custom

microarrays and analyzer is high but falling; commercialDNA arrays retail for a few hundreds dollars

_ Replication of experiments to minimize statisticalvariability of results can be cost prohibitive

_ Use of fabrication processes and procedures from thesemiconductor industry are helping bring the cost down

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Summary_ DNA microarrays give researchers the opportunity to

analyze vast amounts of genetic information in a singleexperiment.

_ The use of DNA microarrays holds great promise in ourunderstanding of genes and their impact on disease,drug discovery and development

_ DNA microarrays like any other emerging technologyhas shortcoming and drawbacks that have beenidentified and addressed by the biotechnologycommunity

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References1. “Glass slides to DNA Microarrays”, by Samuel D. Conzone and Carlo G. Pantano,

Materials Today, March 2004

2. “Vital Statistics”, by Claire Tilstone, Nature, August 2003

3. “Microarray reality checks in the context of a complex disease”, George L GaborMiklos, Nature Biotechnology 22, 615 - 621 (2004)

4. “Challenges in the Application of DNA Microarrays to Biomedical Research”, byRichard Simon, National Cancer Institute, Bethesda, Maryland, USA

5. “State of Microarray: Challenges and concerns”, by Aileen Constans, TheScientist, February, 2003.

6. Gene Expression Studies using microarrays: principles, problems,a andprospects”, by David Murphy, Advances In Physiology Education 26:256-270,2002.

7. National Human Genome Research Institute website: http://www.nhgri.nih.gov/

8. Affymetrix website: www.affymetrix.com