INTRODUCTION

A DNA microarray is a multiplex technology used in molecular biology and in

medicine. It consists of an arrayed series of thousands of microscopic spots of DNA
oligonucleotides, called features, each containing picomoles of a specific DNA sequence.
This can be a short section of a gene or other DNA element that are used as probes to
hybridize a cDNA or cRNA sample called target! under high"stringency conditions.
#robe"target hybridization is usually detected and quantified by fluorescence"based
detection of fluorophore"labeled targets to determine relati$e abundance of nucleic acid
sequences in the target.
In standard microarrays, the probes are attached to a solid surface by a co$alent bond to a
chemical matrix $ia epoxy"silane, amino"silane, lysine, polyacrylamide or others!. The
solid surface can be glass or a silicon chip, in %hich case they are commonly &no%n as
gene chip or colloquially Affy chip %hen an Affymetrix chip is used. 'ther microarray
platforms, such as Illumina, use microscopic beads, instead of the large solid support.
DNA arrays are different from other types of microarray only in that they either measure
DNA or use DNA as part of its detection system.
(icroarray technology e$ol$ed from )outhern blotting, %here fragmented DNA is
attached to a substrate and then probed %ith a &no%n gene or fragment. The use of a
collection of distinct DNAs in arrays for expression profiling %as first described in *+,-,
and the arrayed DNAs %ere used to identify genes %hose expression is modulated by
interferon. These early gene arrays %ere made by spotting cDNAs onto filter paper %ith a
pin"spotting de$ice. The use of miniaturized microarrays for gene expression profiling
%as first reported in *++.,

and a complete eu&aryotic genome Saccharomyce cerevisiae!
on a microarray %as published in *++-.
#RIN/I#01
The microarray technology consists of spotting #/R products or long
oligonucleotides .2mer"-2mer! on glass slides at densities of up to 3222 spots 4
cm5. These slides are hybridised using fluorescent targets cDNAs or genomic
DNAs!. The fluorescent molecules most commonly used are members of the
cyanine family, /y6 et /y.. After hybridisation, the signals are detected using a
fluorescence scanner. The use of t%o different fluorochromes allo%s the
determination of hybridisation signals from t%o distinct strains in one single
experiment.
'ne the fluorescent intensities ha$e been obtained, the ma7or part of the %or& is
the analysis of the data in order to extract the biological information.
This analysis can be di$ided into fi$e steps 8
Target preparation
Hybridization
Slide scanning
Data analysis
Expression profile clustering
MATERIALS
DNA sources
About .522 human cDNA clones of the I(A91 library %ere obtained

from the R:#D
Resource /entre ;erlin, 9ermany!. )ome 5* 222

random shotgun clones representing
the genome of Trypanosoma

brucei %ere pro$ided by Na7ib 1l")ayed of the Institute for
9enomic Research TI9R, Roc&$ille, <)A!. Nearly =..2 shotgun

clones co$ering the
entire genome of Pseudomonas putida as a

minimal tiling path %ere obtained from
>elmut >ilbert of ?iagen

>ilden, 9ermany!. #/R products for some 5* 222 predicted
open

reading frames 'R@s! of Drosophila melanogaster %ere produced

directly from
genomic DNA. The template for some -622 'R@"specific

#/R products of Candida
albicans %as strain )/.6*= /an*=!.

PCR amplification
#/R amplifications %ere performed in 6,=" or +3"%ell microtitre

plates. @or #/R on the
cDNA and shotgun clones, 2.5 A(

of the respecti$e, $ector"specific primer pairs dT/A
/A/A99AAA/A9/TAT9A/!

and d9TAAAA/9A/99//A9T9! human clones!,
dTT9TAAAA/9A/99//A9T9!

and d9/99ATAA/AATTT/A/A/A99A!
T.brucei! or dT/99AT//A/TA9TAA/9!

and d99//9//A9T9T9AT9!
P.putida! all from Interacti$a, <lm,

9ermany! %ere used. The reactions %ere started by
inoculating

5. or *22 Al of #/R mix, usually in *2 m( TrisB>/l,

p> ,.6, 5.5. m(
(g/l
5
, .2 m( C/l, 2.5 m( each dAT#, dTT#, d9T#

and d/T#, *.. ( betaine, 2.* m(
cresol red and 5 < Taq polymerase,

%ith a fe% Escherichia coli cells transferred from a
gro%th

culture using a plastic 6,=" or +3"pin gadget 9enetix, Ne%

(ilton, <C!. The
plates %ere incubated for 6 min at +=D/,

before 6. cycles of denaturation at +=D/ for 62
s, annealing

at .*D/ for 62 s and elongation at -5D/ for +2 s %ere

performed, follo%ed by
a final elongation phase at -5D/

for *2 min. In some cases, the #/R %as performed
%ithout betaine.

The Drosophila 'R@s %ere initially amplified on *22 ng genomic

DNA
%ith some =6 222 gene"specific primers, all of %hich contained

one of se$eral common
tag sequences of *. nt length at their

.E"ends. )ubsequent re"amplification %as carried out
using the

fitting primer pair. #/R products of C.albicans 'R@s %ere produced

on 52 ng
genomic DNA %ith -622 specific primer pairs.

Microarray production process
DNA fragments amplified by #/R technique are spotted on a microscopic glass slide
coated %ith polylysine prior to spotting process. The polylysine coating goal is to ensure
DNA fixation through electrostatic interactions. #/R fragments are in our case the
expressed part 'R@! of the 3522 Saccharomyces cerevisae genes ba&er yeast!. )lide
preparation is achie$ed by bloc&ing the polylysine not fixed to DNA in order to a$oid
target binding. #rior to hybridisation, DNA is denatured to obtained a single strand DNA
on the microarray, this %ill allo% the probe to bind to the complementary strand from the
target.
Tar!et preparation
RNA are extracted from t%o yeast cultures from %hich %e %ant to compare
expression le$el. (essengers RNA are then transformed in cDNA by re$erse
transcription. 'n this stage, DNA from the first culture %ith a green dye, %hereas DNA
from the second culture is labelled %ith a red dye.
The a$ailable target"preparation methods can be di$ided into t%o groups8 first"strand
cDNA that is labeled or tagged %ith a capture sequence, or the generation of antisense
RNA aRNA! from double"stranded cDNA during an in $itro transcription IFT!
reaction. 0abeled cDNA can be prepared $ia direct The incorporation of a fluorophore"
labeled nucleotide or through incorporation of an aminoallyl"labeled nucleotide, follo%ed
by coupling to a fluorophore containing an amine"reacti$e group to the aminoallyl
nucleotide )chena et al. *++.G for re$ie%, see 0oc&hart and Hinzeler 5222!.
Alternati$ely, the first"strand cDNA can be tagged %ith a capture sequence that is used
for subsequent detection steps )tears et al. 5222!. DNA microarrays containing short
oligonucleotide probes I6. nucleotides long! require more target for each hybridization,
%hich requires an amplification method %ith smaller sample sizes. Typically, the
generation of aRNA aRNA is also commonly called complementary RNA or cRNA! is
preceded by first"strand synthesis of cDNA using an oligonucleotide primer containing a
bacteriophage T- RNA polymerase promoter proximal to an oligodT! sequence $an
9elder et al. *++2G1ber%ine et al. *++5G 0oc&hart et al. *++3!. After second"strand cDNA
synthesis and cDNA purification, an IFT reaction is performed using T- RNA
polymerase in the presence of labeled nucleotides. Alternati$es to this labeling strategy
produce unlabeled aRNA, follo%ed by a cDNA synthesis in the presence of a
fluorophore"labeled nucleotide Hang et al. 5222!. Any target preparation method
requires a linear amplification of the a$ailable transcripts to be representati$e of the
transcript population.
"y#ridisation
9reen labelled cDNA and red labelled ones are mixed together call the target! and
put on the matrix of spotted single strand DNA call the probe!. The chip is then
incubated one night at 32 degrees. At this temperature, a DNA strand that encounter the
complementary strand and match together to create a double strand DNA. The fluorescent
DNA %ill then hybridise on the spotted ones.
The discrepancies in microarray results are a consequence of

differences in microarray
measures, such as accuracy Ji.e. Kthe

degree of conformity of the measured quantity to its
actual

true! $alueLG sensiti$ity Ji.e. Kthe concentration

range of target molecules in %hich
accurate measurements can

be madeLG reproducibility Ji.e. Kthe degree

to %hich repeated
measurements of the same quantity %ill sho%

the same or similar resultsLG and specificity
Ji.e.

Kthe ability of a probe to pro$ide a signal that is influenced

only by the presence of
the target moleculeL.

Accuracy, sensiti$ity and reproducibility may be affected by

se$eral effectors. These
measures and their effectors are discussed

by Duf$a and Draghici et al. , and %ill not be
detailed

here. An example for an effector of sensiti$ity, reproducibility

and accuracy is the
type of microarray platform8 oligonucleotide

arrays ha$e been found to be more
reproducible and sensiti$e

than cDNA arrays , and some oligonucleotide arrays ha$e been
found to be more accurate than others. )ensiti$ity is also

affected by probe density i.e.
the number of different probes

that are fabricated in a gi$en area!, %hich has been sho%n
to

be an effector for the a$ailability of probes for hybridizationG this a$ailability may also
be affected by the steric

restrictions imposed by the solid microarray surface. A

higher
a$ailability of probes for hybridization has been demonstrated

to increase sensiti$ity. In
addition, sensiti$ity is affected

by the hybridization signal"to"noise ratio i.e. the ratio
bet%een

the spot signal and that of the bac&ground!8 a lo% bac&ground

increases
microarray hybridization sensiti$ity
0o% specificity of microarray hybridizations has been suggested

to be one of the prime
measures affecting discrepancies in gene"expression

profiles bet%een different probes
targeting the same region

of a gi$en transcript or bet%een different microarray

platformsG
in the present re$ie%, %e %ill highlight

the issue of microarray " hybridization specificity
as a &ey measure

that once impro$ed, may increase the $alidity of microarray

results.

(icroarrays consist of multiple probes. >ence, a prime &ey for

specificity during
microarray hybridiation, for either short"oligomer

or cDNA microarraysG is the ability of
the probe to

discriminate bet%een different target molecules.

#robes are designed to be complementary to the target molecule

according to the HatsonB
/ric& rules of binding. Therefore,

a probe %ith high specificity to its target molecule
should

pro$ide a signal influenced only by the presence of the target

molecule.
Ne$ertheless, a perfect match in terms of sequence"similarity"based

complementarity
bet%een a probe and its target molecule does

not guarantee specificity. This is due to the
presence of thousands

of target molecules during microarray hybridizationMeach

target
molecule being composed of tens of hundreds or thousands

of four"nucleotide bases, and
to the effect of different effectors

discussed subsequently! of hybridization specificity,
%hich

may alter the ability of a probe to bind to a target molecule.

>ence, there is often
some degree of microarray"probe hybridization

to a target molecule %hich is not strictly
complementary to

it or vice versa, a $ariable number of target molecules that

are
hybridized to a microarray probe %hich is not exactly complementary

to them.

$OUR LE%ELS O$ "&'RIDI(ATION SPECI$ICIT&
He define four le$els of hybridization specificity in the context

of microarray
hybridization. The first is of hybridization bet%een

a single probe molecule and a single
target molecule.

The t%o molecules may exhibit perfect hybridization,

partial
hybridization i.e. the target molecule is only partially

hybridized to the probeG or no
hybridization.
The second le$el of specificity is of a spot. At

this le$el, multiple probe molecules that
compose one spot are

hybridized to multiple target molecules. The spot probes may
exhibit perfect, partial or no hybridization %ith the target

molecules.

Notably, at this le$el,
partial hybridization may ha$e one or

both of t%o forms8 only some of the probes may be
hybridized

to the target molecule, or probes may be hybridized to only

some of the target
molecules. This partial hybridization, at

the spot le$el, may be a result of cross"
hybridization i.e. hybridization

bet%een sequences that are not strictly complementary,
due to the presence and hybridization of nontarget

molecules %ith sequences similar to
that of the spot probes.

)ince a spot is composed of multiple probes, a single spot may
simultaneously bear all combinations of one to four of the presented

probe"target
molecule types of binding.

The third le$el of specificity is of a spot"set Jor, in Affymetrix

terminology, Kprobe"setL, in
%hich multiple spots represent different segments of the same

reference sequence e.g.
different exons of a gene!. At this

le$el, different spots of a spot"set may exhibit perfect
hybridization

%ith the target moleculeG partial hybridization

%ith the target molecule due
to the presence of

probes %ith mismatches to the target molecule as a result of,

for
example, an annotation error in the gene sequence, or intended

mismatches introduced to
quantify nonspecific hybridizationG

no hybridization due to, for example, alternati$e
splicing of a transcript, leading to probes %ith no match to

the target moleculeG cross
hybridization due to,

for example, a spot, %ithin a spot"set that represents an
e$olutionarily

conser$ed gene segment, %hich hybridizes %ith nontarget molecules
deri$ed from $arious gene"family members.

The fourth le$el of specificity is that of the microarray, in

%hich a $ariable number of
spot"sets may exhibit different forms

of hybridization %ith target sequences perfect
hybridization i.e. all target molecules are hybridized to their

representati$e spot"sets and
all spot"sets are hybridized to

the target molecules they represent!, partial hybridization

in
either direction, no hybridization i.e. target molecules

are not hybridized to any spot"set
or spot"sets do not match

any target molecules! or cross" hybridization e.g. target
molecules

of different genes hybridize to the same spot"set or target

molecules of a
particular gene hybridize to se$eral different

genesL spot"sets!. These different forms may
exist for

a large number of different target molecules or spot"sets.
Slide scannin!
A laser excites each spot and the fluorescent emission gather through a photo"
multiplicator #(T! coupled to a confocal microscope. He obtained t%o images %here
grey scales represent fluorescent intensities read. If %e replace grey scales by green
scales for the first image and red scales for the second one, %e obtained by
superimposing the t%o images one image composed of spots going from green ones
%here only DNA from the first condition is fixed! to red %here only DNA from the
second condition is fixed! passing through the yello% colour %here DNA from the t%o
conditions are fixed on equal amount!.
Data analysis
He ha$e no% t%o microarray images from %hich %e ha$e to calculate the number of
DNA molecules in each experimental condition. To dos o, %e measure the signal amount
in the green dye emission %a$elength and the signal amount in the red dye emission
%a$elength. Then %e normalise these amount according to $arious parameters yeast
amount in each culture condition, emission po%er of each dye, N!. He suppose that the
amount of fluorescent DNA fixed is proportional to the mRNA amount present in each
cell at the beginning and %e calculate the red4green fluorescence ratio. If this ratio is
greater than * red on the image!, the gene expression is greater in the second
experimental condition, if this ration is smaller than * green on the image!, the gene
expression is greater in the first condition. He can $isualize these differences in
expression using soft%are as the one de$eloped in the laboratory call Array#lot cf belo%
image!. This soft%are allo%s from the intensities list of spot to display the red intensities
of each spot as a function of the green intensities.
$a#rication
(icroarrays can be manufactured in different %ays, depending on the number of probes
under examination, costs, customization requirements, and the type of scientific question
being as&ed. Arrays may ha$e as fe% as *2 probes to up to 5.* million micrometre"scale
probes from commercial $endors.
Surface en!ineerin!
The first step of DNA microarray fabrication in$ol$es surface engineering of a substrate
in order to obtain desirable surface properties for the application of interest. 'ptimal
surface properties are those %hich produce high signal to noise ratios for the DNA targets
of interest. 9enerally, this in$ol$es maximizing the probe surface density and acti$ity
%hile minimizing the non"specific binding of the targets of interest. (ethods of surface
engineering $ary depending on the platform material, design, and application.
Spotted )s* oli!onucleotide arrays
(icroarrays can be fabricated using a $ariety of technologies, including printing %ith
fine"pointed pins onto glass slides, photolithography using pre"made mas&s,
photolithography using dynamic micromirror de$ices, in&"7et printing,

or
electrochemistry on microelectrode arrays.
In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of
#/R products that correspond to mRNAs. The probes are synthesized prior to deposition
on the array surface and are then OspottedO onto glass. A common approach utilizes an
array of fine pins or needles controlled by a robotic arm that is dipped into %ells
containing DNA probes and then depositing each probe at designated locations on the
array surface. The resulting OgridO of probes represents the nucleic acid profiles of the
prepared probes and is ready to recei$e complementary cDNA or cRNA OtargetsO deri$ed
from experimental or clinical samples. This technique is used by research scientists
around the %orld to produce Oin"houseO printed microarrays from their o%n labs. These
arrays may be easily customized for each experiment, because researchers can choose the
probes and printing locations on the arrays, synthesize the probes in their o%n lab or
collaborating facility!, and spot the arrays. They can then generate their o%n labeled
samples for hybridization, hybridize the samples to the array, and finally scan the arrays
%ith their o%n equipment. This pro$ides a relati$ely lo%"cost microarray that may be
customized for each study, and a$oids the costs of purchasing often more expensi$e
commercial arrays that may represent $ast numbers of genes that are not of interest to the
in$estigator. #ublications exist %hich indicate in"house spotted microarrays may not
pro$ide the same le$el of sensiti$ity compared to commercial oligonucleotide arrays,
possibly o%ing to the small batch sizes and reduced printing efficiencies %hen compared
to industrial manufactures of oligo arrays.
In oligonucleotide microarrays, the probes are short sequences designed to match parts of
the sequence of &no%n or predicted open reading frames. Although oligonucleotide
probes are often used in OspottedO microarrays, the term Ooligonucleotide arrayO most
often refers to a specific technique of manufacturing. 'ligonucleotide arrays are
produced by printing short oligonucleotide sequences designed to represent a single gene
or family of gene splice"$ariants by synthesizing this sequence directly onto the array
surface instead of depositing intact sequences. )equences may be longer 32"mer probes
such as the Agilent design! or shorter 5."mer probes produced by Affymetrix! depending
on the desired purposeG longer probes are more specific to indi$idual target genes, shorter
probes may be spotted in higher density across the array and are cheaper to manufacture.
'ne technique used to produce oligonucleotide arrays include photolithographic
synthesis Agilent and Affymetrix! on a silica substrate %here light and light"sensiti$e
mas&ing agents are used to ObuildO a sequence one nucleotide at a time across the entire
array. 1ach applicable probe is selecti$ely Ounmas&edO prior to bathing the array in a
solution of a single nucleotide, then a mas&ing reaction ta&es place and the next set of
probes are unmas&ed in preparation for a different nucleotide exposure. After many
repetitions, the sequences of e$ery probe become fully constructed. (ore recently,
(as&less Array )ynthesis from Nimble9en )ystems has combined flexibility %ith large
numbers of probes.
T+o,c-annel )s* one,c-annel detection
Diagram of typical dual"colour microarray experiment.
Twocolor microarrays or twochannel microarrays are typically hybridized %ith cDNA
prepared from t%o samples to be compared e.g. diseased tissue $ersus healthy tissue!
and that are labeled %ith t%o different fluorophores. @luorescent dyes commonly used for
cDNA labelling include /y6, %hich has a fluorescence emission %a$elength of .-2 nm
corresponding to the green part of the light spectrum!, and /y. %ith a fluorescence
emission %a$elength of 3-2 nm corresponding to the red part of the light spectrum!. The
t%o /y"labelled cDNA samples are mixed and hybridized to a single microarray that is
then scanned in a microarray scanner to $isualize fluorescence of the t%o fluorophores
after excitation %ith a laser beam of a defined %a$elength. Relati$e intensities of each
fluorophore may then be used in ratio"based analysis to identify up"regulated and do%n"
regulated genes.
'ligonucleotide microarrays often contain control probes designed to hybridize %ith
RNA spi&e"ins. The degree of hybridization bet%een the spi&e"ins and the control probes
is used to normalize the hybridization measurements for the target probes. Although
absolute le$els of gene expression may be determined in the t%o"color array, the relati$e
differences in expression among different spots %ithin a sample and bet%een samples is
the preferred method of data analysis for the t%o"color system. 1xamples of pro$iders for
such microarrays includes Agilent %ith their Dual"(ode platform, 1ppendorf %ith their
Dual/hip platform for fluorescence labeling, and Tele/hem International %ith Arrayit.
In singlechannel microarrays or onecolor microarrays, the arrays are designed to gi$e
estimations of the absolute le$els of gene expression. Therefore the comparison of t%o
conditions requires t%o separate single"dye hybridizations. As only a single dye is used,
the data collected represent absolute $alues of gene expression. These may be compared
to other genes %ithin a sample or to reference OnormalizingO probes used to calibrate data
across the entire array and across multiple arrays. Three popular single"channel systems
are the Affymetrix O9ene /hipO, the Applied (icroarrays O/ode0in&O arrays, and the
1ppendorf ODual/hip P )il$erquantO. 'ne strength of the single"dye system lies in the
fact that an aberrant sample cannot affect the ra% data deri$ed from other samples,
because each array chip is exposed to only one sample as opposed to a t%o"color system
in %hich a single lo%"quality sample may drastically impinge on o$erall data precision
e$en if the other sample %as of high quality!. Another benefit is that data are more easily
compared to arrays from different experimentsG the absolute $alues of gene expression
may be compared bet%een studies conducted months or years apart. A dra%bac& to the
one"color system is that, %hen compared to the t%o"color system, t%ice as many
microarrays are needed to compare samples %ithin an experiment.
E.pression profile clusterin!
Then %e can try to gather genes that share the same expression profile on se$eral
experiments. This clustering can be done gradually as for phylogenetic analysis, %hich
consist in calculating similarity criteria bet%een expression profiles and gather the most
similar ones. He can also use more complex techniques as principal component analysis
or neuronal net%or&s.
At the end hierarchical clustering is usually displayed as a matrix %here each column
represent one experiment and each ro% a gene. Ratios are displayed than&s to a colour
scale going from green repressed genes! to red induced genes!.
Uses and types
Arrays of DNA can be spatially arranged, as in the commonly &no%n gene chip also
called genome chip, D!A chip or gene array!, or can be specific DNA sequences labelled
such that they can be independently identified in solution. The traditional solid"phase
array is a collection of microscopic DNA spots attached to a solid surface, such as glass,
plastic or silicon biochip. The affixed DNA segments are &no%n as probes although
some sources use different terms such as reporters!. Thousands of them can be placed in
&no%n locations on a single DNA microarray.
DNA microarrays can be used to detect DNA as in comparati$e genomic hybridization!,
or detect RNA most commonly as cDNA after re$erse transcription!that may or may not
be translated into proteins. The process of measuring gene expression $ia cDNA is called
expression analysis or expression profiling.
)ince an array can contain tens of thousands of probes, a microarray experiment can
accomplish that many genetic tests in parallel. Therefore arrays ha$e dramatically
accelerated many types of in$estigation.
Applications include
Tec-nolo!y or
Application
Synopsis
9ene expression
profiling
In an mRNAor gene expression profiling experiment the
expression le$els of thousands of genes are simultaneously
monitored to study the effects of certain treatments, diseases, and
de$elopmental stages on gene expression. @or example,
microarray"based gene expression profiling can be used to
identify genes %hose expression is changed in response to
pathogens or other organisms by comparing gene expression in
infected to that in uninfected cells or tissues.
/omparati$e genomic
hybridization
Assessing genome content in different cells or closely related
organisms.
/hromatin
immunoprecipitation
on /hip
DNA sequences bound to a particular protein can be isolated by
immunoprecipitating that protein />I#!, these fragments can be
then hybridized to a microarray such as a tiling array! allo%ing
the determination of protein binding site occupancy throughout
the genome. 1xample protein to immunoprecipitate are histone
modifications >6C5-me6, >6C=me5, >6C+me6, etc!,
#olycomb"group protein #R/58)uz*5, #R/*8QQ*! and
trithorax"group protein Ash*! to study the epigenetic landscape
or RNA #olymerase II to study the transcription lanscape.
)N# detection
Identifying single nucleotide polymorphism among alleles %ithin
or bet%een populations. )e$eral applications of microarrays
ma&e use of )N# detection, including 9enotyping, forensic
analysis, measuring predisposition to disease, identifying drug"
candidates, e$aluating germline mutations in indi$iduals or
somatic mutations in cancers, assessing loss of heterozygosity, or
genetic lin&age analysis.
Alternati$e splicing
detection
An Ee"on #unction array design uses probes specific to the
expected or potential splice sites of predicted exons for a gene. It
is of intermediate density, or co$erage, to a typical gene
expression array %ith *"6 probes per gene! and a genomic tiling
array %ith hundreds or thousands of probes per gene!. It is used
to assay the expression of alternati$e splice forms of a gene.
1xon arrays ha$e a different design, employing probes designed
to detect each indi$idual exon for &no%n or predicted genes, and
can be used for detecting different splicing isoforms.
Tiling array
9enome tiling arrays consist of o$erlapping probes designed to
densely represent a genomic region of interest, sometimes as
large as an entire human chromosome. The purpose is to
empirically detect expression of transcripts or alternati$ely splice
forms %hich may not ha$e been pre$iously &no%n or predicted.