Micro arrays

Department of Biotechnology
SNIST

Definition

A microscopic spot containing identical

single-stranded polymeric molecules of
deoxyribonucleotides, usually
oligonucleotides or complementary
DNAs, attached to a solid support (such
as a membrane, a polymer, or glass)
used to simultaneously analyze the
expression levels of the corresponding
genes.

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Introduction

Microarray analysis has emerged in

the last few years as a flexible method
for analyzing large numbers of nucleic
acid fragments in parallel.

Its origins can be traced to several
different disciplines and techniques.

Introduction

Microarrays can be seen as a continued
development of molecular biology
hybridization methods,

As an extension of the use of fluorescence
microscopy in cell biology.

As well as a diagnostic assay using capture
to solid surface as a way to reduce the
amount of analytes needed.

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Introduction

The convergence of ideas and
principles utilized in these fields.

Together with technological
advancements in preparing
miniaturized collections of nucleic
acids on solid supports, have all
contributed to the emergence of
microarray and microchip
technologies.

Principles of microarray
analysis

Despite the variety of technical

solutions that have been developed
for performing microarray analysis.

All Microarrays are miniaturized
hybridization assays.

For studying thousands of nucleic acid
fragments simultaneously

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Key features of any Microarray

All microarray systems share the

following key components:

The array, which contains immobilized
nucleic acid sequences, or ‘targets’

one or more labelled samples or
‘probes’, that are hybridized with the
microarray

A detection system that quantitates
the hybridization signal.

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Commercial Microarray

Nomenclature for microarrays

•Targets are the immobilized nucleic acids on the slide

•surface.
• A probe consisting of two identical populations of nucleic acids
labelled with different fluorescent dyes

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 Fabrication of Microarrays

Making of an array

Introduction

Microarrays consist of a collection of

nucleic acid sequences immobilized
onto a solid support so that each
unique sequence forms a tiny feature,
called a ‘spot’ or ‘target’.

Historically, Microarrays have been
produced nylon membrane or glass
slides.

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 Genetic Content

The genetic content of microarrays resides in the

immobilized nucleic acid sequences on the
microarray.

The identity of these sequences determines what
information can be obtained from array
experiments.

Nucleic acids can be synthesized directly on the
microarray or they can be purified cDNA clones,

Other DNA fragments or oligonucleotides, which
are deposited onto the array by a printing
process.

Source of microarray target

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Desired properties of DNA targets

An optimal length for DNA targets is

between 300–800 nucleotides.

With increasing length, the concentration of
DNA required to guarantee the deposition of
a sufficiently high number of
targetmolecules within a spot increases.

DNA targets should not contain repetitive
sequences, and they should contain
sequences that are unique to one particular
gene.

Desired properties of DNA targets

DNA targets do not need to be single-

stranded. Spotting from denaturing
solutions is enough to render even
double-stranded targets available for
hybridization

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MANUFACTURING OF
MICROARRAY S L I D E S

Microarray analysis is invariably

performed on a glass slide.

Glass slide enables the performance
of high-throughput miniaturized
hybridization assays with fluorescently
labelled samples.

Microarray manufacture

Microarray manufacture requires three

distinct components:

Production method

Microarray slide

Target genetic content

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 Production methods- Oligo
synthesis

Two parallel approaches have been

developed for the production of
microarray slides.

Nucleic acid targets can either be
synthesized directly onto the
microarray slide.

Purified targets can be deposited onto
a solid surface that is capable of
binding nucleic acids.

Microarray Manufacturing using

Photolithography.

This method produces arrays of small

features that are anchored at their 3' ends to
the array surface.

Each feature is made up of oligonucleotides
that all have the same nucleotide sequence.

These arrays have a high density: an area of
1.6 cm can contain up to 400 000 features.

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How do we manufacture a
microarray?

Cheung et al. 1999

How do we manufacture a
microarray?

Cheung et al. 1999

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 Example

Yeast

Grow in aerobic and anaerobic
environment

Different genes will be activated in
order to adapt to each environment

Extract mRNA

Convert mRNA into colored cDNA

(fluorescently labeled)

Example (cont.)

Mix cDNA together

Hybridize cDNA with array

Each cDNA sequence hybridizes

specifically with the corresponding gene
sequence in the array

Wash unhybridized cDNA off

Read array with laser

Analyze images

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 Overview of Example

Brown & Botstein, 1999

Reading an array

Laser scans array and produces images

One laser for each color, e.g. one for green, one for
red

Image analysis, main tasks:
 Noise suppression
 Spot localization and detection, including the
extraction of the background intensity, the spot
position, and the spot boundary and size
 Data quantification and quality assessment

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Gene expression analysis
with microarrays

A typical microarray gene expression

analysis experiment compares the
relative expression levels of specific
transcripts in two samples.

One of these samples is a control and the
other is derived from cells whose
response or status is being investigated.

Gene expression analysis

with microarrays

Each of these samples is labelled with a

different fluorescent dye, and equal
amounts of the labelled samples are
combined and hybridized with the
microarray.

The fluorescent signals corresponding to
the two dyes are measured independently
from each spot after hybridization.

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 Gene expression analysis
with microarrays

After normalization, the intensity of the two

hybridization signals can be compared.

Microarray analysis does not give information
about absolute gene expression levels in the
samples.

This is because the intensity of the fluorescent
signals is not only proportional to the number of
hybridized fragments but also to the length of
these fragments.

The number of fluorescent labels each fragment
carries, i.e. labelling density.

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