DNA MICROARRAY

A DNA microarray (also commonly known as DNA

chip or biochip) is a collection of microscopic DNA
spots attached to a solid surface. Scientists use DNA
microarrays to measure the expression levels of
large numbers of genes simultaneously or
to genotype multiple regions of a genome. Each DNA
spot contains picomoles (10−12 moles) of a specific
DNA sequence, known as probes (or reporters or
oligos). These can be a short section of a gene or
other DNA element that are used
to hybridize a cDNA or cRNA (also called anti-sense
RNA) sample (called target) under high-stringency
conditions. Probe-target hybridization is usually
detected and quantified by detection of fluorophore-,
silver-, orchemiluminescence-labeled targets to
determine relative abundance of nucleic acid
sequences in the target.

THE BASIC MICRO ARRAY

Since an array can contain tens of thousands of

probes, a microarray experiment can accomplish
many genetic tests in parallel. Therefore arrays have
dramatically accelerated many types of
investigation. In standard microarrays, the probes
are synthesized and then attached via surface
engineering to a solid surface by a covalent bond to
a chemical matrix (via epoxy-silane, amino-
silane, lysine, polyacrylamide or others). The solid
surface can be glass or a silicon chip, in which case
they are colloquially known as an Affy chipwhen
an Affymetrix chip is used. Other microarray
platforms, such as Illumina, use microscopic beads,
instead of the large solid support. Alternatively,
microarrays can be constructed by the
direct synthesis of oligonucleotide probes on solid
surfaces. DNA arrays are different from other types
of microarray only in that they either measure DNA
or use DNA as part of its detection system.

DNA microarrays can be used to measure changes

in expression levels, to detect single nucleotide
polymorphisms (SNPs), or to genotype or targeted
resequencing (see uses and types section).
Microarrays also differ in fabrication, workings,
accuracy, efficiency, and cost
(see fabrication section). Additional factors for
microarray experiments are the experimental design
and the methods of analyzing the data
(see Bioinformatics section).

History

Microarray technology evolved from Southern

blotting, where fragmented DNA is attached to
a substrate and then probed with a known DNA
sequence.[1] The first reported use of this approach
was the analysis of 378 arrayed lysed bacterial
colonies each harboring a different sequence which
were assayed in multiple replicas for expression of
the genes in multiple normal and tumor tissue. [2] This
was expanded to an analysis of more than 4000
human sequences with computer driven scanning
and image processing for quantitative analysis of the
sequences in human colonic tumors and normal
tissue [3]
and then to comparison of colonic tissues at
different genetic risk.[4] The use of a collection of
distinct DNAs in arrays for expression profiling was
also described in 1987, and the arrayed DNAs were
used to identify genes whose expression is
modulated by interferon.[5] These early gene arrays
were made by spotting cDNAs onto filter paper with a
pin-spotting device. The use of miniaturized
microarrays for gene expression profiling was first
reported in 1995,[6]and a
complete eukaryotic genome (Saccharomyces
cerevisiae) on a microarray was published in 1997.[7]

Principle

Hybridization of the target to the probe

For more details on this topic, see DNA microarray
experiment.
The core principle behind microarrays is hybridization
between two DNA strands, the property
ofcomplementary nucleic acid sequences to
specifically pair with each other by forming hydrogen
bondsbetween complementary nucleotide base pairs.
A high number of complementary base pairs in a
nucleotide sequence means tighter non-
covalent bonding between the two strands. After
washing off of non-specific bonding sequences, only
strongly paired strands will remain hybridized.
Fluorescently labeled target sequences that bind to a
probe sequence generate a signal that depends on
the hybridization conditions (such as temperature),
and washing after hybridization. Total strength of the
signal, from a spot (feature), depends upon the
amount of target sample binding to the probes
present on that spot. Microarrays use relative
quantization in which the intensity of a feature is
compared to the intensity of the same feature under
a different condition, and the identity of the feature
is known by its position.
 The steps required in a microarray experiment.
Uses and types

Two Affymetrix chips. Amatch is shown at bottom left

for size comparison.
Many types of arrays exist and the broadest
distinction is whether they are spatially arranged on
a surface or on coded beads:
 The traditional solid-phase array is a collection of
orderly microscopic "spots", called features, each
with a thousands of identical and specific probes
attached to a solid surface, such
as glass, plastic or silicon biochip (commonly
known as a genome chip, DNA chip or gene array).
Thousands of these features can be placed in
known locations on a single DNA microarray.
 The alternative bead array is a collection of
microscopic polystyrene beads, each with a specific
probe and a ratio of two or more dyes, which do
not interfere with the fluorescent dyes used on the
target sequence.

DNA microarrays can be used to detect DNA (as

in comparative genomic hybridization), or detect RNA
(most commonly as cDNA after reverse transcription)
that may or may not be translated into proteins. The
process of measuring gene expression via cDNA is
called expression analysis orexpression profiling.
Micro arrays and bioinformatics

Gene expression values from microarray

experiments can be represented as heat maps to
visualize the result of data analysis.
The advent of inexpensive microarray experiments
created several specific bioinformatics challenges:

 the multiple levels of replication in experimental

design (Experimental design)
 the number of platforms and independent groups
and data format (Standardization)
 the treatment of the data (Statistical analysis)
 accuracy and precision (Relation between probe
and gene)
 the sheer volume of data and the ability to share
it (Data warehousing)
Experimental design
Due to the biological complexity of gene expression,
the considerations of experimental design that are
discussed in the expression profiling article are of
critical importance if statistically and biologically
valid conclusions are to be drawn from the data.

There are three main elements to consider when

designing a microarray experiment. First, replication
of the biological samples is essential for drawing
conclusions from the experiment. Second, technical
replicates (two RNA samples obtained from each
experimental unit) help to ensure precision and allow
for testing differences within treatment groups. The
biological replicates include independent RNA
extractions and technical replicates may be
two aliquots of the same extraction. Third, spots of
each cDNA clone or oligonucleotide are present as
replicates (at least duplicates) on the microarray
slide, to provide a measure of technical precision in
each hybridization. It is critical that information
about the sample preparation and handling is
discussed, in order to help identify the independent
units in the experiment and to avoid inflated
estimates of statistical significance

Standardization
Microarray data is difficult to exchange due to the
lack of standardization in platform fabrication, assay
protocols, and analysis methods. This presents
an interoperability problem inbioinformatics.
Various grass-roots open-source projects are trying
to ease the exchange and analysis of data produced
with non-proprietary chips:

 For example, the "Minimum Information About a

Microarray Experiment" (MIAME) checklist helps
define the level of detail that should exist and is
being adopted by manyjournals as a requirement
for the submission of papers incorporating
microarray results. But MIAME does not describe
the format for the information, so while many
formats can support the MIAME requirements, as of
 2007 no format permits verification of complete
semantic compliance.
 The "MicroArray Quality Control (MAQC) Project"
is being conducted by the US Food and Drug
Administration (FDA) to develop standards and
quality control metrics which will eventually allow
the use of Micro Array data in drug discovery,
clinical practice and regulatory decision-making. [20]
 The MGED Society has developed standards for
the representation of gene expression experiment
results and relevant annotations.

Relation between probe and gene

The relation between a probe and the mRNA that it is
expected to detect is not trivial. Some mRNAs may
cross-hybridize probes in the array that are supposed
to detect another mRNA. In addition, mRNAs may
experience amplification bias that is sequence or
molecule-specific. Thirdly, probes that are designed
to detect the mRNA of a particular gene may be
relying on genomic EST information that is
incorrectly associated with that gene.