DNA microarrays: Types, Strategies, Applications and their future

PRESENTED TO: DR. SAIMA SADAFPRESENTED BY: AMINA HUSSAIN

Ph.D. BIOTECHNOLOGY 2N D SEMESTER

University of the Punjab Lahore

INTRODUCTION: DNA MICROARRAY TECHNOLOGY• DNA arrays are a group of

technologies in which specific DNA sequences are either deposited or synthesized (in a 2-D or sometimes 3-D array) on a surface.

• The DNA is either covalently or non-covalently attached to the surface. simplified view of a DNA array:The upper rectangles show

two spots of DNA on a solid surface (sequences “A” and “B”) prior to and after hybridization. The lower rectangles show highly idealized side views of the same surfaces.

The core principle behind microarrays is hybridization between two DNA strands.

Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases.

INTRODUCTION

Fig- Array hybridization

Four Major Steps In A Typical Microarray Experiment.

Sample preparation

and labeling

Hybridization

Washing

Image acquisition

and Data analysis

• Original DNA array was created with the colony hybridization method of Grunstein and Hogness (1) they produced a random and unordered collectionof DNA spots represented the cloned fragments.

• In 1979, this approach was adapted to create ordered arrays by Gergen et. al. (Gergen et al.,1979)

• In the late 1980’s and early 1990’s Hans Lehrach’s group automated these processes by using robotic systems to rapidly array clones from microtiter plates onto filters.

Grunstein M, Hogness DS. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proceedings of the National Academy of Sciences of the United States of America. 1975; 72:3961–3965. [PubMed: 1105573]

HISTORY

• In the late 90’s and 2000’s, DNA array technology progressed rapidly as both new methods of production and fluorescent detection were adapted.

• Manufacture of slides or chips containing thousands of DNA probes arrayed within a small surface area by Dr. Patrick Brown and colleagues at Stanford University

HISTORY

1991 - Photolithographic printing (Affymetrix)1994 - First cDNA collections are developed at Stanford.1996 - Commercialization of arrays (Affymetrix) 1997- Genome-wide expression monitoring in S. cerevisiae (yeast) 2003 - Introduction to clinical practice2004-Whole human genome on one microarray

Singh, A., & Kumar, N. (2013). A review on DNA microarray Technology. International Journal of Current Research and Review, 5(22), 01-05.

HISTORY

TYPES OF ARRAYSThree basic types of arrays came into play during this time frame,

1. spotted arrays on glass,

2. in-situ synthesized arrays and

3. self assembled arrays

SPOTTED ARRAYS ON GLASS With spotted arrays, a “pen” (or multiple pens)

are dipped into solutions containing the DNA of interest and physically deposited on a glass slide.

Typically the glass slide surface is coated with something to help retain the DNA such as polylysine {1}, a silane {2} or a chemically reactive surface {3} (to which chemically reactive oligos or PCR products would be added). (A) Spotted arrays on glass

{1.DeRisi, 1997 ; 2. Call, 2001; 3. Rogers, 1999;}

Self assembled arrays• Self assembled arrays can be

created by applying a collection of beads containing a diverse set of oligos to a surface with pits the size of the beads.

• After the array is constructed, a series of hybridizations determine which oligo is in what position on each unique array

(Ferguson et al., 2000; Michael et al., 1998; Steemers et al., 2000; Walt, 2000) (Gunderson et al., 2004).

Self assembled arrays

In-situ synthesized arrays• C1. In-situ synthesized

arrays can be produced by inkjet oligo synthesis methods

OR

• C2. By photolithographic methods such as used by Affymetrix

Fodor et al., 1991

Strategies for Attaching Oligonucleotides to Solid SupportsTo obtain accurate, precise, and reliable results, considerable effort in choosing and designing synthetic oligonucleotides is essential.

Basically two types of solid surfaces are used to immobilized modified oligo's• Two dimensional solid surfaces (microarray slides)• Three-dimensional Surfaces (Micro-spheres)

Two-dimensional Surfaces (Microarray slides)

• Amino and thiol modifications have been

routinely used to construct oligonucleotide

arrays.

1. Substrates for arrays are usually silicon chips or glass

microscope slides.

2. The glass surface is chemically modified to facilitate

attachment of the oligo.

Note: Silianized oligonucleotides can also be covalently linked to an unmodified

glass surface

Different modifications allow immobilization onto different surfaces

Preparation of Modified Two-dimensional SurfaceThe two-dimensional surface is typically prepared by treating the glass or silicon surface with an amino silane which results in a uniform layer of primary amines or epoxides (Figure 1)

• The Oligonucleotides modified with an NH2 group can be immobilized onto epoxy silane-derivatized or isothiocyanate coated glass slides .

Examples : Binding of modified surface + modified oligo

Coupling of 5' amino-modified oligos reaction to the phenylisothiocyanate groups follows, resulting in the covalent attachment of the oligonucleotide to modified surface.

Guo Z, Guilfoyle RA, et al. (1994) Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res, 22(24): 54565465.

Three-dimensional Surfaces (Micro-spheres)1. Micro-spheres are ideal substrates

2. In these micro-sphere-based assays, each oligonucleotide is

attached to a micro-sphere.

3. They are amenable to multiple assay formats, multiplexing,

rapid assay development and have distinct cost advantages

over two-dimensional glass or silicon chip arrays for

characterizing populations of nucleic acids.

Several different types of micro-spheres are available:Polystyrene micro-spheres: fluorescently labelled through internal dye entrapment or surface attachment.

• To entrap the fluorescent dye, the polymeric micro-spheres are swelled in an organic solvent or dye solution. The water-insoluble dye diffuses into the polymer matrix and is trapped when the solvent is removed from the micro-spheres.

Magnetic micro-spheres 1.These have been used for many years in

radioimmunoassays, ELISAs, and cell separation assays.

2.Magnetic micro-spheres are synthesized by dispersing ferrite crystals in a suspension of styrene / divinylbenzene monomers and polymerizing this cocktail into micro-spheres.

3.A popular use for this type of micro-sphere has been to capture mRNA from a cell lysate using beads coupled to oligo.

Attachment• Nucleic acids can be

covalently attached to micro-spheres with any of several methods.

• Listed here are several reactive groups that can be incorporated on the micro-sphere surface for covalent coupling.

Amine-modified oligo's can be reacted with carboxylate-modified micro-spheres with carbodiimide chemistry in a one-step process at pH 6–8 (Figure).

Example

1-ethyl-3-(3-dimethyl aminoproply)carbodiimide hydrochloride (EDAC)

Using this reagent may yield a matrix of crosslinked oligos and micro-spheres.

Applications of Microarray Technology

MICROARRAY AS A GENE

EXPRESSION PROFILING TOOL

MICROARRAY AS A COMPARATIVE

GENOMICS TOOL

DISEASE DIAGNOSIS

DRUG DISCOVERY

TOXICOLOGICAL RESEARCH

Advantages of Microarrays• Small volume deposition (nL)• Minimal wasted reagents• Access many genes / proteins

simultaneously• Can be automated

Limitations of Microarrays• Relatively new technology (10

years old)

• Still has technical problems (background)

• Poor reproducibility between investigators

• Still mostly manual procedure

• Relatively expensive

ConclusionDNA Microarray analysis has provided scientists with a tool to

screen thousands of DNA and protein samples. After the

completion of Human Genome Project, DNA Microarrays have

been used for clinical diagnosis of gene expression patterns.

DNA Microarray technology has proved boon to industrial fields

serving in clinical diagnosis.

The Future of DNA arraysThe advent of next generation sequencing technologies combined with the rapid decrease in the cost of sequencing has now made sequencing cost competitive with microarrays for all assays with the possible exception of genotyping. • Sequencing is a relatively unbiased approach. • Not dependent on prior knowledge of which nucleic acids • Independently detect closely related gene sequences,

novel splice forms or RNA editing, missed due to cross hybridization on DNA microarrays. Wold B, Myers RM. Sequence census methods for functional genomics. Nature methods. 2008; 5:19– 21. [PubMed: 18165803]

• As a result of these advantages and the decreasing cost of sequencing, DNA arrays are being rapidly replaced by sequencing for nearly every assay.

• As the cost of sequencing is currently dropping by a factor of two every five months, it’s likely that DNA arrays will be fully replaced by sequencing methods within the next 5-10 years.

The Future of DNA arrays

References 1. Bumgarner, R. (2013). Overview of DNA microarrays: types, applications, and their

future. Current Protocols in Molecular Biology, 22-1.2. Guo Z, Guilfoyle RA, et al. (1994) Direct fluorescence analysis of genetic polymorphisms by

hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res, 22(24): 5456-5465.

3. Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable4. parallel chemical synthesis. Science. 1991; 251:767–773. [PubMed: 1990438].5. Ferguson JA, Steemers FJ, Walt DR. High-density fiber-optic DNA random microsphere array.6. Analytical chemistry. 2000; 72:5618–5624. [PubMed: 11101240].7. Grunstein M, Hogness DS. Colony hybridization: a method for the isolation of cloned DNAs

that contain a specific gene. Proceedings of the National Academy of Sciences of the United States of America. 1975; 72:3961–3965. [PubMed: 1105573].

8. Singh, A., & Kumar, N. (2013). A review on DNA microarray Technology. International Journal of Current Research and Review, 5(22), 01-05.

9. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM. Use of a10.cDNA microarray to analyse gene expression patterns in human cancer. Nature genetics.

1996;11.14:457–460. [PubMed: 8944026].