Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
METHOD OF LABELING cRNAs FOR PROBING OLIGO.-BASED
MICROARRAYS
FIELD OF THE INVENTION
The invention relates in part to novel methods for producing cDNA and cRNA
from a
biological sample. Also included are methods for comparing the presence or
amount of
mRNA in a biological sample. The invention also includes random
oligonucleotide primers
which comprise an RNA polymerase promoter.
BACKGROUND OF THE INVENTION
Gene expression is important for understanding a wide range of biological
phenomena, including development, differentiation, senescence, oncogenesis,
and many other
medically important processes. Recently, changes in gene expression have also
been used to
assess the activity of new drug candidates and to identify new targets for
drug development.
The latter objective is accomplished by correlating the expression of a gene
or genes known
to be affected by a particular drug with the expression profile of other genes
of unknown
function when exposed to that same drug. Genes of unknown function that
exhibit the same
pattern of regulation, or signature, in response to the drug are likely to
represent novel targets
for pharmaceutical development.
Generally, the level of expression of the protein product of a gene and its
messenger
RNA (mRNA) transcript are correlated, so that measuring one provides reliable
information
about the other. Since in most instances it is technically easier to measure
levels of RNA than
protein, variations in mRNA levels are commonly employed to assess gene
expression in
different cells and tissues or in the same cells and tissues at different
stages of disease or
development or exposed to different stimuli. One particularly useful method of
assaying gene
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
expression at the level of transcription employs DNA microarrays (Ramsay,
Nature
Biotechnol. 16: 40-44, 1998; Marshall and Hodgson, Nature Biotechnol. 16: 27-
31, 1998;
Lashkari et al., Proc. Natl. Acad. Sci. (USA) 94: 130-157, 1997; DeRisi et
al., Science 278:
680-6, 1997).
Mammalian cells contain as many as 1 x105 to 3 x 105 different mRNA molecules,
each of which varies in abundance (or frequency) within a given cell. The most
abundant
mRNAs are typically present at many thousands of copies per cell, while others
may be
present in as few as one copy or less per cell. Techniques for analyzing gene
expression at the
level of transcription typically require tens to hundreds of micrograms of
mRNA, or as much
mRNA as might be found in 10' -10~ mammalian cells. Oftentimes, it is
impractical to obtain
this number of cells from a tissue of interest. For example, a blood sample
typically contains
106 nucleated cells/ml. Hence, to obtain 10~ cells for analysis would
necessitate taking a
1000 ml blood sample, which is clearly impractical in most instances.
Various methods have been described in the literature for amplifying the
amount of a
nucleic acid, such as DNA and RNA, present in a sample. Among these, the most
widely
practiced is the polymerase chain reaction (PCR), described in U.S. Pat. No.
4,683,195 and
U.S. Pat. No. 4,683,202, herein incorporated by reference. Briefly, PCR
consists of
amplifying denatured, complementary strands of target nucleic acid by
annealing each strand
to a short oligonucleotide primer, wherein the primers are chosen so as to
flank the sequence
of interest. The primers are then extended by a polymerise enzyme to yield
extension
products that are themselves complementary to the primers and hence serve as
templates for
synthesis of additional copies of the target sequence. Each successive cycle
of denaturation,
primer annealing, and primer extension essentially doubles the amount of
target synthesized
in the previous cycle, resulting in exponential accumulation of the target.
When PCR is used to amplify mRNA into double-stranded (ds) DNA, the
polyadenylated (poly(A)+) fraction is first selected, then a complementary DNA
(cDNA)
copy of the mRNA is made using reverse transcriptase and an oligo(dT) primer.
The products
of this reaction can then be amplified directly using random priming (Ausubel
et al., eds.,
1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols
Publishing, New
York).
cDNA is a deoxyribonucleic acid that contains the information coding for the
synthesis of proteins, but lacks the intervening introns present in genomic
DNA. The
synthesis and/or use of cDNA and/or libraries thereof plays a critical role in
a variety of
2
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
different applications in biotechnology and related fields. Applications in
which cDNAs
and/or libraries thereof are employed include gene discovery, differential
gene expression
analysis, and the like. A variety of protocols have been developed to prepare
cDNA and
libraries thereof, where such methods are continually being modified.
PCR methodologies in general suffer from several limitations that are well-
known in
the art. See U.S. Pat. No. 5,716,785 and Innis et al., eds. (1990, PCR
Protocols: A Guide to
Methods and Applications, Academic Press Inc., San Diego, Calif.) for review
and discussion
of limitations. One such limitation results from the poor fidelity of commonly
used,
thermostable polymerase enzymes, such as Taq. This results in nucleotide base
misincorporations that are propagated from one cycle to the next. It is
estimated that such
misincorporations may occur as often as once per one thousand bases of
incorporation. A
second limitation is that different cDNAs are amplified with different
efficiencies, resulting
in under representation of some cDNA sequences and overrepresentation of
others in the
amplified product. Even a small difference in efficiency may result in a
several-thousand fold
differential in the representation of these cDNAs in the product after only 30
cycles of
amplification.
An alternative method of mRNA synthesis and amplification is known as reverse
transcription and is described in U.S. Pat. No. 5,716,785, herein incorporated
by reference.
Unlike PCR, reverse transcription does not result in geometric amplification,
but rather in
linear amplification. In reverse transcription, an oligo(dT) primer that is
extended at the 3'-
end with a bacteriophage T7 RNA polymerase promoter is used to prime the poly-
A+ mRNA
population for cDNA synthesis. After synthesis of the first-strand cDNA, the
second-strand
cDNA is made using the method of Gubler and Hoffman (Gene 25:263-69, 1983).
Addition
of RNA polymerase results in reverse transcription and linear amplification of
mRNA that is
anti-sense to the poly-A+ RNA. This method has several limitations. It is not
as sensitive as
PCR and requires a much larger sample of mRNA to generate the same amount of
material
and thus displays low efficiency.
Moreover, conventional reverse transcription cannot produce full length cDNAs
from
mRNAs because the conventional reverse transcription method cannot complete
reverse
transcription to the end cap sites of mRNAs.
That is, current techniques for reverse transcription have a technical
limitation that the
reaction ends prematurely because of a stable secondary structure of mRNA and
thus the
probability of complete transcription over the whole transcription unit
including its 5' end is
3
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
extremely low. In many instances, the 5' information is of great interest.
This technical
limitation affects the quality of libraries. That is, most cloned cDNAs
synthesized from the
poly A at the 3' end using oligo (dT) as a primer have only the 3' end and do
not cover the full
mRNA length because of premature termination of synthesis. Several attempts
have been
made to overcome this problem. While some of these techniques are effective
for increasing
efficiency of the synthesis of the first chain to some extent, they are not
yet sufficient to
efficiently obtain full length cDNAs. In particular, they show particularly
low efficiency for
the reverse transcription of long mRNAs of several kbp or more.
Parallel quantification of large numbers of mRNA transcripts using microarray
technology promises to provide detailed insight into cellular processes
involved in the
regulation of gene expression. This allows new understanding of signaling
networks that
operate in the cell and of the molecular basis and classification of disease.
The ability of
DNA microarrays to monitor gene expression, simultaneously in a large-scale
format is
helping to usher in a post-genomic age, where simple constructs about the role
of nature
versus nurture are being replaced by a functional understanding of gene
expression in living
organisms. At present, high-density DNA microarrays allow researchers to
quickly and
accurately quantify gene-expression changes in a massively parallel manner.
Microarray
technology is a rapidly advancing area, which is gaining popularity in many
biological
disciplines from drug target identification to predictive toxicology. Over the
past few years,
there has been a dramatic increase in the number of methods and techniques
available for
carrying out this form of gene expression analysis. The techniques and
associated peripherals,
such as slide types, deposition methods, robotics, and scanning equipment, are
undergoing
constant improvement, helping to drive the technology forward in terms of
robustness and
ease of use.
Microarrays can be divided into two groups based on what is immobilized: A)
cDNAs
or B) oligodeoxyribonucleotides (oligos). For oligo-based microarrays, one can
use sense
oligos i.e. oligos having the same polarity as the mRNA/cDNA one wishes to
study). This
makes oligo design and matching oligos to cDNA records easy. The probe mixture
then has
to be made with anti-sense polarity, so that it can hybridize to the sense
oligos. The anti-sense
probe mixture is generally made as follows: A total RNA (or mRNA) preparation
from a
given tissue is subjected to reverse transcription using an oligo-dT primer
with a dangling T7
promoter in the 5' end, and reverse transcriptase. Second strand cDNA
synthesis is performed
in the classical way using DNA polymerise I and DNA ligase. Next, the double-
stranded
4
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
cDNA mixture is transcribed in vitro using T7 RNA polymerase, including
labeling
nucleotides, i.e. biotinylated nucleotides, producing a biotin-labeled, anti-
sense; cRNA probe
mixture. The cRNA probe mix is hybridized to the oligo microarray, containing
sense
orientation oligos representing a collection of cDNA sequences. In the example
of a biotin
label, a three-step sandwiched detection procedure can be applied, such as one
involving
streptavidin that binds biotin, a primary antibody to streptavidin, and a
fluorophore-labeled
secondary antibody, i.e. Cy3, to the primary antibody, with high-stringency
washes between
each step. The fluorescence of specific Cy3 signals are detected by a confocal
scanner and
digitized. These primary data are then normalized using background controls
and internal
standards. The same general procedure may also be used with cRNAs directly
labeled with
Cy3 or other commonly used fluorophore labels. In this case, longer oligos (50-
60-mers) are
used. With the sandwich method described above, sensitivity is greater, and
one can use 30-
mers.
The current methods of synthesizing cRNA probes hinges on the use of an oligo-
dT-
T7 primer for reverse transcription. This primer is intended to hybridize to
the poly-A tail of
most mRNA. However, the problem with this approach is two-fold: 1) some mRNAs
don't
have a poly-A tail; 2) some mRNA have a long 3' UTR and thus the reverse
transcriptase
may have difficulties copying the entire mRNA, and may produce truncated cDNAs
that
mostly represent non-coding sequences.
Thus, there exists a need in the art for improved methods of synthesizing and
amplifying nucleic acids, especially mRNA, which methods can achieve a high
degree of
synthesis and amplification from a limited amount of mRNA and which
simultaneously avoid
the infidelity with respect to sequence and representation often introduced by
other synthesis
and amplification methods.
SU11~IARY OF THE INVENTION
In one aspect, the present invention provides a method of production of double-
stranded molecules from a single-stranded ribonucleic acid molecule, which
involves three
steps. The first step involves contacting the ribonucleic acid molecule with
one or more first
random oligonucleotide primers containing an RNA polymerase promoter and
reverse
transcriptase under conditions sufficient for template-driven enzymatic
deoxyribonucleic acid
synthesis to occur, whereby a first deoxyribonucleic acid molecule is
produced. The second
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
step involves contacting this first deoxyribonucleic acid molecule with a
second random
primer which does not comprise an RNA polymerase promoter, and DNA polyinerase
under
conditions sufficient for template driven enzymatic deoxyribonucleic acid
synthesis to occur,
whereby a second deoxyribonucleic acid molecule is produced. The third step
involves
isolating and purifying the resulting double-stranded deoxyribonucleic acid
molecule, such
that a double-stranded deoxyribonucleic acid molecule is produced.
In one embodiment, the single-stranded ribonucleic acid molecule used to
produce
double-stranded DNA molecules is an mRNA. In another embodiment, the molecule
produced from the RNA of interest is a cDNA. In various embodiments, the RNA
polymerase used is T3, T7 or SP6 polymerases. In other embodiments, the DNA
polymerase
used is E. coli DNA polymerase I, the Klenow fragment of E. coli DNA
polymerase I, or T4
DNA polymerase. In another embodiment, the double-stranded DNA produced by
reverse
transcription of the mRNA of interest is introduced into a vector. In one
embodiment, the
primers are each 4-50 nucleotides in length.
Another aspect of the present invention provides a method for synthesizing at
least
one cRNA from a sample containing a plurality of different mRNAs. First strand
cDNA is
synthesized by contacting at least one mRNA in the sample with one or more
random
oligonucleotide primers including an RNA polymerase promoter that is
sufficiently
complementary to a sequence in the mRNA so as to prime first strand cDNA
synthesis, and
reverse transcriptase under conditions sufficient for reverse transcriptase
activity to occur.
Next, double-stranded cDNA is synthesized by contacting the first strand cDNA
with a
second random primer that does not contain an RNA polymerase promoter but is
sufficiently
complementary to a sequence in the first strand cDNA so to prime second strand
cDNA
synthesis, and with a DNA polymerase under conditions sufficient for RNA
polymerase
activity to occur. The resulting double-stranded cDNA is isolated and
purified, and then
subjected to in vitro transcription under conditions su~cient for template
driven RNA
transcription to occur, such that cRNA is produced.
In one embodiment, the single-stranded ribonucleic acid molecule used to
produce
double-stranded DNA molecules is an mRNA. In another embodiment, the molecule
produced from the RNA of interest is a cDNA. In various embodiments, the RNA
polymerase used is T3, T7 or SP6 polymerases. In other embodiments, the DNA
polymerase
used is E. coli DNA polymerase I, the I~lenow fragment of E. coli DNA
polymerase I, or T4
DNA polymerase. In another embodiment, the double-stranded DNA produced by
reverse
6
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
transcription of the mRNA of interest is introduced into a vector. In one
embodiment, the
primers are each 4-50 nucleotides in length. In various embodiments, the
samples contain
total RNA or mRNA from cells. In separate embodiments, the cells are
prokaryotic, yeast,
eukaryotic, mammalian and/or human cells. In one embodiment, the sample
contains total
RNA from 1 x 106 cells or less. In another embodiment, the sample contains at
least 10,000
different mRNAs.
Another aspect of the present invention discloses a method for comparing the
presence or amount of at least one mRNA of interest in a first sample and in a
second sample,
where these first and second samples each contain a plurality of different
mRNAs from one
or more cells. First strand cDNA is synthesized by contacting at least one
mRNA in the
sample with one or more random oligonucleotide primers including an RNA
polymerase
promoter that is sufficiently complementary to a sequence in the mRNA so as to
prime first
strand cDNA synthesis, and reverse transcriptase under conditions sufficient
for reverse
transcriptase activity to occur. Next, double-stranded cDNA is synthesized by
contacting the
first strand cDNA with a second random primer that does not contain an RNA
polymerase
promoter but is sufficiently complementary to a sequence in the first strand
cDNA so to
prime second strand cDNA synthesis, and with a DNA polymerase under conditions
sufficient for RNA polymerase activity to occur. The resulting double-stranded
cDNA is
isolated and purified, and then subjected to in vitro transcription under
conditions sufficient
for template driven RNA transcription to occur, such that cRNA is produced.
The cRNA
from the first sample is labeled with a first label; the cRNA produced using
the second
sample is labeled with a second label distinguishable from the first label.
The mRNA of
interest in the first sample is detected or measured by contacting the first
cRNA labeled with
the first label with a polynucleotide probe capable of hybridizing to the
first cRNA of the
mRNA of interest under conditions conducive to hybridization; and detecting
any
hybridization that occurs between said probe and said first cRNA. The mRNA of
interest in
the second sample is detected or measured by contacting the second cRNA,
labeled with
second label, with polynucleotide probe capable of hybridizing to second cRNA
of the
mRNA of interest under conditions conducive to hybridization; and detecting
any
hybridization that occurs between probe and second cRNA. The levels of the
mRNA of
interest detected or measured in the first sample are compared to levels of
the mRNA of
interest detected or measured in the second sample.
7
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In one embodiment, the single-stranded ribonucleic acid molecule used to
produce
double-stranded DNA molecules is an mRNA. In another embodiment, the molecule
produced from the RNA of interest is a cDNA. In various embodiments, the RNA
polymerise used is T3, T7 or SP6 polymerises. In other embodiments, the DNA
polymerise
used is E. coli DNA polymerise I, the I~lenow fragment of E. coli DNA
polymerise I, or T4
DNA polymerise. In another embodiment, the double-stranded DNA produced by
reverse
transcription of the mRNA of interest is introduced into a vector. In one
embodiment, the
primers are each 4-50 nucleotides in length. In various embodiments, the
samples contain
total RNA or mRNA from cells. In separate embodiments, the cells are
prokaryotic, yeast,
eukaryotic, mammalian andlor human cells. In one embodiment, the sample
contains total
RNA from 1 x 106 cells or less. In another embodiment, the sample contains at
least 10,000
different mRNAs.
In another aspect, the present invention provides a method for producing a 5'
enriched
cDNA library from a sample of mRNA molecules. The mRNA is contacted with one
or more
first random oligonucleotide primers including an RNA polymerise promoter, and
reverse
transcriptase under conditions sufficient for template driven enzymatic
deoxyribonucleic acid
synthesis to occur, whereby a first population of cDNA molecules is produced.
This first
population of cDNA molecules is contacted with a second random primer not
comprising an
RNA polymerise promoter, and a DNA polymerise under conditions sufficient for
template
driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a second
population of
cDNA molecules is produced. This second population of double-stranded cDNA
molecules
is isolated in order to produce a 5' enriched library. In one embodiment, the
mRNA contains
a poly-adenylated tail. In another embodiment, the rnRNA is contacted with a
primer mixture
that includes one or more first random oligonucleotide primers containing an
RNA
polymerise promoter and oligo (dT) primers containing an RNA polymerise
promoter. In a
further embodiment, the oligo (dT) primer is stably associated with the
surface of a solid
support. In yet another embodiment, the first and second labels are
fluorescent, radioactive,
enzymatic, hapten, biotin or digoxygenin labels. In a further embodiment, the
fluorescent
label is fluorescein isothiocyanate, lissamine, Cy3, CyS, or rhodamine 110. In
another
embodiment, a first aliquot of the first cRNA is labeled with a first
fluorophore having a first
emission spectrum, and a second aliquot of the second cRNA is labeled with a
second
fluorophore with a second emission spectrum differing from that of the first
emission
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
spectrum. In another embodiment, the first fluorophore is Cy3 and the second
fluorophore is
CyS.
Still another aspect of the present invention discloses a leit including, in
one or more
containers a mixture of first random oligonucleotide primers, where each first
primer contains
a 3' end sequence of 10-50 nucleotides that contains an A, a G, a T, or a C
nucleotide
randomly present in each position, and a 5' end sequence of 10-50 nucleotides
that includes
an RNA polymerise promoter, a mixture of oligo (dT) primers, where each oligo
(dT) primer
contains a 3' end sequence of 10-50 nucleotides that includes a T nucleotide
present in each
position; and also includes a 5' end sequence of 10-50 nucleotides containing
an RNA
polymerise promoter and a mixture of second random oligonucleotide primers,
each second
primer containing a sequence of 10-50 nucleotides that comprises an A, a G, a
T, or a C
nucleotide randomly present in each position.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method capable of reverse transcription of
mRNA
over its full length, particularly the 5' region of the cDNA, and hence
capable of providing a
full length cDNA even if a long chain mRNA is used as a template. The present
invention
also provides a method for in vitro transcription of double-stranded cDNA
molecule to
produce a labeled cRNA molecule for use in various methodologies including
microarray
technology.
The present invention relates to random oligonucleotide primers that contain
an RNA
polymerise promoter for synthesis of nucleic acids. The invention further
relates to methods
of use of the oligonucleotide primers of the invention for the synthesis of
nucleic acid
sequences in a population of cells, which methods preserve fidelity with
respect to sequence
and transcript representation.
More specifically, the present invention incorporates a change in the methods
used for
priming of the mRNA. Instead of using only oligo-dT primers in conjunction
with T7
promoters, random oligonucleotide primers of varying lengths (i.e. hexamer or
nanomer) are
employed that have RNA polymerise promoters in their 5' ends. Using these
random primers
containing the RNA polymerise promoters, alone or in combination with an oligo-
dT primer,
will increase probe coverage towards the 5' end of the template mRNA, as
compared to
methods using only an oligo-dT primer.
9
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
Use of the random primers of the invention, comprising an RNA polymerase
promoter, and subsequent second strand cDNA synthesis generates a library of
~cDNAs of
different lengths and different start and stop positions. This results in the
generation of a tiled
network, completely covering the mRNA population; in particular the cDNAs
generated
provide coverage across the full length of most of the individual mRNA
sequences. The
ensuing in vitro transcription of these cDNAs generates a complete, full-
coverage set of
either directly labeled or biotin-tagged cRNA probes. The present invention
does not
necessitate any changes in existing chip oligo design, hybridization, or
detection protocols.
Thus it is a stream-lined improvement to existing procedures.
For clarity of disclosure, and not by way of limitation, the detailed
description of the
invention is divided into the subsections set forth below.
OLIGONUCLEOTIDES
The oligonucleotide primers (i.e. random primers or oligo (dT) primers) for
use in the
methods of the invention can be of any suitable size, and are preferably 4-50
nucleotides in
length. The terms "oligonucleotide" and "oligo" as used herein denotes single
stranded
nucleotide multimers of from about 10 to about 100 nucleotides in length. The
term
"polynucleotide" as used herein refers to a single- or double-stranded polymer
composed of
nucleotide monomers of greater than about 100 nucleotides in length up to
about 1000
nucleotides in length.
An oligo (dT) primer for use in the subject methods is sufficiently long to
provide for
efficient hybridization to the polyA tail of mRNA. Generally, the length of
the oligo (dT)
primer ranges from about 10 to 50 nt in length, preferably from about 10 to 30
nt in length,
and more preferably from about 20 to 25 nt length. The oligo (dT) primer
contains a T
nucleotide present in each position of the oligo (dT) primer sequence. In many
embodiments,
the oligo (dT) primer is stably attached to the surface of a solid support.
The solid support
may be any convenient solid support known to those skilled in the art. A
variety of different
solid-phases are suitable for use in the subject methods, such phases being
known in the art
and commercially available. Specific solid-phases of interest include
polymeric supports
including, but not limited to, polystyrene, pegs, sheets, beads, magnetic
beads, and the like.
By "stably attached" is meant that the oligo (dT) primer remains associated
with the surface
of the solid support under at least conditions of enzymatic template driven
nucleic acid
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
synthesis. The oligo (dT) primer may be covalently or non-covalently bonded to
the solid
phase. The oligo (dT) primer is contacted with the mRNA under conditions
sufficient for
template driven enzymatic deoxyribonucleic acid synthesis to occur, where the
mRNA
molecule serves as the template molecule.
A random oligonucleotide primer for use in the subject methods is sufficiently
long to
provide for efficient hybridization to a complementary nucleic acid. By
"random
oligonucleotide primer" or "random primer" is meant a primer of random base
sequence
relative to the single-stranded RNA of the first hybrid, i.e. a primer that is
not known with
certainty to hybridize to the mRNA component of the first hybrid or duplex
prior to use. In
other words, the random primer has a sequence that is not known for certainty
to have a
complementary region in the single-stranded portion of the RNA of the hybrid
prior to
contact with the random primer. The random primer is sufficiently long to
provide for
efficient hybridization to a complementary region of the mRNA component, where
the
random primer typically ranges from about 4 to 50 nt, preferably from about 10
to 30 nt and
more preferably from about 20 to 30 nt in length. Primers contain random
arrangements of A,
T, C, G, U or I nucleotides, such that each random primer contains an A, a G,
a T, a C or an I
nucleotide randomly present in each position of the sequence. In preferred
embodiments, the
nucleotides are deoxyribonucleotide triphosphates (dNTPs) i.e. dATP, dTTP,
dCTP, dGTP,
or dITP or ribonucleotide triphosphates (rNTPs) i.e., rATP, rUTP, rCTP, rGTP,
or rTTP.
Specific primers of interest include those that have from about 5 to 15,
preferably from about
6 to 9 nt random nts fused in one embodiment to a restriction enzyme cut site
or more
preferably fused to an RNA polymerase promoter from about 5 to 20 nt,
preferably from
about 8 to 16 nt, e.g. 3'(N)9GCGGCCGCGCGGCCGCS', where N is any nucleotide.
Hexamer or nanomer oligonucleotides are preferred embodiments for random
primers. In
another preferred embodiment, the primers of the present invention are present
in a kit.
The oligonucleotide primers can be DNA, RNA, chimeric mixtures or derivatives
or
modified nucleic acid versions thereof, so long as it is still capable of
priming the desired
reaction. The oligonucleotide primer can be modified at the base moiety, sugar
moiety, or
phosphate backbone, and may include other appending groups or labels, so long
as it is still
capable of priming the desired amplification reaction. The term "nucleic acid"
as used herein
means a polymer composed of nucleotides, e.g. deoxyribonucleotides or
ribonucleotides. °The
terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of
ribonucleorides. RNA can be single-stranded or in a duplex. In a preferred
embodiment,
11
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
single-stranded RNA is message RNA (mRNA). The terms "deoxyribonucleic acid"
and
"DNA" as used herein mean a polymer composed of deoxyribonucleotides.
Deoxyribonucleic
acid can be single-stranded or double-stranded. In a preferred embodiment, DNA
is
complementary DNA (cDNA).
In a preferred embodiment, the random oligonucleotide primer contains an RNA
polymerase promoter. Preferably, the RNA polymerase promoter can be at the 5'
end of the
random oligonucleotide primer. The RNA polymerase promoter can be any RNA
polymerase promoter sequence known in the art. In preferred embodiments, the
RNA
polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA
polymerase
promoter sequence, or a SP6 RNA polymerase promoter sequence; T7 RNA
polymerase, T3
RNA polymerase and SP6 RNA polymerase can be used to initiate RNA synthesis
from these
promoters, respectively.
In one embodiment, a random oligonucleotide primer contains a 3' end sequence
of
10-50 nucleotides, said 3' end sequence having an A, a G, a T, a C, or a I
nucleotide randomly
present in each position of said 3' end sequence; and contains a 5' end
sequence of 10-50
nucleotides, said 5' end sequence having an RNA polymerase promoter (i.e. any
RNA
polymerase promoter sequence known in the art). More preferably, the RNA
polymerase
promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase
promoter
sequence, or a SP6 RNA polymerase promoter sequence.
In another embodiment, an oligo (dT) primer contains a 3' end sequence of 10-
50
nucleotides, said 3' end sequence having a T nucleotide present in each
position of said 3' end
sequence; and contains a 5' end sequence of 10-50 nucleotides, said 5' end
sequence having
an RNA polymerase promoter (i. e. any RNA polymerase promoter sequence known
in the
art). More preferably, the RNA polymerase promoter is a T7 RNA polymerase
promoter
sequence, a T3 RNA polymerase promoter sequence, or a SP6 RNA polymerase
promoter
sequence.
In another embodiment, a random oligonucleotide primer, contains a sequence of
10-
50 nucleotides, said sequence having an A, a G, a T, a C, or a I nucleotide
randomly present
in each position of said sequence.
In another embodiment, the oligonucleotide primer may contain at least one
modified
base moiety, including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-
chlorouracil, 5-
iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil,
12
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-
methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-
rriethylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-
methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-
5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-
methyl-2-
thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic
acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-
carboxypropyl) uracil,
(acp3)w, and 2,6-diaminopurine.
In another embodiment, the oligonucleotide primer contains at least one
modified
sugar moiety selected from the group including, but not limited to, arabinose,
2-
fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the oligonucleotide primer contains at least one
modified
phosphate backbone selected from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or analog
thereof.
The oligonucleotide primers of the present invention may be derived by
cleavage of a
larger nucleic acid fragment using non-specific nucleic acid cleaving
chemicals or enzymes
or site-specific restriction endonucleases; or synthesized by standard methods
known in the
art, e.g. by use of an automated DNA synthesizer (such as are commercially
available from
Biosearch, Applied Biosystems) and standard phosphoramidite chemistry.
Phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl.
Acids Res.
16:3209-3221), methylphosphonate oligonucleotides can be prepared by use of
controlled
pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451),
etc.
Once the desired oligonucleotide is synthesized, it is cleaved from the solid
support
on which it was synthesized and treated, by methods known in the art, to
remove any
protecting groups present. The oligonucleotide may then be purified by any
method known in
the art, including extraction and gel purification. The concentration and
purity of the
oligonucleotide may be determined by examining oligonucieotide that has been
separated on
an acrylamide gel, or by measuring the optical density at 260 nm in a
spectrophotometer.
METHODS OF LABELING OF NUCLEIC ACID PRODUCTS
13
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
Nucleic acid synthesis or amplification products such as synthesize RNA (i.e.
cRNA)
may be labeled with any art-known detectable marker, including radioactive
labels such as 32
P, 3s S, 3I3, and the like; fluorophores; chemiluminescers; or enzymatic
markers. In a
preferred embodiment, the label is fluorescent.
Exemplary suitable fluorophore moieties that can be used as labels are
selected from
the group including but not limited to: 4-acetamido-4'-isothiocyanatostilbene-
2,2'disulfonic
acid acridine and derivatives: acridine, acridine isothiocyanate, 5-(2'-
aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-
vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), -(4-
anilino-1-
naphthyl)maleimide, anthranilamide, Brilliant Yellow; coumarin and
derivatives: coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-
trifluoromethylcoumarin
(Coumarin 151), Cy3, CyS, cyanosine, 4',6-diaminidino-2-phenylindole (DAPI),
5',5"-
dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red), 7-diethylamino-3-(4'-
isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4,4'-
diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-
diisothiocyanatostilbene-2,2'-
disulfonic acid, 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride),
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), 4-
dimethylaminophenylazophenyl-
4'-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin
isothiocyanate; erythrosin
and derivatives: erythrosin B, erythrosin isothiocyanate, ethidium;
fluorescein and
derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-
yl)aminofluorescein
(DTAF), 2'T-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein,
fluorescein
isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, Malachite Green
isothiocyanate, 4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,
pararosaniline,
Phenol Red,
B-phycoerythrin, o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene
butyrate,
succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron Brilliant Red 3B-A);
rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G),
lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 110,
rhodamine
123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,
sulfonyl chloride
derivative of sulforhodamine i01 (Texas Red), N,N,N'N'-tetramethyl-6-
carboxyrhodamine
(TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC),
riboflavin, rosolic acid, terbium chelate derivatives.
14
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In a preferred embodiment, synthesized cRNA is labeled with a label, i.e., any
label
known in the art and as described herein. In other preferred embodiments, the
synthesized
cRNA is labeled with a label wherein the label is a fluorescent, radioactive,
enzymatic,
hapten, biotin, or digoxygenin label, more preferably the label is
fluorescent. The preferred
method of labeling in this invention is with a fluorophore, such as
fluorescein isothiocyanate,
lissamine, Cy3, CyS, and rhodamine 110, with Cy3 and Cy5 particularly
preferred.
In addition to fluorophores, chemiluminescers and enzymes, among others, may
also
be used as labels. In another embodiment, the oligonucleotide may be labeled
with an
enzymatic marker that produces a detectable signal when a particular chemical
reaction is
conducted, such as alkaline phosphatase or horseradish peroxidase. Such
enzymatic markers
are preferably heat stable, so as to survive the denaturing steps of the
amplification process.
Oligonucleotides may also be indirectly labeled by incorporating a nucleotide
linked
covalently to a hapten or to a molecule such as biotin, to which a labeled
avidin molecule or
streptavidin may be bound, or digoxygenin, to which a labeled anti-digoxygenin
antibody
may be bound.
Oligonucleotides of the invention may be labeled with labeling moieties during
chemical synthesis or the label may be attached after synthesis by methods
known in the art.
LABELING OF RNA
In one embodiment, the synthesized RNA of the invention (i.e. cRNA) is labeled
during synthesis to facilitate its detection in subsequent steps. The RNA may
be labeled with
any art-known detectable marker, including but not limited to radioactive
labels such as 32 P,
3s S, 3H, and the like; fluorophores; chemiluminescers; or enzymatic markers.
Labeling of RNA is preferably accomplished by including one or more labeled
NTPs
in the in vitro transcription reaction mixture (See, Example 1). NTPs may be
directly labeled
with a radioisotope, such as 32 P, 3s S, 3 H. Radiolabeled NTPs are available
from several
sources, including New England Nuclear (Boston, Mass.) and Amersham. NTPs may
be
directly labeled with a fluorescent label such as Cy3 or CyS. In one
embodiment, biotinylated
or allylamine-derivatized NTPs are incorporated during the in vitr~
transcription reaction and
the resultant cRNAs thereafter labeled indirectly, for example, by the
addition of fluorophore-
conjugated avidin (in the case of biotin) or the NHS ester of a fluorophore
(in the case of
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
allylamine). In another embodiment, fluorescently labeled NTPs may be
incorporated during
the in vitro transcription reaction, which fluorescently labels the resultant
cRNAs directly.
Exemplary fluorophore moieties that can be used as labels are set forth
herein. The
preferred label in the methods of this invention is a fluorophore, such as
fluorescein
isothiocyanate, lissamine, Cy3, CyS, and rhodamine 110, with Cy3 and Cy5
particularly
preferred.
Not only fluorophores, but also chemiluminescers and enzymes, among others,
may
be used as labels. In yet another embodiment, the RNA may be labeled with an
enzymatic
marker that produces a detectable signal when a particular chemical reaction
is conducted,
such as alkaline phosphatase or horseradish peroxidase. Such enzymatic markers
are
preferably heat stable, so as to survive the denaturing steps of the
amplification process.
RNA may also be indirectly labeled by incorporating a nucleotide linked
covalently to
a hapten or to a molecule such as biotin, to which a labeled avidin molecule
may be bound, or
digoxygenin, to which a labeled anti-digoxygenin antibody may be bound. RNA
may be
labeled with labeling moieties during chemical synthesis or the label may be
attached after
synthesis by methods known in the art.
Labeling of RNA is preferably accomplished by preparing cRNA that is
fluorescently
labeled with NHS-esters. Most preferably, labeling of RNA is accomplished in a
two-step
procedure in which allylamine-derivatized UTP is incorporated during in vitro
transcription.
Following the in vitro transcription reaction, unincorporated nucleotides are
removed and the
allylamine-containing RNAs are conjugated to the N-hydroxysuccinimide (NHS)
esters of
Cy3 or CyS.
This two-step method of preparing fluorescent-labeled cRNA is relatively
inexpensive
and suitable for use in two color hybridizations to DNA microarrays. In the
first step,
aminoallyl (AA)-labeled nucleic acids are prepared by incorporation of AA-
nucleotides. AA-
UTP (Sigma A-5660) may be used for labeling cRNA. AA-cRNA is prepared using
the
Ambion MegaScript T7 RNA polymerase in vitro transcription kit, with AA-UTP
substituted
at 50-100% of the total UTP concentration. It is essential to remove all
traces of amine-
containing buffers such as Tris prior to derivatizing the AA-nucleic acids. AA-
nucleic acids
prepared in enzymatic reactions are preferably cleaned up on appropriate
QIAGEN columns:
RNeasy Mini kit (for RNA) or QIAquick PCR Purification kit (for DNA) (QIAGEN
Inc.--
LTSA, Valencia, Calif.). For the QIAGEN columns, samples are applied twice.
For washes,
80% EtOH is preferably substituted for the buffer provided with the QIAGEN
kit. Samples
16
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
are eluted twice with 50 p1 volumes of 70° C. H20. Alternatively (but
less preferably),
samples may be cleaned up by repeated cycles of dilution and concentration
on~Microcon-30
filters.
In the second step, AA-nucleic acids are derivatized with NHS-esters,
preferably Cy 3
or Cy 5. Preferably, 2-6 p,g of AA-labeled nucleic acid are aliquoted into a
microfuge tube,
adjusting the total volume to 12 ~.l with H20. The NHS-ester is dissolved at a
concentration
of ~15 mM in anhydrous DMSO 0200 nmoles in 13 p,1). 27 ~.l of 0.1 M sodium
carbonate
buffer, pH 9, are added. 12 (..t1 of the dye mix (containing ~60 nmoles dye-
NHS ester) are then
immediately added to the AA-labeled nucleic acid (~6-20 pmoles of a 1 kb
molecule). The
samples are then incubated in the dark at 23 ° C for 1 hour. The
coupling reaction is stopped
by adding 5 p.1 of a 4M solution of hydroxylamine. Incubation is continued at
23 ° C for an
additional 0.25 hr. Dye-coupled nucleic acid is separated from unincorporated
dye on an
RNeasy Mini kit or QIAquick PCR Purification kit (QIAGEN Inc.--USA, Valencia,
Calif.).
Samples are washed with 80% EtOH instead of buffer, as described above, and
eluted twice
with 50 ~1 volumes of 70 ° C HBO.
The spectrum of the labeled nucleic acid is preferably measured from 220 nm-
700
nm. The percent recovery of nucleic acid and molar incorporation of dye is
calculated from
extinction coefficients and absorbance values at 1~. Recovery of nucleic acid
is typically
~80%. The mole percent of dye incorporated per nucleotide ranges from 1.5-5%
of total
nucleotides.
In a preferred embodiment, in vitro transcribed RNA is labeled with biotin
(See,
Example 1) or other labels, as described above. Most preferably the label is
fluorescent and
the label is incorporated by procedures commonly used in the art, as well as
by the procedure
described in Example 1 for a biotin label.
Often, it is desirable to compare gene expression in two different populations
of cells,
perhaps derived from different tissues or perhaps the same tissue exposed to
different stimuli
(i.e. comparison of a diseased tissue sample with a normal tissue sample or
the comparison of
a drug-treated tissue with a untreated tissue). Such comparisons are
facilitated by labeling the
RNAs from one population with a first fluorophore and the RNAs from the other
population,
with a second fluorophore, where the two fluorophores have distinct emission
spectra. Again,
Cy3 and Cy5 are particularly preferred fluorophores for use in comparing gene
expression
between two different populations of cells.
17
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
METHODS OF PREPARATION OF RNA
The methods of the invention are applicable to nucleic acid sequences derived
from
both eukaryotic and prokaryotic cells (i.e. yeast cells), although they are
preferably used with
eukaryotic cells, more preferably, with mammalian cells and most preferably,
with human
cells. Among cells that may serve as sources of DNA or RNA are nucleated blood
cells,
established cell lines, tumor cells, and tissue biopsy specimens, among others
that will be
readily apparent to those of skill in the art.
Although the synthesis methods of the invention can synthesize DNA, it is
preferred
to utilize the methods to synthesize RNA (i.e. cRNA) from a population of
cells. Total
cellular RNA, cytoplasmic RNA, mRNA, or poly(A)+ RNA may be used. Methods for
preparing total and poly(A)+ RNA are well known and are described generally in
Sambrook
et al. (1989, Molecular Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3,
Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1994,
Current
Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New
York).
RNA may be isolated from eukaryotic cells by procedures that involve lysis of
the
cells and denaturation of the proteins contained therein. Cells of interest
include wild-type
cells, drug-exposed wild-type cells, diseased cells, modified cells, and drug-
exposed modified
cells.
Additional steps may be employed to remove DNA. Cell lysis may be accomplished
with a nonionic detergent, followed by microcentrifugation to remove the
nuclei and hence
the bulk of the cellular DNA. In one embodiment, RNA is extracted from cells
of the various
types of interest using guanidinium thiocyanate lysis followed by CsCI
centrifugation to
separate the RNA from DNA (Chirgwin et al., 1979, Biochemistry 18:5294-5299).
Poly(A)+
RNA is selected by selection with oligo-dT cellulose (see Sambrook et al.,
1989, Molecular
Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, Cold
Spring Harbor, N.Y.). Alternatively, separation of RNA from DNA can be
accomplished by
organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl
alcohol.
If desired, RNase inhibitors may be added to the lysis buffer. Likewise, for
certain
cell types, it may be desirable to add a protein denaturation/digestion step
to the protocol.
For many applications, it is desirable to preferentially enrich mRNA with
respect to
other cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA).
Most
18
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched
by affinity
chromatography, for example, using oligo(dT) or poly(U) coupled to a solid
support, such as
cellulose or Sephadex~ (see Ausubel et al., eds., 1994, Current Protocols in
Molecular
Biology, vol. 2, Current Protocols Publishing, New York). Once bound, poly(A)+
mRNA is
eluted from the affinity column using 2 mM EDTA/0.1 % SDS.
The sample of RNA can contain a plurality of different mRNA molecules, each
different mRNA molecule having a different nucleotide sequence. In a specific
embodiment,
the mRNA molecules in the RNA sample contain at least 100 different nucleotide
sequences.
More preferably, the mRNA molecules of the RNA sample contain at least 500,
1,000, 5,000,
10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000 or
100,000 different
nucleotide sequences. In another specific embodiment, the RNA sample is a
mammalian
RNA sample (i.e. human RNA sample), the mRNA molecules of the mammalian RNA
sample containing about 20,000 to 30,000 different nucleotide sequences.
In a specific embodiment, total RNA or mRNA from cells are used in the methods
of
the invention. The source of the RNA can be cells of a plant or animal, human,
mammal,
primate, non-human animal, dog, cat, mouse, rat, bird, yeast, eukaryote,
prokaryote, etc. In
specific embodiments, the method of the invention is used with a sample
containing total
mRNA or total RNA from 1 x 106 cells or less.
METHODS OF DOUBLE-STRANDED CDNA SYNTHESIS
In the broadest sense, the subject invention is directed to methods of
preparing
double-stranded nucleic acid molecules from single-stranded nucleic acid
molecules, and
specifically to methods of preparing double-stranded deoxyribonucleic acid
molecules from
single-stranded ribonucleic acid molecules. Generally, the single-stranded
ribonucleic acid
molecule is an mRNA molecule having a polyA tail while the double-stranded
deoxyribonucleic acid molecule is a cDNA molecule. However, the mRNA molecule
need
not contain a polyA tail. In particular, the subject invention is directed to
methods of
producing double-stranded cDNA molecules that include the sequence information
from the
5' end of the template mRNA from which they are prepared. The length of the
cDNA
molecules prepared by the subject methods typically ranges from about 0.5 to
3.0 kb, usually
from about 1.0 to 2.0 kb and more usually from about 1.0 to 1.5 kb.
19
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
The first step in the subject methods is to prepare a first deoxyribonucleic
acid strand
(i.e. cDNA). This first DNA strand is prepared from the initial single-
stranded RNA
molecule, e.g. the mRNA. This step is generally accomplished by contacting the
initial RNA
with a primer and a reverse transcriptase under conditions sufficient for
template driven
enzymatic DNA synthesis to occur. The terms "sufficient for" and "conducive
to" as used
herein refers to a quantity or condition that can fulfill a need or
requirement but without being
abundant, i.e. an circumstance, situation or environment which allows the
method, procedure
or protocol of the invention to occur. In a preferred embodiment, this first
step involves
contacting a single stranded RNA molecule, i.e. mRNA, with one or more first
random
primers containing an RNA polymerase promoter and reverse transcriptase under
conditions
sufficient for template driven enzymatic DNA synthesis to occur, where the
mRNA molecule
serves as the template molecule. In preferred embodiments, the RNA polymerase
promoter is
a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence
or a
SP6 RNA polymerase promoter sequence and T7 RNA polymerase, T3 RNA polymerase
and
SP6 RNA polymerase are used to initiate RNA or DNA synthesis from these
promoters,
respectively. In a further embodiment, where the initial single-stranded RNA
molecule
contains a polyadenylated tail, one or more random primers containing an RNA
polymerase
promoter contacts the mRNA in combination with an oligo (dT) primer containing
an RNA
polymerase promoter under conditions for template driven enzymatic DNA
synthesis to
occur. In another embodiment the oligo (dT) primer is stably associated with
the surface of a
solid support.
In a preferred embodiment, contact of the initial single stranded RNA, i.e.
mRNA,
with one or more first random primers containing an RNA polymerase promoter
alone or in
combination with an oligo (dT) primer containing an RNA polymerase promoter
under
conditions sufficient for template driven enzymatic DNA synthesis to occur
produces a first
deoxyribonucleic acid molecule, i.e. first strand cDNA. In another embodiment,
the primers
used in the method of the invention are each 4-50 nucleotides in length.
Suitable
oligonucleotide primers are described herein and can be modified as necessary
by the skilled
artisan.
The initial mRNA that serves as template in the first step may be present in a
variety
of different samples, where the sample will typically be derived from a
physiological source.
The physiological source may be derived from a variety of eukaryotic sources,
with
physiological sources of interest including sources derived from single celled
organisms such
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
as yeast and multicellular organisms, including plants and animals,
particularly mammals,
where the physiological sources from multicellular organisms may be derived
from particular
organs or tissues of the multicellular organism, or from isolated cells
derived therefrom. In
obtaining the sample of RNAs to be analyzed from the physiological source from
which it is
derived, the physiological source may be subjected to a number of different
processing steps,
where such processing steps might include tissue homogenization, cell
isolation and
cytoplasmic extraction, nucleic acid extraction and the like, where such
processing steps are
known to those of skill in the art. Methods of isolating RNA from cells,
tissues, organs or
whole organisms are known to those of skill in the art and are described
herein and in
Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Press)(1989).
In addition to the mRNA and the primer, reverse transcriptase, RNA polymerise
and
other reagents necessary for primer extension are present. In preferred
embodiments, the
RNA polymerise is any know RNA polymerise but most preferably the RNA
polymerise is
T7 RNA polymerise, T3 RNA polymerise and SP6 RNA polymerise. In other
embodiments,
a reverse transcription or first strand DNA synthesis buffer, dithiothreitol
(DTT), a mixture of
deoxyribonucleotide triphosphates (dNTPs) i.e. dATP, dTTP, dCTP, dGTP, or
dTTP, and
nuclease-free H20 are present. An illustrative procedure for first strand cDNA
synthesis as
provided in Example 1. The order in which the reagents are combined may be
modified as
desired, and the entire illustrative procedure described therein can be
modified by the skilled
artisan.
Second strand cDNA synthesis is carried out by any method known in the art, as
illustrated in Example 1. In a preferred embodiment, the second step of the
subject methods is
to contact the first deoxyribonucleic acid molecule with a second random
primer which does
not contain an RNA polymerise promoter, and DNA polymerise under conditions
sufficient
for template driven enzymatic deoxyribonucleic acid synthesis to occur,
whereby a second
deoxyribonucleic acid molecule is produced resulting in a double-stranded
deoxyribonucleic
acid molecule (cDNA). In another embodiment, the double-stranded
deoxyribonucleic acid
molecule can be isolated and purified according to any method known in the art
and
illustrated in Example 1. In specific embodiments, the DNA polymerise used for
second
strand cDNA synthesis is any DNA polyrrierase known in the art and more
preferably E. coli
DNA polymerise I, HIenow fragment of E. coli DNA polymerise I, or T4 DNA
polymerise.
In another embodiment, the primers used in the method of the invention are
each 4-50
nucleotides in length. Suitable oligonucleotide primers are described herein
and can be
21
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
modified by the skilled artisan. In other embodiments, a second strand DNA
synthesis buffer,
DNA ligase, a mixture of deoxyribonucleotide triphosphates (dNTPs) i.e. dATP,
dTTP,
dCTP, dGTP, or dITP, RNase H (to degrade any remaining RNA molecules), and
nuclease-
free HZO are present (See, Example 1).
Contact of the first strand DNA with the second random primer under conditions
sufficient for template driven enzymatic DNA synthesis (as described above),
results in the
production of double-stranded cDNA molecules with different lengths and
different start and
stop positions. Of particular interest are double-stranded cDNA molecules in
which the
cDNA molecule includes the genetic information of the 5' terminus of the RNA,
e.g. mRNA.
The length of the double-stranded cDNA molecule typically ranges from about
500 to 3000
nt, preferably from about 1000 to 2000 nt and more preferably from about 1500
to 2000 nt.
In another embodiment, the present invention is directed to a method for
producing a
5' enriched cDNA library from a sample of mRNA molecules, said method
containing: (a)
contacting said mRNA with (i) one or more first random oligonucleotide primers
containing
an RNA polymerase promoter, and (ii) reverse transcriptase under conditions
sufficient for
template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a
first
population of cDNA molecules is produced; (b) contacting said first population
of cDNA
molecules (i} with a second random primer not containing an RNA polymerase
promoter, and
(ii) DNA polymerase under conditions sufficient for template driven enzymatic
deoxyribonucleic acid synthesis to occur, whereby a second population of cDNA
molecules
is produced; and (c) isolating said second population of double-stranded cDNA
molecules;
such that said 5' enriched library is produced. By "5' enriched" is meant that
a significant
proportion of the cDNAs in the library contain the nucleotide sequence
information of the 5'
end of the mRNAs from which the cDNAs are derived. The 5' enriched cDNA
libraries of the
subject invention contain at least 5, usually at least 50 and more usually at
least 100 distinct
cDNAs (i.e. cDNAs that differ in sequence from each other), where the number
of distinct
cDNAs in the library may be as high as 10,000 or higher. A significant portion
of the cDNA
constituents of the library include the 5' sequence information of their
corresponding mRNA
from which they were derived, where the percentage of distinct cDNAs in the
library that
include 5' sequence information from their corresponding mRNAs is at least
about 10%,
usually at least about 20% and more usually at least about 25%, where the
percentage may be
as high as 30% or higher, including in certain embodiments, 40%, 50%, 60% 70%
80%, 90%
or higher. The initial mRNA that serves as template in the first step may be
present in a
22
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
variety of different samples, where the sample will typically be derived from
a physiological
source. In preferred embodiments, the physiological source is prokaryotic
tissue or cell (i.e.
yeast cells) and eukaryotic tissue or cell. In more preferred embodiments, the
sample source
is a mammalian tissue or cell and most preferably the sample source is a human
tissue or cell.
In another embodiment, the sample contains total RNA or total mRNA from the
tissue or
cells described herein. In a preferred embodiments the sample contains total
RNA from 1 x
106 cells or less and the sample contains at least 10,000 different mRNAs. In
preferred
embodiments, the RNA polymerise promoter is a T7 RNA polymerise promoter
sequence, a
T3 RNA polymerise promoter sequence or a SP6 RNA polymerise promoter sequence
and
T7 RNA polymerise, T3 RNA polymerise and SP6 RNA polymerise are used to
initiate
RNA or DNA synthesis from these promoters, respectively. In specific
embodiments, the
DNA polymerise used for second strand cDNA synthesis is any DNA polymerise
known in
the art and more preferably E. coli DNA polymerise I, Klenow fragment of E.
coli DNA
polymerise I, or T4 DNA polymerise. In another embodiment, the primers used in
the
method of the invention are each 4-50 nucleotides in length. Suitable
oligonucleotide primers
are described herein and can be modified by the skilled artisan. In a further
embodiment,
where the initial single-stranded RNA molecule contains a polyadenylated tail,
one or more
random primers containing an RNA polymerise promoter contacts the mRNA in
combination
with an oligo (dT) primer containing an RNA polymerise promoter under
conditions for
template driven enzymatic DNA synthesis to occur. In another embodiment the
oligo (dT)
primer is stably associated with the surface of a solid support. In another
embodiment, the
second population of double-stranded cDNA molecules can be introduced into a
vector as
described below.
In another embodiment, the resultant isolated double-stranded cDNA may then be
ligated into an appropriate vector for propagation and subsequent use, as
desired, using
methods well known to those of skill in the art. Appropriate vectors include
viral, phagemid
and plasmid vectors. Generally, this step involves contact of the vector with
the double-
stranded cDNA under conditions sufficient for ligation of the cDNA with the
vector to occur.
Typically, the vector and the cDNA will have been treated to produce
complementary or
"sticky-ends" with at least one, and preferably.two different restriction
endonucleases. This
pretreatment step may further include ligation of linker or adapter sequences
onto the ends of
the vector andlor ds cDNA in order to introduce desired restriction sites,
etc.
23
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
METHODS OF CRNA SYNTHESIS
In a preferred embodiment, the present invention provides a method for
synthesizing
at least one cRNA from a sample containing a plurality of different mRNAs,
said method
containing: (a) synthesizing first strand cDNA by contacting at least one mRNA
in said
sample with (i) one or more random oligonucleotide primers containing an RNA
polymerise
promoter that is sufficiently complementary to a sequence in the mRNA so as to
prime first
strand cDNA synthesis, and (ii) reverse transcriptase under conditions
sufficient for reverse
transcriptase activity to occur; (b) synthesizing double-stranded cDNA by
contacting the first
strand cDNA with (i) a second random primer not containing an RNA polymerise
promoter
wherein said second random primer is sufficiently complementary to a sequence
in the first
strand cDNA so to prime second strand cDNA synthesis, and (ii) a DNA
polymerise under
conditions sufficient for RNA polymerise activity to occur; (c) isolating and
purifying said
double-stranded cDNA; and
(d) subjecting said purified double-stranded cDNA to in vitro transcription
under conditions
sufficient for template driven DNA transcription to occur, such that cRNA is
produced.
In vitro transcription can be accomplished by any method well known in the
art, i.e.,
Example l; Ausubel et al., eds., 1994, Current Protocols in Molecular Biology,
vol. 2,
Current Protocols Publishing, New York. Prior to subjecting double-stranded
cDNA to in
vitro transcription, it may be necessary to introduce the double-stranded cDNA
molecule into
a vector using any methods well know to those of skill in the art (Ausubel et
al., eds., 1994,
Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing,
New York) and
as described herein.
In preferred embodiments, the physiological source is prokaryotic tissue or
cell (i.e.
yeast cell) and eukaryotic tissue or cell. In more preferred embodiments, the
sample source is
a mammalian tissue or cell and most preferably the sample source is a human
tissue or cell. In
another embodiment, the sample contains total RNA or total mRNA from the
tissue or cells
described herein. In a preferred embodiments the sample contains total RNA
from up to 1 x
106 cells and the sample contains at least 10,000 different mRNAs. In
preferred
embodiments, the RNA polymerise promoter is a T7 RNA polymerise promoter
sequence, a
T3 RNA polymerise promoter sequence or a SP6 RNA polymerise promoter sequence
and
T7 RNA polymerise, T3 RNA polymerise and SP6 RNA polymerise are used to
initiate
RNA or DNA synthesis from these promoters, respectively. In specific
embodiments, the
24
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
DNA polymerase used for second strand cDNA synthesis is any DNA polymerase
known in
the art and more preferably E. coli DNA polymerase I, Klenow fragment of E.
~coli DNA
polymerase I, or T4 DNA polymerase. In another embodiment, the primers used in
the
method of the invention are each 4-SO nucleotides in length. Suitable
oligonucleotide primers
are described herein and can be modified by the skilled artisan. In a further
embodiment,
where the initial single-stranded RNA molecule contains a polyadenylated tail,
one or more
random primers containing an RNA polymerase promoter contacts the mRNA in
combination
with an oligo (dT) primer containing an RNA polymerase promoter under
conditions for
template driven enzymatic DNA synthesis to occur. In another embodiment, the
oligo (dT)
primer is stably associated with the surface of a solid support.
In a preferred embodiment, synthesized cRNA is labeled with a label, i.e., any
label
known in the art and as described herein. In other preferred embodiments, the
synthesized
cRNA is labeled with a label, wherein the label is a fluorescent, radioactive,
enzymatic,
hapten, biotin, or digoxygenin label. More preferably, the label is
fluorescent. The preferred
label in the methods of this invention is a fluorophore, such as fluorescein
isothiocyanate,
lissamine, Cy3, CyS, and rhodamine 110, with Cy3 and Cy5 particularly
preferred.
In additional embodiments, the present invention provides a method for
determining
the presence or absence of a target mRNA from a sample and determining the
effect of drug
treatment on a target mRNA in a sample following the in vitro transcription
step and
production of cRNA
In another embodiment, the present invention is directed to a method wherein
the
mRNA is extracted from at least one cell of interest, and further containing
(e) contacting the
cRNA produced in step (d) described above with an array containing one or more
species of
polynucleotide positioned at preselected sites on the array, under conditions
conducive to
hybridization; and (f) detecting any hybridization that occurs between said
one or more
species of polynucleotide and said cRNA. Methods of preparing and using
microarrays are
described herein.
METHODS FOR DETERMINING BIOLOGICAL RESPONSE PROFILES
In one embodiment, the invention is directed to a method for comparing the
presence
or amount of at least one mRNA of interest in a first sample and in a second
sample, said first
sample and said second sample each containing a plurality of different mRNAs
from one or
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
more cells, said method containing: (a) synthesizing first strand cDNA by
contacting at least
one mRNA in said sample with (i) one or more first random oligonucleotide
primers
containing an RNA polymerase promoter that is sufficiently complementary to a
sequence in
the mRNA so as to prime first strand cDNA synthesis, and (ii) reverse
transcriptase under
conditions sufficient for reverse transcriptase activity to occur; (b)
synthesizing double-
stranded cDNA by contacting the first strand cDNA with (i) a second random
primer not
containing an RNA polymerase promoter that is sufficiently complementary to a
sequence in
the first strand cDNA so to prime second strand cDNA synthesis, and (ii) a DNA
polymerase
under conditions sufficient for DNA polymerase activity to occur; (c)
isolating and purifying
said double-stranded cDNA; (d) subjecting said purified double-stranded cDNA
to i~z vitro
transcription under conditions sufficient for template driven DNA
transcription to occur, such
that cRNA is produced; (e) labeling the cRNA produced in step (d) with a first
label; (f)
repeating steps (a)-(d) with said second sample; (g) labeling the cRNA
produced in step (f)
with a second label distinguishable from said first label; (h) detecting or
measuring the
mRNA of interest in the first sample by contacting the first cRNA labeled with
said first label
with a polynucleotide probe capable of hybridizing to said first cRNA of the
mRNA of
interest under conditions conducive to hybridization; and detecting any
hybridization that
occurs between said probe and said first cRNA; (i) detecting or measuring the
mRNA of
interest in the second sample by contacting the second cRNA labeled with said
second label
with said polynucleotide probe capable of hybridizing to said second cRNA of
the mRNA of
interest under conditions conducive to hybridization; and detecting any
hybridization that
occurs between said probe and said second cRNA; and (j) comparing the levels
of the mRNA
of interest detected or measured in said first sample with levels of the mRNA
of interest
detected or measured in said second sample.
The initial mRNA that serves as template in the first step may be present in a
variety
of different samples, where the sample will typically be derived from a
physiological source.
In preferred embodiments, the physiological source is prokaryotic tissue or
cell (i.e. yeast
cell) and eukaryotic tissue or cell. In more preferred embodiments, the sample
source is a
mammalian tissue or cell and most preferably the sample source is a human
tissue or cell. In
another embodiment, the sample contains total RNA or total mRNA from the
tissue or cells
described herein. In a preferred embodiments the sample contains total RNA
from 1 x 106
cells or less and the sample contains at least 10,000 different mRNAs. In
preferred
embodiments, the RNA polymerase promoter is a T7 RNA polymerase promoter
sequence, a
26
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
T3 RNA polymerise promoter sequence or a SP6 RNA polymerise promoter sequence
and
T7 RNA polymerise, T3 RNA polymerise and SP6 RNA polymerise are used ~to
initiate
RNA or DNA synthesis from these promoters, respectively. In specific
embodiments, the
DNA polymerise used for second strand cDNA synthesis is any DNA polymerise
known in
the art and more preferably E. cola DNA polymerise I, Klenow fragment of E.
cola DNA
polymerise I, or T4 DNA polymerise. In another embodiment, the primers used in
the
method of the invention are each 4-50 nucleotides in length. Suitable
oligonucleotide primers
are described herein and can be modified by the skilled artisan. In a further
embodiment,
where the initial single-stranded RNA molecule contains a polyadenylated tail,
one or more
random primers containing an RNA polymerise promoter contacts the mRNA in
combination
with an oligo (dT) primer containing an RNA polymerise promoter under
conditions for
template driven enzymatic DNA synthesis to occur. In another embodiment the
oligo (dT)
primer is stably associated with the surface of a solid support
In a preferred embodiment, synthesized first and second cRNAs is labeled with
a
label, i.e., any label known in the art and described herein. In other
preferred embodiments,
the synthesized first and second cRNAs is labeled with a label wherein the
label is a
fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label.
Preferably the label
is fluorescent. The preferred label in the methods of this invention is a
fluorophore, such as
fluorescein isothiocyanate, lissamine, Cy3, CyS, and rhodamine 110, with Cy3
and Cy5
particularly preferred.
In a preferred embodiment, a first aliquot of the first cRNA is labeled with a
first
fluorophore having a first emission spectrum, and a second aliquot of the
second cRNA is
labeled with a second fluorophore with a second emission spectrum differing
from that of the
first emission spectrum; more preferably the first fluorophore is Cy3 and the
second
fluorophore is CyS.
In another embodiment, the present invention is directed to a method wherein
in steps
(h) and (a) as described above, the steps of contacting the first cRNA labeled
with said first
label with said polynucleotide probe, and contacting the second cRNA labeled
with said
second label with said polynucleotide probe, are carned out concurrently or
sequentially. The
term "concurrently" refers to two events occurnng at the same time and the
term
"sequentially" and refers to succeeding or following in a particular order.
In preferred embodiments, the first sample contains mRNAs from cells that are
diseased and wherein said second sample contains mRNAs from normal cells; or
first sample
27
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
contains mRNAs from drug-treated cells and wherein said second sample contains
mKNAs
from untreated cells, wherein said levels of said mRNAs from these cells can
be compared.
In another embodiment, the present invention is directed to a method wherein
in steps
(h) and (i) are carned out by a method containing contacting said first and
second cRNAs
with an array containing one or more species of polynucleotide probe
positioned at
preselected sites on the array, under conditions sufficient for hybridization
to occur; and
detecting any hybridization that occurs between said polynucleotide probes and
said cRNAs.
Methods of preparing and using microarrays are described herein.
The invention utilizes the ability to measure the responses of a biological
system to a
large variety of perturbations. Exemplary methods for measuring biological
responses are
described herein. One of skill in the art will appreciate that this invention
is not limited to the
following specific methods for measuring the responses of a biological system.
In particular,
the presence of cRNA(s) of interest (and thus mRNA(s) of interest in the
sample) can be
detected or measured by procedures including but not limited to Northern
blotting or using
bead-bound oligonucleotides as probes, or the use of polynucleotide
microarrays. In
additional embodiments, the synthesized labeled cRNA probes of the invention
can be
utilized for various other~methodologies, including but not limited to dot
blot analysis,
differential hybridization, RT-PCR, and anti-sense RNA studies.
Dot blot analysis: For any type of dot blot experiment that employs cDNA,
genomic
DNA, mRNA, or oligos, one skilled in the art can use the invention to
synthesize full-length
coverage cRNA probes. For genomic DNA, this method can be used to ask
evolutionary-type
questions such as: "are there exons present in this organism that are similar
to exons from this
other organism, but less so for this one?"
Differential hybridization: When studying expression differences between cell
types
or tissues, it is sometimes useful to employ differential hybridization. A
cDNA library
displayed as bacteriophage plaques may be hybridized with the cRNA probes,
synthesized
and labeled by the methods of the present invention, from the cell type or
tissue one wants to
compare.
RT PCR and anti-sense RNA studies: A reverse transcription reaction of an mRNA
pool from a certain tissue can be primed using the random oligonucleotide
primer of the
present invention containing an RNA polymerase promoter. In an illustrative
and non-
limiting example, using a random-T7 primers, the next step, PCR, one would use
a gene-
specific upper primer directed at the 5 prime end of the desired mRNA, and a
T7 promoter-
28
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
complementary primer. If used during stringent conditions (a long (27-30-mer)
upper primer,
high annealing temperature, or preferably a touch-down PCR protocol) one cari
isolate the
full-length cDNA of a gene of choice. In addition, this cDNA is ready to be in
vitro
transcribed in the anti-sense direction using T7 RNA polymerise. The anti-
sense RNA can be
used in anti-sense RNA studies for a) use in secondary structure determination
of the anti-
sense RNA; b) use directly labeled with radioactivity or biotin as an anti-
sense RNA probe
for in situ hybridization; c) use in RNA Interference experiments in duplex
with the
corresponding sense mRNA, produced by conventional means; d) other anti-sense
RNA
experiments. Furthermore, a single reverse transcription reaction can be used
for multiple
different genes only by changing the upper primer. This is particularly useful
if the tissue or
cell type under study is scarce.
In one specific embodiment of the invention, one or more labels is introduced
into the
RNA during the ira vitro transcription step, as described herein, to
facilitate gene expression
profiling. Gene expression can be profiled in any of several ways. The
preferred method is to
probe a DNA microarray with the labeled RNA transcripts generated as described
above. A
DNA microarray, or chip, is a microscopic array of DNA fragments or synthetic
oligonucleotides, disposed in a defined pattern on a solid support, wherein
they are amenable
to analysis by standard hybridization methods (Schena, BioEssays 18: 427,
1996).
The DNA in a microarray may be derived from genomic or cDNA libraries, from
fully sequenced clones, or from partially sequenced cDNAs known as expressed
sequence
tags (ESTs). Methods for obtaining such DNA molecules are generally known in
the art (See,
e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol.
2, Current
Protocols Publishing, New York). Alternatively, oligonucleotides may be
synthesized by
conventional methods, such as phosphoramidite-based synthesis.
Gene expression profiling can be done for purposes of screening, diagnosis,
staging of
a disease, monitoring response to therapy, as well as for identifying genetic
targets of drugs
and of pathogens.
Tltarrsclurr ASSAY UsirrG DNA A~xays
The methods of this invention are particularly useful for the analysis of gene
expression profiles. For expression profiling, DNA microarrays are typically
probed using
mRNA, extracted and synthesized to cRNA from the cells whose gene expression
profile it is
29
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
desired to analyze, using the methods of the invention (i.e. producing double-
stranded DNA,
synthesizing at least one cRNA molecule, labeling at least one synthesized
cRNA molecule,
producing 5' enriched cDNA library). To facilitate comparison between any two
samples of
interest, the mRNAs are typically labeled separately with fluorescent dyes
that emit at
different wavelengths, as described above.
Some embodiments of this invention are based on measuring the transcriptional
rate
of genes. Transcriptional rates can be measured by techniques of hybridization
to arrays of
nucleic acid or nucleic acid mimic probes. However measured, the result is
either the
absolute, relative amounts of transcripts or response data, including values
representing RNA
abundance ratios, which usually reflect DNA expression ratios (in the absence
of differences
in RNA degradation rates).
In various alternative embodiments of the present invention, aspects of the
biological
state other than the transcriptional state, such as the translational state,
the activity state, or
mixed aspects can be measured. Preferably, measurement of the transcriptional
state is made
by hybridization to transcript arrays, which are described herein. Certain
other methods of
transcriptional state measurement are also described herein.
In a preferred embodiment the present invention makes use of "transcript
arrays" (also
referred to herein as "microarrays"). Transcript arrays can be employed for
analyzing the
transcriptional state in a biological sample and especially for measuring the
transcriptional
states of a biological sample exposed to graded levels of a drug of interest
or to graded
perturbations to a biological pathway of interest.
In one embodiment, transcript arrays are produced by hybridizing detectably
labeled
polynucleotides representing the mRNA transcripts present in a cell (e.g.,
fluorescently
labeled cRNA that is synthesized by the methods of the present invention) to a
microarray. A
microarray is a surface with an ordered array of binding (e.g., hybridization)
sites for
products of many of the genes in the genome of a cell or organism, preferably
most or almost
all of the genes. Microarrays can be made in a number of ways. However
produced,
microarrays share certain preferred characteristics. Specifically, the arrays
are reproducible,
allowing multiple copies of a given array to be produced and easily compared
with each
other. Preferably the microarrays are small, usually smaller than 5 cm2, and
they are made
from materials that are stable under binding (e.g., nucleic acid
hybridization) conditions. A
given binding site or unique set of binding sites in the microarray will
specifically bind the
product of a single gene in the cell. Although there may be more than one
physical binding
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
site (hereinafter "site") per specific mRNA, for the sake of clarity the
discussion below will
assume that there is a single site.
In one embodiment, the microarray is an array of polynucleotide probes, the
array
containing a support with at least one surface and at least 100 different
polynucleotide
probes, each different polynucleotide probe containing a different nucleotide
sequence and
being attached to the surface of the support in a different location on the
surface. Preferably,
the nucleotide sequence of each of the different polynucleotide probes is in
the range of 40 to
80 nucleotides in length. More preferably, the nucleotide sequence of each of
the different
polynucleotide probes is in the range of 50 to 70 nucleotides in length. Even
more preferably,
the nucleotide sequence of each of the different polynucleotide probes is in
the range of 50 to
60 nucleotides in length.
In specific embodiments, the array contains polynucleotide probes of at least
2,000,
4,000, 10,000, 15,000, 20,000, 50,000, 80,000, or 100,000 different nucleotide
sequences.
In another embodiment, the nucleotide sequence of each polynucleotide probe in
the
array is specific for a particular target polynucleotide sequence. In yet
another embodiment,
the target polynucleotide sequences contain expressed polynucleotide sequences
of a cell or
organism. In a specific embodiment, the cell or organism is a mammalian cell
or organism. In
another specific embodiment, the cell or organism is a human cell or organism.
In specific embodiments, the nucleotide sequences of the different
polynucleotide
probes of the array are specific for at least 50%, at least 75%, at least 80%,
at least 85%, at
least 90%, at least 95%, or at least 99% of the genes in the genome of the
cell or organism.
Most preferably, the nucleotide sequences of the different polynucleotide
probes of the array
are specific for all of the genes in the genome of the cell or organism.
In specific embodiments, the polynucleotide probes of the array hybridize
specifically
and distinguishably to at least 10,000, to at least 20,000, to at least
50,000, different
polynucleotide sequences, to at least 80,000, or to at least 100,000 different
polynucleotide
sequences.
In other specific embodiments, the polynucleotide probes of the array
hybridize
specifically and distinguishably to at least 90%, at least 95%, or at least
99% of the genes or
gene transcripts of the genome of a cell or organism. Most preferably, the
polynucleotide
probes of the array hybridize specifically and distinguishably to the genes or
gene transcripts
of the entire genome of a cell or organism.
31
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In specific embodiments, the array has at least 100, at least 250, at least
1,000, or at
least 2,500 probes per 1 cm2, preferably all or at least 25% or 50% of which
are different
from each other. In another embodiment, the array is a positionally
addressable array (in that
the sequence of the polynucleotide probe at each position is known).
In another embodiment, the nucleotide sequence of each polynucleotide probe in
the
array is a DNA sequence. In another embodiment, the DNA sequence is a single-
stranded
DNA sequence. The DNA sequence may be, e.g., a cDNA sequence, or a synthetic
sequence.
In a preferred embodiment, the nucleotide sequence of each polynucleotide
probe in the
array is a oligodeoxyribonucleotides sequence.
It will be appreciated by those skilled in the art that when cRNA
complementary to
the RNA of a cell is synthesized and hybridized to a microarray under suitable
hybridization
conditions, the level of hybridization to the site in the array corresponding
to any particular
gene will reflect the prevalence in the cell of mRNA transcribed from that
gene. For example,
when detectably labeled (e.g., with a fluorophore) cRNA complementary to the
total cellular
mRNA is hybridized to a microarray, the site on the array corresponding to a
gene (i.e.,
capable of specifically binding the product of the gene) that is not
transcribed in the cell will
have little or no signal (e.g., fluorescent signal), and a gene for which the
encoded mRNA is
prevalent will have a relatively strong signal.
In preferred embodiments, cRNAs from two different cells are hybridized to the
binding sites of the microarray. In the case of monitoring drug responses one
biological
sample is exposed to a drug and another biological sample of the same type is
not exposed to
the drug. In the case of pathway responses one cell is exposed to a pathway
perturbation and
another cell of the same type is not exposed to the pathway perturbation. The
cRNA derived
from each of the two cell types are differently labeled so that they can be
distinguished. In
one embodiment, for example, cRNA from a cell treated with a drug (or exposed
to a
pathway perturbation) is synthesized using a fluorescein-labeled NTP, and cRNA
from a
second cell, not drug-exposed, is synthesized using a rhodamine-labeled NTP.
When the two
cRNAs are mixed and hybridized to the microarray, the relative intensity of
signal from each
cRNA set is determined for each site on the array, and any relative difference
in abundance of
a particular mRNA detected.
In the example described above, the cRNA from the drug-treated (or pathway
perturbed) cell will fluoresce green when the fluorophore is stimulated and
the cRNA from
the untreated cell will fluoresce red. As a result, when the drug treatment
has no effect, either
32
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
directly or indirectly, on the relative abundance of a particular mRNA in a
cell, the mRNA
will be equally prevalent in both cells and, upon reverse transcription, red-
labeled and green-
labeled cRNA will be equally prevalent. When hybridized to the microarray, the
binding
sites) for that species of RNA will emit wavelengths characteristic of both
fluorophores (and
appear brown in combination). In contrast, when the drug-exposed cell is
treated with a drug
that, directly or indirectly, increases the prevalence of the mRNA in the
cell, the ratio of
green to red fluorescence will increase. When the drug decreases the mRNA
prevalence, the
ratio will decrease.
The use of a two-color fluorescence labeling and detection scheme to define
alterations in gene expression has been described, e.g., in Schena et al.,
1995, Science
270:467-470, which is incorporated herein by reference in its entirety. An
advantage of using
cRNA labeled with two different fluorophores is that a direct and internally
controlled
comparison of the mRNA levels corresponding to each arrayed gene in two cell
states can be
made, and variations due to minor differences in experimental conditions
(e.g., hybridization
conditions) will not affect subsequent analyses. However, it will be
recognized that it is also
possible to use cRNA from a single cell, and compare, for example, the
absolute amount of a
particular mRNA in, e.g., a drub treated or pathway-perturbed cell and an
untreated cell.
In a preferred embodiment, cRNA from two different cells are hybridized to the
binding sites of a microarray. In this embodiment, one biological sample is
from a diseased
tissue or cell and the other biological sample of the same type is from a
normal non-diseased
tissue or cell. As described herein, these samples can be labeled in one
embodiment and
subjected to the two-color fluorescence labeling and detection scheme to
define alterations in
gene expression. Thus, synthesis of a labeled cRNA molecule using the random
oligonucleotides of the invention containing an RNA polymerase promoter can be
used in
methods of diagnosis, wherein a synthesized cRNA sequence is complementary to
a sequence
(e.g., genomic) of an infectious disease agent, e.g. of human disease
including but not limited
to viruses, bacteria, parasites, and fungi, thereby diagnosing the presence of
the infectious
agent in a sample of nucleic acid from a patient. The target nucleic acid can
be genomic or
cDNA or mRNA or synthetic, human or animal, or of a microorganism, etc. In
another
embodiment that can be used in the diagnosis or prognosis of a disease or
disorder, the target
sequence is a wild type human genomic or RNA or cDNA sequence, mutation of
which is
implicated in the presence of a human disease or disorder, or alternatively,
can be the mutated
sequence. In such an embodiment, the mutation can be an insertion,
substitution, and/or
33
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
deletion of one or more nucleotides, or a translocation.
PREPARATION OF MICROARRAYS
Microarrays are known in the art and consist of a surface to which probes that
correspond in sequence to gene products (e.g., cDNAs, mRNAs, cRNAs,
polypeptides, and
fragments thereof), can be specifically hybridized or bound at a known
position. In one
embodiment, the microarray is an array (i.e., a matrix) in which each position
represents a
discrete binding site for a product encoded by a gene (e.g., a protein or
RNA), and in which
binding sites are present for products of most or almost all of the genes in
the organism's
genome. In a preferred embodiment, the "binding site" (hereinafter, "site") is
a nucleic acid or
nucleic acid analogue to which a particular cognate cRNA can specifically
hybridize. The
nucleic acid or analogue of the binding site can be, e.g., a synthetic
oligomer, a full Length
cRNA, a less-than full length cRNA, or a gene fragment.
In one embodiment, the microarray contains binding sites for products of all
or almost
all genes in the target organism's genome. This microarray will have binding
sites
corresponding to at least about 50% of the genes in the genome, often at least
about 75%,
more often at least about ~5%, even more often more than about 90%, and most
often at least
about 99%.
Such comprehensiveness, however, is not necessarily required. In another
embodiment, the microarray contains binding sites for products of human genes.
This
microarray will have binding sites corresponding to at least about 5-10% of
the genes in the
genome, preferably at least about 10-15%, and more preferably at least about
40%.
Preferably, the microarray has binding sites for genes relevant to the action
of a drug
of interest, disease of interest, or in a biological pathway of interest. A
"gene" is identified as
an open reading frame (ORF) of preferably at least 50, 75, or 99 amino acids
from which a
mRNA is transcribed in the organism (e.g., if a single cell) or in some cell
in a multicellular
organism. The number of genes in a genome can be estimated from the number of
mRNAs
expressed by the organism, or by extrapolation from a well-characterized
portion of the
genome. When the genome of the organism of interest has been sequenced, the
number of
ORFs can be determined and mRNA coding regions identified by analysis of the
DNA
sequence. For example, the Saccharomyces cerevisiae genome has been completely
sequenced and is reported to have approximately 6275 open reading frames
(ORFs) longer
34
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
than 99 amino acids. Analysis of these ORFs indicates that there are 5885 ORFs
that are
likely to specify protein products (Goffeau et al., 1996, Science 274:546-567,
which is
incorporated by reference in its entirety). In contrast, the human genome is
estimated to
contain approximately 105 genes.
PREPARATION OF NUCLEIC ACIDS FOR MICROARRAYS
As noted above, the "binding site" to which a particular cognate cRNA
specifically
hybridizes is usually a nucleic acid or nucleic acid analogue attached at that
binding site. In
one embodiment, the binding sites of the microarray are DNA polynucleotides
corresponding
to at least a portion of each gene in an organism's genome. These DNAs can be
obtained by,
e.g., polymerase chain reaction (PCR) amplification of gene segments from
genomic DNA,
cDNA (e.g., by RT-PCR), or cloned sequences. PCR primers are chosen, based on
the known
sequence of the genes or cDNA, that result in amplification of unique
fragments (i.e.,
fragments that do not share more than bases of contiguous identical sequence
with any other
fragment on the microarray). Computer programs are useful in the design of
primers with the
required specificity and optimal amplification properties. See, e.g., Oligo
version 5.0
(National Biosciences). In the case of binding sites corresponding to very
long genes, it will
sometimes be desirable to amplify segments near the 3' end of the gene so that
when oligo-dT
primed cDNA probes are hybridized to the microarray, less-than-full-length
probes will bind
efficiently. Typically each gene fragment on the microarray will be between
about 50 by and
about 2000 bp, more typically between about 100 by and about 1000 bp, and
usually between
about 300 by and about 800 by in length.
PCR methods are well known and are described, for example, in Innis et al.,
eds.,
1990, PCR Protocols: A Guide to Methods and Applications, Academic Press Inc.,
San
Diego, Calif., which is incorporated herein by reference in its entirety. It
will be apparent that
computer controlled robotic systems are useful for isolating and amplifying
nucleic acids.
An alternative means for generating the nucleic acid for the microarray is by
synthesis
of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or
phosphoramidite chemistries (e.g., Froehler et al., 1986, Nucleic Acid Res
14:5399-5407.).
Synthetic sequences are between about 15 and about 100 bases in length,
preferably between
about 20 and about 50 bases.
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In some embodiments, synthetic nucleic acids include non-natural bases, e.g.,
inosine.
Where the particular base in a given sequence is unknown or is polymorphic,
a'universal
base, such as inosine or 5-nitroindole, may be substituted. Additionally, it
is possible to vary
the charge on the phosphate backbone of the oligonucleotide, for example, by
thiolation or
methylation, or even to use a peptide rather than a phosphate backbone. The
making of such
modifications is within the skill of one trained in the art.
As noted above, nucleic acid analogues may be used as binding sites for
hybridization. An example of a suitable nucleic acid analogue is peptide
nucleic acid (see,
e.g., Egholm et al., 1993, Nature 365:566-568 and U.S. Pat. No. 5,539,083).
In an alternative embodiment, the binding (hybridization) sites are made from
plasmid
or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts
therefrom
(Nguyen et al., 1995, Genomics 29:207-209). In yet another embodiment, the
polynucleotide
of the binding sites is RNA.
ATTACHING NUCLEIC ACIDS TO THE SOLID SURFACE
The nucleic acid or analog is attached to a solid support, which may be made
from
glass, silicon, plastic (e.g., polypropylene, nylon, polyester),
polyacrylamide, nitrocellulose,
cellulose acetate or other materials. In general, non-porous supports, and
glass in particular,
are preferred. The solid support may also be treated in such a way as to
enhance binding of
oligonucleotides thereto, or to reduce non-specific binding of unwanted
substances thereto.
Preferably, the glass support is treated with polylysine or silane to
facilitate attachment of
oligonucleotides to the slide.
Methods of immobilizing DNA on the solid support may include direct touch,
micropipetting (Yershov et al., Proc. Natl. Acad. Sci. USA (1996) 93(10):4913-
4918), or the
use of controlled electric fields to direct a given oligonucleotide to a
specific spot in the array
(U.S. Pat. No. 5,605,662,). DNA is typically immobilized at a density of 100
to 10,000
oligonucleotides per cm2 and preferably at a density of about 1000
oligonucleotides per cm2.
A preferred method for attaching the nucleic acids to a surface is by printing
on glass
plates, as is described generally by Schena et al., 1995, Science 270:467-470.
This method is
especially useful for preparing microarrays of cDNA. See also DeRisi et al.,
1996, Nature
Genetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena et
al., Proc.
Natl. Acad. Sci. USA, 1996, 93(20):10614-19.)
36
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In a preferred alternative to immobilizing pre-fabricated oligonucleotides
onto a solid
support, it is possible to synthesize oligonucleotides directly on the support
(Maskos et al.,
Nucl. Acids Res. 21: 2269-70, 1993; Fodor et al., Science 251: 767-73, 1991;
Lipshutz et al.,
1999, Nat. Genet. 21(1 Suppl):20-4). Among methods of synthesizing
oligonucleotides
directly on a solid support, particularly preferred methods include
photolithography (see
Fodor et al., Science 251: 767-73, 1991; McGall et al., Proc. Natl. Acad. Sci.
(USA) 93:
13555-60, 1996) and piezoelectric printing (Lipshutz et al., 1999, Nat. Genet.
21(1 Suppl):20-
4), with the piezoelectric method being the most preferred.
In one embodiment, a high-density oligonucleotide array is employed.
Techniques are
known for producing arrays containing thousands of oligonucleotides
complementary to
defined sequences, at defined locations on a surface using photolithographic
techniques for
synthesis in situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al.,
1994, Proc. Natl.
Acad. Sci. USA 91:5022-5026; Lockhart et al., 1996, Nature Biotechnol. 14:1675-
80; U.S.
Pat. No. 5,578,832,; U.S. Pat. No. 5,556,752; and U.S. Pat. No. 5,510,270;
each of which is
incorporated by reference in its entirety) or other methods for rapid
synthesis and deposition
of defined oligonucleotides (Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-
4.)
When these methods are used, oligonucleotides (e.g., 20-mers) of known
sequence are
synthesized directly on a surface such as a derivatized glass slide. Usually,
the array produced
contains multiple probes against each target transcript. Oligonucleotide
probes can be chosen
to detect alternatively spliced mRNAs or to serve as various type of control.
Another preferred method of making microarrays is by use of an inkjet printing
process to synthesize oligonucleotides directly on a solid phase, as
described, e.g., in U.S.
Pat. No. 6,419,883, which is incorporated by reference herein in its entirety.
Other methods for making microarrays, e.g., by masking (Maskos and Southern,
1992, Nuc. Acids Res. 20:1679-1684), may also be used. In principal, any type
of array, for
example, dot blots on a nylon hybridization membrane (see Sambrook et al.,
1989, Molecular
Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, Cold
Spring Harbor, N.Y.), could be used, although, as will be recognized by those
of skill in the
art, very small arrays will be preferred because hybridization volumes will be
smaller.
HYBRIDIZATION TO MICROARRAYS
37
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
Nucleic acid hybridization and wash conditions are optimally chosen so
that.the probe
"specifically binds" or "specifically hybridizes" to a specific array site,
i.e., the 'probe
hybridizes, duplexes or binds to a sequence array site with a complementary
nucleic acid
sequence but does not hybridize to a site with a non-complementary nucleic
acid sequence.
As used herein, one polynucleotide sequence is considered complementary to
another when,
if the shorter of the polynucleotides is less than or equal to 25 bases, there
are no mismatches
using standard base-pairing rules or, if the shorter of the polynucleotides is
longer than 25
bases, there is no more than a 5% mismatch. Preferably, the polynucleotides
are perfectly
complementary (i.e. no mismatches). It can easily be demonstrated that
specific hybridization
conditions result in specific hybridization by carrying out a hybridization
assay including
negative controls (see, e.g., Shalon et al., 1996, Genome Research 6:639-645,
and Chee et al.,
1996, Science 274:610-614).
Optimal hybridization conditions will depend on the length (e.g., oligomer
versus
polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of
labeled probe
and immobilized polynucleotide or oligonucleotide. General parameters for
specific (i.e.,
stringent) hybridization conditions for nucleic acids are described in
Sambrook et al. (I989,
Molecular Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring
Harbor
Laboratory, Cold Spring Harbor, N.Y.) and in Ausubel et al. (1987, Current
Protocols in
Molecular Biology, Greene Publishing, Media, Pa., and Wiley-Interscience, New
York).
When the cDNA microarrays of Schena et al. (1996, Proc. Natl. Acad. Sci. USA,
93:10614-
19) are used, typical hybridization conditions are hybridization in 533 SSC
plus 0.2% SDS at
65° C. for 4 hours followed by washes at 25 ° C in low
stringency wash buffer (lxSSC plus
0.2% SDS) followed by 10 minutes at 25 ° C in high stringency wash
buffer (O.IxSSC plus
0.2% SDS) (Schena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19).
Useful
hybridization conditions are also provided in, e.g., Tijssen, 1993,
Hybridization With Nucleic
Acid Probes, Elsevier Science Publishers B.V., Amsterdam and New York, and
Kricka, 1992,
Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Calif.
Although simultaneous hybridization of differentially labeled mRNA samples is
preferred, it is also possible to use a single label and to perform
hybridizations sequentially
rather than simultaneously.
SINGLE DETECTION AND DATA ANALYSIS
38
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
When fluorescently labeled probes are used, the fluorescence emissions at each
site of
a transcript array can preferably be detected by scanning confocal laser
microscopy. In one
embodiment, a separate scan, using the appropriate excitation line, is carried
out for each of
the two fluorophores used. Alternatively, a laser can be used that allows
simultaneous
specimen illumination at wavelengths specific to the two fluorophores and
emissions from
the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996,
Genome
Research 6:639-645, which is incorporated by reference in its entirety for all
purposes). In a
preferred embodiment, the arrays are scanned with a laser fluorescent scanner
with a
computer controlled X-Y stage and a microscope objective. Sequential
excitation of the two
fluorophores is achieved with a mufti-line, mixed gas laser and the emitted
light is split by
wavelength and detected with two photomultiplier tubes. Fluorescence laser
scanning devices
are described in Shalon et al., 1996, Genome Res. 6:639-645 and in other
references cited
herein. Alternatively, the fiber-optic bundle described by Ferguson et al.,
1996, Nature
Biotechnol. 14:1681-1684, may be used to monitor mRNA abundance levels at a
large
number of sites simultaneously.
Signals are recorded and, in a preferred embodiment, analyzed by computer,
e.g.,
using a 12 bit analog to digital board. In one embodiment the scanned image is
despeckled
using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using
an image
gridding program that creates a spreadsheet of the average hybridization at
each wavelength
at each site. If necessary, an experimentally determined correction for "cross
talk" (or
overlap) between the channels for the two fluors may be made. For any
particular
hybridization site on the transcript array, a ratio of the emission of the two
fluorophores can
be calculated. T'he ratio is independent of the absolute expression level of
the cognate gene,
but is useful for genes whose expression is significantly modulated by drug
administration,
gene deletion, or any other tested event.
According to the method of the invention, the relative abundance of an mRNA in
two
biological samples is scored as a perturbation and its magnitude determined
(i.e., the
abundance is different in the two sources of mRNA tested), or as not perturbed
(i.e., the
relative abundance is the same). In various embodiments, a difference between
the two
sources of RNA of at least a factor of about 25% (RNA from one source is 25%
more
abundant in one source than the other source), more usually about 50%, even
more often by a
factor of about 2 (twice as abundant), 3 (three times as abundant) or 5 (five
times as
abundant) is scored as a perturbation.
39
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
Preferably, in addition to identifying a perturbation as positive or negative,
it is
advantageous to determine the magnitude of the perturbation. This can be
carried out, as
noted above, by calculating the ratio of the emission of the two fluorophores
used for
differential labeling, or by analogous methods that will be readily apparent
to those of skill in
the art.
In one embodiment, two samples, each labeled with a different fluor, are
hybridized
simultaneously to permit differential expression measurements. If neither
sample hybridizes
to a given spot in the array, no fluorescence will be seen. If only one
hybridizes to a given
spot, the color of the resulting fluorescence will correspond to that of the
fluor used to label
the hybridizing sample (for example, green if the sample was labeled with Cy3,
or red, if the
sample was labeled with Cy5). If both samples hybridize to the same spot, an
intermediate
color is produced (for example, yellow if the samples were labeled with
fluorescein and
rhodamine). Then, applying methods of pattern recognition and data analysis
known in the
art, it is possible to quantify differences in gene expression between the
samples. Methods of
pattern recognition and data analysis are described in e.g., U.S. Pat. No.
6,203,569 and U.S.
Pat. No. 6,271,002; which are incorporated herein by reference in their
entireties.
FITS FOR THE SYNTHESIS OF NUCLEOTIDE SEQUENCES
An additional aspect provided by the subject invention are kits for carrying
out the
subject methods. In specific embodiments, the kits contain in one or more
containers: (a) a
mixture of first random oligonucleotide primers, each first primer containing
a 3' end
sequence of 10-SO nucleotides, said 3' end sequence having an A, a G, a T, or
a C nucleotide
randomly present in each position of said 3' end sequence; and each first
primer having a 5'
end sequence of 10-50 nucleotides, said 5' end sequence containing an RNA
polymerase
promoter; (b) a mixture of oligo (dT7 primers, each oligo (dT) primer
containing a 3' end
sequence of 10-50 nucleotides, said 3' end sequence having a T nucleotide
present in each
position of said 3' end sequence; and each oligo (dT) primer having a 5' end
sequence of 10-
50 nucleotides, said 5' end sequence containing an RNA polymerase promoter;
and (c) a
mixture of second random oligonucleotide primers, each second primer having a
sequence of
10-50 nucleotides, said sequence containing an A, a G, a T, or a C nucleotide
randomly
present in each position of said sequence. Oligonucleotide primers can be in
any form, e.g.,
lyophilized, or in solution (e.g., a distilled water or buffered solution),
etc. Oligonucleotides
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
for use in the same reaction can be combined in a single container or can be
in separate
containers.
In another embodiment, the kit contains in a separate container an RNA
polymerise
specific to said RNA polymerise promoter. In a another embodiment, the kit
contains and
RNA polymerise for in vitro transcription of double-stranded cDNA. More
preferably, the
RNA polymerise is T7 RNA polymerise, T3 RNA polymerise or SP6 RNA polymerise.
In another embodiment, the kit contains in a separate container a DNA
polymerise.
More preferably, the DNA polymerise is E. coli DNA polymerise I, Klenow
fragment of E.
coli DNA polymerise I, or T4 DNA polymerise.
In another embodiment, the kit contains a buffer for reverse transcription or
first
strand DNA synthesis, a buffer for DNA synthesis, a buffer for in vitro
transcription of
double-stranded cDNA, DNA ligase, an RNase (i.e. RNase H).
In another embodiment, the kit contains deoxyribonucleotide triphosphate
molecules
(dNTPs) and ribonucleotide triphosphate molecules (rNTPs). More preferably the
dNTPs are
dATP, dCTP, dTTP and dGTP and the rNTPs are rATP, rCTP, rUTP and rGTP. In a
preferred embodiment, the dNTPs and rNTPs are labeled, i.e., any label known
in the art and
as described herein. In other preferred embodiments, the label is a
fluorescent, radioactive,
enzymatic, hapten, biotin, or digoxygenin label, more preferably the label is
fluorescent. The
preferred label is a fluorophore, such as fluorescein isothiocyanate,
lissamine, Cy3, CyS, and
rhodamine 110, with Cy3 and Cy5 particularly preferred.
The kit optionally further contains a set of directions for carrying out
reverse
transcription or first strand cDNA synthesis and second strand cDNA synthesis;
as well as a
set of directions for carrying out the in vitro transcription of double-
stranded cDNA into
cRNA. The instructions may be associated with a package insert and/or the
packaging of the
kit or the components thereof.
The kit optionally further contains a control nucleic acid, and/or a
microarray, and/or
means for stimulating and detecting fluorescent light emissions from
fluorescently labeled
RNA, and/or expression profile projection and analysis software capable of
being loaded into
the memory of a computer system.
ANALYTIC KIT IMPLEMENTATION
41
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
In a preferred embodiment, the methods of this invention can be implemented by
use
of kits containing random oligonucleotide primers of the invention containing
an RNA
polymerase promoter and microarrays. The microarrays contained in such kits
contain a solid
phase, e.g., a surface, to which probes are hybridized or bound at a known
location of the
solid phase. Preferably, these probes consist of nucleic acids of known,
different sequence,
with each nucleic acid being capable of hybridizing to an RNA species or to a
cDNA species
derived therefrom. In particular, the probes contained in the kits of this
invention are nucleic
acids capable of hybridizing specifically to nucleic acid sequences derived
from RNA species
which are known to increase or decrease in response to perturbations to the
particular protein
whose activity is determined by the kit. The probes contained in the kits of
this invention
preferably substantially exclude nucleic acids which hybridize to RNA species
that are not
increased in response to perturbations to the particular protein whose
activity is determined
by the kit.
In another preferred embodiment, the kits of the invention further contains
expression
profile projection and analysis software capable of being loaded into the
memory of a
computer system. An example of such a system is described in reference in U.S.
Pat. No.
6,271,002, which is incorporated herein by reference in its entirety.
Preferably, the expression
profile analysis software contained in the kits of this invention, is
essentially identical to the
expression profile analysis software 512 described in reference in U.S. Pat.
No. 6,271,002.
Alternative kits for implementing the analytic methods of this invention will
be
apparent to those of skill in the art and are intended to be comprehended
within the
accompanying claims. In particular, the accompanying claims are intended to
include the
alternative program structures for implementing the methods of this invention
that will be
readily apparent to one of skill in the art.
The invention is further defined by reference to the following examples. It is
understood that the foregoing detailed description and the following examples
are illustrative
only and are not to be taken as limitations upon the scope of the invention.
It will be apparent
to those skilled in the art that many modifications, both to the materials and
methods, may be
practiced without departing from the purpose and interest of the invention.
Further, all
patents, patent applications and publications cited herein are incorporated
herein by
reference.
42
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
EXAMPLES
Example 1.
Labeling Protocol:
1. First Strand cDNA Synthesis
This 40 ~l reaction converts a single-stranded RNA molecule (i.e. mRNA
sequences) to first
strand cDNA from up to 5 pg of mRNA (or up to 30 p,g total RNA). For best
results use only
high quality RNA and nuclease-free reagents and materials. RNA can be isolated
according to
any method well known in the art and as described herein. Primers are random
oligonucleotide primers containing an RNA polymerise promoter as described
herein.
Random primers can be used alone or in combination with an oligo (dT) primer
containing an
RNA polymerise promoter up to 2 pg total primer. Note that the procedure calls
for a Reverse
Transcriptase with no RNase H activity.
1) Add the following in a 1.5 ml micro-centrifuge tube:
Primers (2 p,g) 4 ~,1
Water, nuclease-free x p,1
mRNA (up to 5 p,g) yJal
Final volume 20 p,1
2) Heat at 68 °C for 10 minutes. Chill on ice-water slush. Spin down
briefly, and keep on ice.
3) In a separate tube, mix the following components (i.e. Superscript II
Reverse Transcriptase
and buffer from G1BC0 BRL/Roche). Do not use nuclease inhibitors, as this will
interfere
with the second strand synthesis. 5X First-Strand buffer comprises 250 mM Tris-
HCl (pH
8.3), 375 mM KCl, 15 mM MgCl2. The volume of water depends on the amount of
Superscript II added in Step 6.
5X First-Strand buffer 8 p1
0.1 M DTT 4 p1
mM dNTP mix 2 ~1
Water (nuclease-free) x p1
4) Mix the contents of the tube by gently vortexing, spin briefly and add to
the Primer/RNA
mixture from step 2.
5) Place the tube at 42 °C for 2 minutes.
6) Add Superscript 1I RT (GIBCO BRIJRoche); the amount of Superscript II RT
varies with
the amount of initial RNA (use 200 units of Superscript II RT for each p,g of
mRNA).
Superscript II RT (200 U/p,l) x .mil
43
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
Final volume: 40 w1
7) Incubate at 42 to 50 °C for 1 hour. Higher temperature dissolves
some secondary RNA
structures.
8) Chill on ice to stop reaction and immediately proceed to second strand
synthesis.
2. Second Strand cDNA Synthesis
The yield of the second strand reaction is typically > 80% relative to the
amount of first strand
cDNA synthesized.
1) On ice, add the following reagents to the first strand reaction, in this
order.
For the composition of lOX Second Strand buffer see New England Biolabs
catalog (same as
DNA ligase buffer).
Water (nuclease-free) 86 p1
lOX Second Strand buffer 15 p.1
mM dNTP 3 ~tl
E. coli DNA ligase (10 U/pl)1 p1
E. coli DNA polymerase I 4 p1
(10 U/pl)
E. coli RNase H (2 U/u,l) 1 u1
Final volume 150 p,1
2) Mix gently, spin down briefly, and incubate at 16 °C for 2-3 hours.
3) Without allowing the temperature, to rise, add 4 p,1 of T4 DNA polymerase
(3 U/p,l) and
incubate at 16 °C for 5 minutes.
4) Stop the reaction with 10 p1 of 0.5 M EDTA and proceed with cDNA
purification
immediately.
The RNA is destroyed by adding 30 p1 of 1 M NaOH l 2 mM EDTA. Incubate at 65
°C for
10 minutes. Neutralize with 30 p,1 of 1N HCI, and 30 ~1 of 1 M Tris, pH 7.
3, cDNA Purification
1) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1; pH 8).
~lortex and
centrifuge at room temperature for 2 minutes at full speed.. Carefully recover
the upper
phase and transfer to a clean 1.5 ml microcentrifuge tube.
44
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
2) Extract with an equal volume of chloroform to remove traces of phenol. ,
Transfer upper
phase to a new 1.5 ml microcentrifuge tube.
3) Add 1/10 volume of sodium acetate (3 M, pH 5.2), 1 ~1 of glycogen (10
p,g/pl), and 2.5
volume of cold 100% ethanol. Vortex and leave at -20 °C for at least 1
hour, preferably over
night.
4) Centrifuge at 13K for 60 minutes at 4 °C.
5) Discard the supernatant and add 200 p,1 of cold 70% ethanol to the pellet.
Carefully decant
and discard the supernatant.
6) Air-dry the double-strand cDNA pellet at room temperature for 10 minutes.
7) Resuspend the pellet in 50 p,1 of MilliQ filtered water.
8) Purify the double-strand cDNA using a Micro-Spin column.
9) Lyophilize the flow-through to 7.5 p,1 in a Speedvac, or precipitate
according to Step 10.
10) Adjust the sample to 50 p,1 final volume with nuclease-free water. Add 5
p1 of sodium
acetate (3 M, pH 5.2), 1 p,1 of the supplied Glycogen solution (10 pg/p,l),
and 125 ~tl of ice-
chilled 100% ethanol. Vortex and precipitate at - 20 °C for at least 1
hour, preferably over
night.
Centrifuge at 13K for 60 minutes at 4 °C. Discard the supernatant and
gently add 200 ~tl of
ice-chilled 70% ethanol (diluted in nuclease-free water) to the pellet.
Gently discard the supernatant and completely air-dry the double-strand cDNA
pellet at room
temperature for 10 minutes in a clean and dust-free environment.
Resuspend pellet in 7.5 p,1 of nuclease-free water by pipeting up and down
several times.
11) Leave the sample on ice and proceed with the transcription procedure, or
keep samples at -80
°C for long-term storage.
4. In vitro Transcription
This protocol produces biotin-labeled cRNA probes from the double strand cDNA
template
synthesized above. One can use a high yield transcription kit such as T7
MEGAscript~ from
Ambion Inc. (cat. #1334). Please see the instructions of the manufacturer.
Biotin-labeling is
meant to be illustrative and not to limit the invention. The procedure can be
modified by one
of ordinary skill in the art to introduce any label as described herein.
Furthermore, it may be
necessary to introduce the double-stranded cDNA into a vector suitable for in
vitro
CA 02450215 2003-12-09
WO 03/016483 PCT/US02/26063
transcription. This can be accomplished by any suitable method and any
suitable vector well
known to the skilled artisan or as described herein.
1) Add the following MEGAscript~ reagents to the ds-DNA sample from Step 11.
Proceed at
room temperature to avoid precipitation of DNA with the spermidine in
Transcription
Buffer. Keep T7 Enzyme Mix on ice before adding to translation mixture:
Final Concentration
Water (nuclease-free) _ p,1
lOX Transcription Buffer3.0 1..t,1
rATP (75 mM) 3.0 p1 7.5 mM
rGTP (75 mM) , 3.0 p1 7.5 mM
rUTP (75 mM) 3.0 p1 7.5 mM
rCTP (15 mM) 3.0 1.t1 1.5 mM
Biotin-rCTP (10 mM) 4.5 p1 1.5 mM
T7 Enzyme Mix 3 O~.t,l
Total volume: 30 p,1
2) Incubate for 4 hours at 37 °C.
3) Adjust the volume of the transcription mixture to 40 p,1 final volume using
water.
4) Purify by using one of the Micro-Spin columns (provided).
5) Adjust flow-through to 50 j..tl final volume with nuclease-free water. Keep
probe at -80°C,
unless used immediately for hybridization.
6) The final yield with Ambion's MEGAscript~ kit is typically 25-35 wg of cRNA
transcript
per 1 p,g of mRNA.
OTFIER EMBODIMENTS
Although particular embodiments have been disclosed herein in detail, this has
been
done by way of example for purposes of illustration only, and is not intended
to be limiting
with respect to the scope of the appended claims, which follow. In particular,
it is
contemplated by the inventors that various substitutions, alterations, and
modifications may
be made to the invention without departing from the spirit and scope of the
invention as
defined by the claims. Other aspects, advantages, and modifications are
considered to be
within the scope of the following claims.
46
