U.S. patent application number 13/372320 was filed with the patent office on 2012-08-16 for random-primed transcriptase in-vitro transcription method for rna amplification.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Colleen P. Davis, Michael ZIMAN.
Application Number | 20120208199 13/372320 |
Document ID | / |
Family ID | 22961105 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120208199 |
Kind Code |
A1 |
ZIMAN; Michael ; et
al. |
August 16, 2012 |
RANDOM-PRIMED TRANSCRIPTASE IN-VITRO TRANSCRIPTION METHOD FOR RNA
AMPLIFICATION
Abstract
A random-primed reverse transcriptase-in vitro transcription
method of linearly amplifying RNA is provided. According to the
methods of the invention, source RNA (or other single-stranded
nucleic acid), preferably, mRNA, is converted to double-stranded
cDNA using two random primers, one of which comprises a RNA
polymerase promoter sequence ("promoter-primer"), to yield a
double-stranded cDNA that comprises a RNA polymerase promoter that
is recognized by a RNA polymerase. Preferably, the primer for
first-strand cDNA synthesis is a promoter-primer and the primer for
second-strand cDNA synthesis is not a promoter-primer. The
double-stranded cDNA is then transcribed into RNA by the RNA
polymerase, optimally in the presence of a reverse transcriptase
that is rendered incapable of RNA-dependent DNA polymerase activity
during this transcription step. The subject methods produce
linearly amplified RNA with little or no 3' bias in the sequences
of the nucleic acid population amplified.
Inventors: |
ZIMAN; Michael; (San
Francisco, CA) ; Davis; Colleen P.; (Seattle,
WA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
22961105 |
Appl. No.: |
13/372320 |
Filed: |
February 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12790234 |
May 28, 2010 |
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13372320 |
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11745386 |
May 7, 2007 |
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12790234 |
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10432176 |
Nov 13, 2003 |
7229765 |
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PCT/US01/44821 |
Nov 28, 2001 |
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11745386 |
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60253641 |
Nov 28, 2000 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12N 15/1096 20130101;
C12Q 1/6865 20130101; C12Q 2525/179 20130101; C12Q 2525/143
20130101; C12Q 1/6865 20130101; C12Q 2521/119 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A kit for use in amplifying one or more single stranded nucleic
acids, said kit comprising in one or more containers: (i) a first
set of oligonucleotides, each of said oligonucleotides in said
first set comprising a promoter sequence operably linked to a
random sequence at least 4 nucleotides in length from a set of
random sequences of at least 4 nucleotides; and (ii) a second set
of oligonucleotides, each of said oligonucleotides in said second
set comprising a random sequence of at least 4 nucleotides in
length from a set of random sequences of at least 4
nucleotides.
2. The kit of claim 1, which further comprises a reverse
transcriptase.
3. The kit of claim 1, which further comprises an RNA polymerase
that recognizes said promoter sequence.
4. The kit of claim 1, wherein the random sequences of the
oligonucleotides in said first set of oligonucleotides are 6 to 9
nucleotides in length.
5. The kit of claim 1, wherein the random sequences of the
oligonucleotides in said second set of oligonucleotides are 6 to 9
nucleotides in length.
6. The kit of claim 1, wherein the random sequences of the
oligonucleotides in said first set of oligonucleotides are 6
nucleotides in length.
7. The kit of claim 1, wherein the random sequences of the
oligonucleotides in said second set of oligonucleotides are 6
nucleotides in length.
8. The kit of claim 1, wherein the oligonucleotides in said second
set of oligonucleotides do not comprise a promoter sequence.
9. The kit of claim 1, wherein each oligonucleotide in said second
set of oligonucleotides consists of a random sequence of at least 4
nucleotides in length from the set of random sequences of at least
4 nucleotides.
10. The kit of claim 1, which further comprises a third set of
oligonucleotides, each of said oligonucleotides in said third set
comprising the promoter sequence operably linked to a polydT
sequence of at least 5 nucleotides.
11. The kit of claim 10, wherein said polydT sequence is 5 to 25
nucleotides.
12. The kit of claim 11, wherein said polydT sequence is 18
nucleotides.
13. The kit of claim 3, wherein said promoter sequence is a T7
promoter sequence, and said RNA polymerase is T7 RNA
polymerase.
14. The kit of claim 1, which further comprises a reverse
transcriptase inhibitor.
15. The kit of claim 14, wherein the reverse transcriptase
inhibitor comprises ddNTP.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of,
and claims priority to, U.S. application Ser. No. 12/790,234 filed
on May 28, 2010, which application claims priority to U.S.
application Ser. No. 11/745,386 filed on May 7, 2007, which
application claims priority to U.S. application Ser. No. 10/432,176
filed on Nov. 13, 2003, now U.S. Pat. No. 7,229,765, which is a
.sctn.371 filing of International Application No. PCT/US01/44821
filed on Nov. 28, 2001, which application claims the benefit of
U.S. Provisional Patent Application Ser. No. 60/253,641 filed on
Nov. 28, 2000. Said applications are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to enzymatic amplification of
nucleic acids using two random primers, one of which contains a RNA
polymerase promoter sequence, to generate a double stranded DNA
template, and in vitro transcription.
BACKGROUND OF THE INVENTION
[0003] The characterization of cellular gene expression finds
application in a variety of disciplines, such as in the analysis of
differential expression between different tissue types, different
stages of cellular growth or between normal and diseased states.
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. One particularly useful method of
assaying gene 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).
[0004] A number of methods for the amplification of nucleic acids
have been described. Such methods include the "polymerase chain
reaction" (PCR) (Mullis et al., U.S. Pat. No. 4,683,195), and a
number of transcription-based amplification methods (Malek et al.,
U.S. Pat. No. 5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491;
Burg et al., U.S. Pat. No. 5,437,990). Each of these methods uses
primer-dependent nucleic acid synthesis to generate a DNA or RNA
product, which serves as a template for subsequent rounds of
primer-dependent nucleic acid synthesis. Each process uses (at
least) two primer sequences complementary to different strands of a
desired nucleic acid sequence and results in an exponential
increase in the number of copies of the target sequence. These
amplification methods can provide enormous amplification (up to
billion-fold). However, these methods have limitations that make
them not amenable for gene expression monitoring applications.
First, each process results in the specific amplification of only
the sequences that are bounded by the primer binding sites. Second,
exponential amplification can introduce significant changes in the
relative amounts of specific target species-small differences in
the yields of specific products (for example, due to differences in
primer binding efficiencies or enzyme processivity) become
amplified with every subsequent round of synthesis.
[0005] Amplification methods that utilize a primer containing a RNA
polymerase promoter sequence ("promoter-primer") are amenable to
the amplification of heterogeneous mRNA populations. The vast
majority of mRNAs carry a homopolymer of 20-250 adenosine residues
on their 3' ends (the poly-A tail), and the use of poly-dT primers
for cDNA synthesis is a fundamental tool of molecular biology.
"Single-primer amplification" protocols have been reported (see
e.g., Kacian et al., U.S. Pat. No. 5,554,516; Van Gelder et al.,
U.S. Pat. No. 5,716,785). The methods reported in these patents
utilize a single promoter-primer containing a RNA polymerase
promoter sequence and a sequence complementary to the 3'-end of the
desired nucleic acid target sequence(s). In both methods, the
promoter-primer is added under conditions in which it hybridizes to
the target sequence(s) and is converted to a substrate for RNA
polymerase. In both methods, the substrate intermediate is
recognized by RNA polymerase, which produces multiple copies of RNA
complementary to the target sequence(s) ("antisense RNA"). Each
method uses, or could be adapted to use, a primer containing
poly-dT for amplification of heterogeneous mRNA populations.
[0006] Amplification methods that proceed linearly during the
course of the amplification reaction are less likely to introduce
bias in the relative levels of different mRNAs than those that
proceed exponentially. In the method described in Kacian et al.,
U.S. Pat. No. 5,554,516, the amplification reaction contains a
nucleic acid target sequence, a promoter-primer, a RNA polymerase,
a reverse transcriptase, and reagent and buffer conditions
sufficient to allow amplification. The amplification proceeds in a
single tube under conditions of constant temperature and ionic
strength. Under these conditions, the antisense RNA products of the
reaction can serve as substrates for further amplification by
non-specific priming and extension by the RNA-dependent DNA
polymerase activity of reverse transcriptase. As such, the
amplification described in U.S. Pat. No. 5,554,516 proceeds
exponentially. In contrast, in specific examples described in Van
Gelder et al., U.S. Pat. No. 5,716,785, cDNA synthesis and
transcription occur in separation reactions separated by
phenol/chloroform extraction and ethanol precipitation (or
dialysis), which may incidentally allow for the amplification to
proceed linearly since the RNA products cannot serve as substrates
for further amplification.
[0007] The method described in U.S. Pat. No. 5,716,785 has been
used to amplify cellular mRNA for gene expression monitoring (for
example, R. N. Van Gelder et al. (1990), Proc. Natl. Acad. Sci. USA
87, 1663; D. J. Lockhart et al. (1996), Nature Biotechnol. 14,
1675). However, this procedure is not readily amenable to high
throughput processing. In preferred embodiments of the method
described in U.S. Pat. No. 5,716,785, poly-A mRNA is primed with a
promoter-primer containing poly-dT and converted into
double-stranded cDNA using a method described by Gubler and Hoffman
(U. Gubler and B. J. Hoffman (1983), Gene 25, 263-269) and
popularized by commercially available kits for cDNA synthesis.
Using this method for cDNA synthesis, first strand synthesis is
performed using reverse transcriptase and second strand cDNA is
synthesized using RNaseH and DNA polymerase I. After
phenol/chloroform extraction and dialysis, double-stranded cDNA is
transcribed by RNA polymerase to yield antisense RNA product. The
phenol/chloroform extractions and buffer exchanges required in this
procedure are labor intensive, and are not readily amenable to
robotic handling.
[0008] A method of linear amplification of mRNA into antisense RNA
has been recently developed, U.S. Pat. No. 6,132,997 issued to
Shannon ("Shannon"), which is incorporated by reference in its
entirety for all purposes. Shannon does not require a reverse
transcriptase separation step and is therefore readily amenable to
high throughput processing. Shannon discloses a method in which
mRNA is converted to cDNA (particularly double-stranded cDNA) using
a promoter-primer having a poly-dT primer site linked to a promoter
sequence so that the resulting cDNA is recognized by a RNA
polymerase. The resultant cDNA is then transcribed into RNA
(particularly antisense RNA) in the presence of a reverse
transcriptase that is rendered incapable of RNA-dependent DNA
polymerase activity during the transcription step.
[0009] A significant drawback of the Shannon method, however, is
that it produces a 3' bias in the amplification of mRNA. Sequences
that are more than 1000 bp from the 3' end to which the primer has
hybridized are underamplified with respect to sequences that are
less than 1000 bp from the 3' end, i.e., the sequences that are
more than 1000 bp from the 3' end are amplified in less than linear
amounts.
[0010] Thus there exists a need in the art for an improved method
of linear amplification of mRNA that is amenable to high throughput
processing, that produces little or no 3' bias, that improves the
ability to detect the 5' ends of mRNA, and therefore achieves good
representation of both the 3' and 5' regions of an original mRNA in
the amplified complementary RNA (cRNA).
SUMMARY OF THE INVENTION
[0011] A random-primed reverse transcriptase-in vitro transcription
method of linearly amplifying RNA is provided. According to the
methods of the invention, source RNA (or other single-stranded
nucleic acid), preferably, mRNA, is converted to double-stranded
cDNA using two random primers, one of which comprises a RNA
polymerase promoter sequence ("promoter-primer"), to yield a
double-stranded cDNA that comprises a RNA polymerase promoter that
is recognized by a RNA polymerase. Preferably, the primer for
first-strand cDNA synthesis is a promoter-primer and the primer for
second-strand cDNA synthesis is not a promoter-primer. The
double-stranded cDNA is then transcribed into RNA by the RNA
polymerase, optimally in the presence of a reverse transcriptase
that is rendered incapable of RNA-dependent DNA polymerase activity
during this transcription step. The subject methods of producing
linearly amplified RNA provide an improvement over prior methods in
that little or no 3' bias in the sequences of the nucleic acid
population amplified is produced, and the ability to detect the 5'
end sequences of the nucleic acids is improved. The methods of the
invention therefore achieve good representation of both the 3' and
5' regions of the source nucleic acid in the amplified
complementary RNA (cRNA). Linear amplification extents of at least
100-fold can be achieved using the subject methods. All of the
benefits of linear amplification are achieved with the subject
methods, such as the production of unbiased antisense RNA libraries
from heterogeneous mRNA mixtures.
[0012] In particular, the invention provides a method for linearly
amplifying one or more single stranded nucleic acids, said method
comprising (a) contacting said one or more single stranded nucleic
acids with a first set of oligonucleotides, each of which comprises
a promoter sequence and a sequence from a set of random sequences
of at least 4 nucleotides (but preferably 6 to 9 nucleotides, more
preferably 9 nucleotides), a second set of oligonucleotides, each
of which comprises (preferably, consists of) of one or a set of
random sequences of at least 4 nucleotides (but preferably 6 to 9
nucleotides, more preferably 6 nucleotides) and one or more enzymes
that alone or in combination catalyze the synthesis of
double-stranded cDNA, under conditions suitable for the production
of double-stranded cDNA; and (b) contacting the double-stranded
cDNA produced in step (a) with a RNA polymerase that recognizes
said promoter sequence and ribonucleotides under conditions
suitable to effect transcription, thereby producing sense or
antisense RNA copies corresponding to said one or more single
stranded nucleic acids. In a preferred embodiment, the second set
of oligonucleotides does not contain a promoter sequence.
Alternatively, the cDNA may be generated in two steps where the
first step is the synthesis of first strand cDNA using the first
set of oligonucleotides and one or more enzymes that catalyze first
strand cDNA synthesis and the second step is the synthesis of
double-stranded cDNA by contacting the first strand cDNA made in
the first step with the second set of oligonucleotides and one or
more enzymes that alone or in combination catalyze second strand
cDNA synthesis. In preferred embodiments, the enzyme used in step
(a) is a reverse transcriptase. In an alternative embodiment, the
single-stranded nucleic acid is also contacted in step (a) with a
promoter-primer containing the same promoter sequence used in the
set of random primer-promoter primers used in step (a) and a polydT
sequence of at least 4 nucleotides (preferably at least 5
nucleotides, more preferably 15 to 25 nucleotides, and most
preferably 18 nucleotides).
[0013] The invention further provides kits for carrying out the
linear amplification methods of the invention containing one or
more components used in the methods of the inventions and
instructions for use. In a particular embodiment, the invention
provides a kit for use in linearly amplifying single stranded
nucleic acids into sense or antisense RNA, said kit comprising a
first set of oligonucleotides each comprising a promoter sequence
and one of a set of random sequences of at least 4 nucleotides; and
a second set of oligonucleotides each of which comprises
(preferably, consists of) of one of a set of random sequences of at
least four nucleotides. In a preferred embodiment the second set of
oligonucleotides does not contain a promoter sequence[.?] In
another embodiment, the kit also contains a reverse transcriptase
and a RNA polymerase. In yet another embodiment, the kit further
contains, in addition to the two sets of random primers,
oligonucleotides containing the same promoter sequence as the
random primer-promoter primer oligonucleotide and a polydT sequence
of at least 5 nucleotides (preferably 18 nucleotides).
DESCRIPTION OF THE FIGURES
[0014] FIGS. 1(A-B). Comparison of profiles obtained from
single-gene analysis using (A) the mRNA amplification method
described in U.S. Pat. No. 6,132,997 (Shannon, issued Oct. 17,
2000) ("Shannon") and (B) the random-primed reverse
transcriptase-in vitro transcription (RT-IVT) method of the
invention. The graphs plot signal intensity (mlavg) of
oligonucleotides in a single gene (X-axis) as a function of the
number of base pairs from the 5' end (Y-axis). The 3' bias of
signal intensity seen when the Shannon method is used cannot be
seen when the random-primed RT-IVT method is used, indicating that
the random-primed RT-IVT method overcomes the 3' bias of the
Shannon method.
[0015] FIG. 2. Intensity difference as a function of distance from
the 3' end. The graph shows the intensity of all oligonucleotides
as a function of distance from the 3' end. The graph plots mlavg
(Shannon method)-mlavg (random-primed RT-IVT method) (X-axis)
versus log.sub.10 of the number of by from the 3' end (Y-axis). The
intensity obtained with the Shannon method is greater than the
intensity obtained with the random-primed RT-IVT method for probes
less than 1000 bp from the 3' end of the message. The intensity
obtained with the Shannon method is less than the intensity
obtained with the random-primed RT-IVT method for probes greater
than 1000 bp from the 3' end of the message.
[0016] FIGS. 3(A-C). Signature differences in the numbers and
percentages of significant data points. The top graph (A) plots the
number of probes (X-axis) versus the log.sub.10 (bp) (Y-axis). The
middle graph (B) plots the number of signatures (X-axis) versus the
log.sub.10 (bp) (Y-axis). The bottom graph (C) plots the fraction
of signatures versus the log.sub.10 (bp) (Y-axis). As can be seen
in the bottom graph, the random-primed RT-IVT method outcompetes
the Shannon method for probes greater than 1000 bp from the 3' end.
Note the black arrow at approximately 700 bp where random-primed
RT-IVT method is more representative than the Shannon method.
Stars: Shannon method. Circles: random-primed RT-IVT method.
[0017] FIGS. 4(A-C). Shows the results obtained when the
amplification methods of the invention were run using a primer
comprising a T7 RNA polymerase promoter site and an poly-dT.sub.18
sequence ("T7-dT.sub.18"), in addition to using random T7-dN.sub.9
and dN.sub.6 primers. The top graph (A) plots the number of probes
(X-axis) versus the log.sub.10, (bp) (Y-axis). The middle graph (B)
plots the number of signatures (X-axis) versus the log.sub.10 (bp)
(Y-axis). The bottom graph (C) plots the fraction ("frac") or
percentage of signatures versus the log.sub.10 (bp) (Y-axis). As
can be seen in the bottom graph, the number of probes at greater
than 1000 base pairs is greater with the random-primed RT-IVT
method. Using both the T7-dT.sub.18 and random T7-dN.sub.9 primers
for first strand cDNA synthesis improves the fraction of
statistically significant probes more efficiently than either the
Shannon method or the method of the invention in which just the
random T7-dN.sub.9 primer is used. Stars: Shannon method. Circles:
random-primed RT-IVT method.
DETAILED DESCRIPTION OF THE INVENTION
[0018] A random-primed reverse transcriptase-in vitro transcription
(RT-IVT) method of linearly amplifying RNA is provided. According
to the methods of the invention, source RNA (or other
single-stranded nucleic acid), preferably, mRNA, is converted to
double-stranded cDNA using two random primers, one of which
comprises a RNA polymerase promoter sequence ("promoter-primer"),
to yield a double-stranded cDNA that comprises a RNA polymerase
promoter that is recognized by a RNA polymerase. Thus, "promoter
sequence" refers to a single-stranded nucleotide sequence that when
double-stranded (i.e., paired with its reverse-complement) forms a
RNA polymerase promoter that is recognized by a RNA polymerase.
Preferably, the primer for first-strand cDNA synthesis is a
promoter-primer and the primer for second-strand cDNA synthesis is
not a promoter-primer. Optionally, the cDNA synthesis reaction
contains a mixture of the random-sequence-promoter primer and an
oligonucleotide containing the promoter sequence and an oligodT
sequence. The double-stranded cDNA is then transcribed into RNA by
the RNA polymerase, optimally in the presence of a reverse
transcriptase that is rendered incapable of RNA-dependent DNA
polymerase activity during this transcription step.
[0019] The subject methods of producing linearly amplified RNA
provide an improvement over prior methods in that little or no 3'
bias in the sequences of the nucleic acid population amplified is
produced, and the ability to detect the 5' end sequences of the
nucleic acids is improved. The methods of the invention therefore
achieve good representation of both the 3' and 5' regions of the
source nucleic acid in the amplified complementary RNA (cRNA).
Linear amplification extents of at least 100-fold can be achieved
using the subject methods. All of the benefits of linear
amplification are achieved with the subject methods, such as the
production of unbiased antisense RNA libraries from heterogeneous
mRNA mixtures.
[0020] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections set forth below.
[0021] 5.1. Methods of Nucleic Acid Amplification
[0022] The invention provides methods for producing amplified
amounts of either sense or antisense RNA from an initial amount of
source single-stranded nucleic acid, preferably poly-A+ RNA or
mRNA. By amplified amounts is meant that for each initial source of
nucleic acid, multiple corresponding sense or antisense RNAs are
produced. The term antisense RNA is defined here as RNA
complementary to the source single-stranded nucleic acid. By
corresponding is meant that the sense or antisense RNA shares a
substantial sequence identity with the sequence of, or the sequence
complementary to (i.e., the complement of the initial source
nucleic acid), the source nucleic acid. Substantial sequence
identity means at least 95%, usually at least 98%, and more usually
at least 99%, and, in certain embodiments, 100% sequence identity,
where sequence identity is determined Using the BLAST algorithm, as
described in Altschul et al. (1990), J. Mol. Biol. 215:403-410
(using the published default setting, i.e., parameters w=4, t=17).
Generally, the number of corresponding antisense RNA molecules
produced for each initial nucleic acid during the subject linear
amplification methods will be at least about 10, usually at least
about 50, more usually at least about 100, and may be as great as
600 or greater, but often does not exceed about 1000.
[0023] The subject methods can be used to produce amplified amounts
of RNA corresponding to substantially all of the nucleic acid
present in the initial sample, or to a proportion or fraction of
the total number of distinct nucleic acids present in the initial
sample. By substantially all of the nucleic acid present in the
sample is meant more than 90%, usually more than 95%, where that
portion not amplified is solely the result of inefficiencies of the
reaction and not intentionally excluded from amplification.
[0024] In a specific embodiment, only a single cycle of reverse
transcription is carried out. In alternative embodiments, more than
one cycle of reverse transcription is performed (with transcription
and denaturation between cycles). For example, in a specific
embodiment, a first cycle of reverse transcription is carried out
wherein one or more single stranded nucleic acids are (a) contacted
with a first set of oligonucleotides, each of which comprises a
promoter sequence and a sequence from a set of random sequences of
at least 4 nucleotides (but preferably 6 to 9 nucleotides, more
preferably 9 nucleotides), a second set of oligonucleotides, each
of which comprises (preferably, consists of) of one or a set of
random sequences of at least 4 nucleotides (but preferably 6 to 9
nucleotides, more preferably 6 nucleotides) and one or more enzymes
that alone or in combination catalyze the synthesis of
double-stranded cDNA, under conditions suitable for the production
of double-stranded cDNA. The resultant double-stranded cDNA is then
(b) contacted with a RNA polymerase that recognizes said promoter
sequence and ribonucleotides under conditions suitable to effect
transcription (i.e., in vitro transcription or "IVT"), thereby
producing sense or antisense RNA copies corresponding to said one
or more single stranded nucleic acids. The resultant sense or
antisense RNA copies are then reverse transcribed in a second cycle
of reverse transcription, as described in step (a) above, and the
resultant double-stranded cDNA is then transcribed via IVT into
sense or antisense RNA copies as described in step (b) above.
Additional cycles of RT-IVT may be performed to obtain the desired
quantity of sense or antisense RNA copies.
[0025] According to the methods of the invention, additional linear
amplification is afforded by a subsequent in vitro transcription
(IVT) step as described below in Section 5.2. During IVT, the
double-stranded cDNA produced in the first step is transcribed by
RNA polymerase to yield RNA that is complementary to the initial
RNA target from which it is amplified. This combination of cDNA
synthesis and IVT enables the generation of a relatively large
amount of cRNA from a very small starting amount of nucleic acid
without loss of fidelity, and particularly, without 3'
amplification bias.
[0026] In one embodiment of the invention (see Example 1, Section
6), 0.2 .mu.g (200 ng) of source mRNA is used.
[0027] In another embodiment of the invention, nucleic acid
amplification is performed in situ, on samples of preserved or
fresh cells or tissues (see, e.g., Nuovo, 1997, PCR In situ
Hybridization: Protocols and Applications, Third Edition,
Lippincott-Raven Press, New York).
[0028] The subject methods may be applied to other amplification
systems in which an oligonucleotide is incorporated into an
amplification product such as polymerase chain reaction (PCR)
systems (U.S. Pat. No. 4,683,195, Mullis et al., entitled "Process
for amplifying, detecting, and/or-cloning nucleic acid sequences,"
issued Jul. 28, 1987; U.S. Pat. No. 4,683,202, Mullis, entitled
"Process for amplifying nucleic acid sequences," issued Jul. 28,
1987).
[0029] 5.1.1. cDNA Synthesis
[0030] Double-stranded cDNA molecules can be synthesized from a
collection of RNAs (or other single-stranded nucleic acids), e.g.,
mRNAs present in a population of cells, by methods well-known in
the art. In order for the cDNAs produced in this step to be useful
in the methods of the invention, it is necessary to incorporate a
RNA polymerase promoter into the cDNA molecules during synthesis.
This enables the cDNA molecules to serve as templates for RNA
transcription. This is accomplished by choosing one or more primers
for the cDNA synthesis reaction that comprise a single-stranded,
synthetic oligonucleotide containing a RNA polymerase promoter
sequence in sense orientation. This "promoter-primer" may be used
to prime either first strand and/or second strand cDNA synthesis.
In preferred embodiments, the "promoter-primer" primes first strand
cDNA synthesis and the promoter is in the appropriate orientation
to promote synthesis of antisense RNA.
[0031] Typically, only one RNA polymerase promoter
sequence-containing primer is used during cDNA synthesis.
Preferably, the promoter-primer is used to prime first strand cDNA
synthesis. Following reverse transcription, the resultant RNA
polymerase promoter-containing double-stranded cDNA is transcribed
into RNA using a RNA polymerase capable of binding to the RNA
polymerase promoter introduced during cDNA synthesis (see below
Section 5.2).
[0032] In a preferred embodiment, the primer for first strand cDNA
synthesis is a mixture of random primers linked to a promoter
sequence that prime synthesis in a direction toward the 5' end of
the nucleic acids (e.g., mRNAs) in the sample, and the primer for
second strand cDNA synthesis is a mixture of random primers that
prime synthesis of double-stranded cDNA from substantially all the
first strand cDNAs thus produced.
[0033] Preferably, the first-strand primer is a random
promoter-primer, wherein the random (poly-dN) sequence is operably
linked to a RNA polymerase promoter sequence. In one aspect, the
first-strand primer is a mixture of primers, each primer comprising
a RNA polymerase promoter sequence and a 3' end or 3' distal
sequence of 6-9 nucleotides, preferably 9 nucleotides.
[0034] The mixture of primers comprises random primers, i.e.,
primers having an A, a G, a C, or a T residue present in each
position of the 3' end sequence or 3' distal sequence (i.e., the
non-promoter sequence). In particular, the random primer for
priming first strand cDNA synthesis is a random promoter-primer
that includes: (a) a poly-dN region for hybridization to the mRNA;
and (b) a RNA polymerase promoter region 5' of the poly-dN region
that is in an orientation capable of directing transcription of
antisense RNA when it primes first strand cDNA synthesis. The
poly-dN region is sufficiently long to provide for efficient
hybridization to the mRNA, where the region typically ranges in
length from 4-50 nucleotides in length, preferably 6-25 nucleotides
in length, more preferably from 6-12, and most preferably, 9
nucleotides in length, i.e., a random 9-mer. In specific
embodiments, the poly-dN region is 4, 5, 6, 7, 8, 9, 10, 11, or 12
nucleotides in length.
[0035] In a preferred embodiment, the random promoter-primer used
to prime first strand cDNA synthesis is a random 9-mer operably
linked to a T7 RNA polymerase promoter sequence (T7-dN.sub.9: (5')
AAT TAA TAC GAC TCA CTA TAG GGA GAT NNN NNN NNN (3') (N=A, T, C or
G) (SEQ ID NO.: 1)).
[0036] In another embodiment, the random promoter primers used to
prime first strand cDNA synthesis are a complete set of all (or
almost all) combinations of random 9-mers, i.e., a total of 4.sup.9
9-mers, linked to a T7 RNA polymerase promoter sequence.
[0037] In another embodiment, a poly-dT primer comprising a RNA
polymerase promoter sequence and a random dN primer comprising a
RNA polymerase promoter sequence are used together to prime first
strand cDNA synthesis. Preferably, the poly-dT-promoter primer and
the random primer-promoter primer contain the same promoter
sequence. In particular embodiments the poly-dT sequence is at
least 5 thymidilate residues, preferably 15 to 25 residues and,
preferably 18 residues. In a preferred embodiment, a T7-dT.sub.18
primer and a T7-dN.sub.9 primer are used to prime first strand cDNA
synthesis.
[0038] A number of RNA polymerase promoters may be used for the
promoter region of the promoter-primer. Suitable promoter regions
will be capable of initiating transcription from an operably linked
DNA sequence in the presence of ribonucleotides and a RNA
polymerase under suitable conditions. The terms "operably linked"
refers to a functional linkage, i.e., the promoter will be linked
in an orientation to permit transcription of sense or antisense
RNA. Preferably the linkage is covalent, most preferably by a
nucleotide bond. Most preferably, the promoter is linked in an
orientation to permit transcription of antisense RNA when the
promoter is incorporated into the first strand of cDNA synthesis. A
linker oligonucleotide between the promoter and the DNA may be
present, and if,[<cut","?] present, will typically comprise
between about 5 and 20 bases, but may be smaller or larger as
desired. The promoter region is of sufficient length to promote
transcription, and will usually comprise between about 15 and 250
nucleotides, preferably between about 17 and 60 nucleotides, from a
naturally occurring RNA polymerase promoter or a consensus promoter
region, as described in Alberts et al. (1989) in Molecular Biology
of the Cell, 2d Ed. (Garland Publishing, Inc.), or any other
variant that promotes transcription. In a specific embodiment, the
promoter region is 36 nucleotides. Preferred promoter regions
include the bacteriophage SP6 and T3 promoters and, most
preferably, T7 promoters.
[0039] The random promoter-primer and/or the random primer may
additionally contain a restriction site, in the middle or at the 5'
distal end of the primer, but preferably not immediately at the 5'
terminus. The restriction site may be used for cloning in to a
vector. Restriction enzymes and the sites they recognize can be
found, for example, in Sambrook et al., 1989, Molecular Cloning--A
Laboratory Manual (2nd Ed.), Vol. 1, Chapter 5, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
[0040] The primers of the invention may be prepared using any
suitable method known in the art, e.g., as described in Section 5.3
infra.
[0041] Preferably both first- and second-strand cDNA synthesis is
produced by reverse transcription, wherein DNA is made from RNA
using the enzyme reverse transcriptase. Reverse transcriptase is
found in all retroviruses and is commonly obtained from avian
myeloblastoma virus or Moloney murine leukemia virus; enzyme from
these sources is commercially available from Life Technologies
(Gaithersburg, Md.) and Boehringer Mannheim (Indianapolis,
Ind.).
[0042] The catalytic activities required to convert the
promoter-primer-mRNA hybrid to double-stranded cDNA are a
RNA-dependent DNA polymerase activity, a RNaseH activity, and a
DNA-dependent DNA polymerase activity. Most reverse transcriptases,
including those derived from Moloney murine leukemia virus
(MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemia
virus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiency
virus (HIV-RT) catalyze each of these activities. These reverse
transcriptases are sufficient to convert a primer-mRNA hybrid to
double-stranded DNA in the presence of additional reagents that
include, but are not limited to: dNTPs; monovalent and divalent
cations, e.g., KCl, MgCl.sub.2; sulfhydryl reagents, e.g.,
dithiothreitol; and buffering agents, e.g., Tris-Cl. Alternatively,
a variety of proteins that catalyze one or two of these activities
can be added to the cDNA synthesis reaction. For example, MMLV
reverse transcriptase lacking RNaseH activity (described in U.S.
Pat. No. 5,405,776) catalyzes RNA-dependent DNA polymerase activity
and DNA-dependent DNA polymerase activity. These proteins may be
added together during a single reaction step, or added sequentially
during two or more substeps. Preferably, MMLV is used for both
first- and second-strand cDNA synthesis. As described above,
preferably the reverse transcriptase is inactivated prior to or
inhibited during the transcription step of the method.
[0043] In general, it is preferable for the RNA-containing sample
to contain purified poly-A+RNA (mRNA). In one embodiment, a random
promoter-primer is hybridized with an initial mRNA (poly-A.sup.+
RNA) sample. The promoter-primer is contacted with the mRNA under
conditions that allow the poly-dN site to hybridize to the mRNA.
The random promoter-primer-mRNA hybrid is then converted to a
double-stranded cDNA product that is recognized by a RNA
polymerase.
[0044] In a preferred embodiment, first-strand cDNA synthesis is
allowed to proceed at a lower temperature (for example, 25.degree.
C.) for a certain period of time (e.g., 10 min) prior to increasing
the temperature (e.g., to 40.degree. C.) for the remainder of the
reverse transcription reaction, which improves annealing of the
first primer (e.g., the promoter-primer) to its target nucleic acid
sequence.
[0045] In the subject methods, conversion of the primer-mRNA hybrid
to double-stranded cDNA proceeds by priming second strand cDNA
synthesis with a random primer in the presence of a DNA-dependent
DNA polymerase activity.
[0046] In another embodiment, the primer for second strand cDNA
synthesis is a mixture of primers consisting of a poly-dN sequence
that is sufficiently long to provide for efficient hybridization to
the mRNA. The sequence typically ranges in length from 4-50,
preferably 6-25, more preferably 6-12 or 6-9 and most preferably 6
degenerate bases, i.e., a random hexamer (dN.sub.6), wherein the
degenerate bases may be A, T, G, or C. (In theory, the primer
should hybridize on average 4.sup.6 or 4096 base pairs from the 3'
priming site of the first-strand cDNA.) In specific embodiments,
the poly-dN sequence is 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides
in length.
[0047] In a specific embodiment, the random primers used to prime
second strand cDNA synthesis will be a complete set of all
combinations of random hexamers, i.e., a total of 4.sup.6 or 4096
hexamers.
[0048] Additional proteins that may enhance the yield of
double-stranded DNA products may also be added to the cDNA
synthesis reaction. These proteins include a variety of DNA
polymerases (such as those derived from E. coli, thermophilic
bacteria, archaebacteria, phage, yeasts, Neurosporas, Drosophilas,
primates and rodents), and DNA ligases (such as those derived from
phage or cellular sources, including T4 DNA ligase and E. coli DNA
ligase).
[0049] The second strand cDNA synthesis results in the creation of
a double-stranded promoter region. The second strand cDNA includes
not only a sequence of nucleotide residues that comprise a DNA copy
of the mRNA template, but also additional sequences at its 3' end
that are complementary to the promoter-primer used to prime first
strand cDNA synthesis. The double-stranded promoter region serves
as a recognition site and transcription initiation site for RNA
polymerase, which uses the second strand cDNA as a template for
multiple rounds of RNA synthesis during the next stage of the
subject methods (see Section 5.2, "Transcription of cDNA,"
below).
[0050] Depending on the particular protocol, the same or different
DNA polymerases may be employed during the cDNA synthesis step. In
a preferred embodiment, a single reverse transcriptase, most
preferably MMLV-RT, is used as a source of all the requisite
activities necessary to convert the primer-mRNA hybrid to
double-stranded cDNA. In another preferred embodiment, the
polymerase employed in first strand cDNA synthesis is different
from that which is employed in second strand cDNA synthesis.
Specifically, a reverse transcriptase lacking RNaseH activity
(e.g., SUPERSCRIPT II.TM.) is combined with the primer-mRNA hybrid
during a first substep for first strand synthesis. A source of
RNaseH activity, such as E. coli RNaseH or MMLV-RT, but most
preferably MMLV-RT, is added during a second substep to initiate
second strand synthesis.
[0051] In yet other embodiments, the requisite activities are
provided by a plurality of distinct enzymes. The manner in which
double-stranded cDNA is produced from the initial mRNA is not
critical to certain embodiments of the invention. However, the
preferred embodiments use MMLV-RT, or a combination of SUPERSCRIPT
II.TM. and MMLV-RT, or a combination of SUPERSCRIPT II.TM. and E.
coli RNaseH, for cDNA synthesis as these embodiments yield certain
desired results. Specifically, in the preferred embodiments,
reaction conditions were chosen so that enzymes present during the
cDNA synthesis do not adversely affect the subsequent transcription
reaction. Potential inhibitors include, but are not limited to,
RNase contaminants of certain enzyme preparations.
[0052] 5.2. Transcription of cDNA
[0053] The next step of the subject method is the preparation of
RNA from the double-stranded cDNA prepared in the first step.
During this step, the double-stranded cDNA produced in the first
step is transcribed by RNA polymerase to yield RNA that, in certain
embodiments, is complementary to the initial nucleic acid target
from which it is amplified. This step is sometimes referred to as
"in vitro transcription" (IVT).
[0054] The promoter regions that find use in the methods of the
invention are regions where RNA polymerase binds tightly to the DNA
and contain the start site and signal for RNA synthesis to begin. A
wide variety of promoters are known and many are very well
characterized. In general, prokaryotic promoters are preferred over
eukaryotic promoters, and phage or virus promoters most preferred.
The RNA polymerase promoter sequence is therefore preferably
derived from a prokaryote such as E. coli or the bacteriophage T7,
SP6, and T3, with the T7 RNA polymerase promoter sequence
particularly preferred. T7, T3 and SP6 promoter regions are
described in Chamberlin and Ryan, The Enzymes (ed. P. Boyer,
Academic Press, New York) (1982) pp 87-108, which excerpt is hereby
incorporated by reference in its entirety.
[0055] The RNA polymerase used for transcription must be capable of
binding to the particular RNA polymerase promoter sequence
contained in the primer; hence usually the RNA polymerase promoter
sequence and the polymerase will be homologous. For example, if the
T7 RNA polymerase promoter sequence is employed in the primer, it
is preferred to use T7 RNA polymerase to drive transcription. T7
polymerase is commercially available from several sources,
including Promega Biotech (Madison, Wis.) and Epicenter
Technologies (Madison, Wis.).
[0056] In a preferred embodiment, the random promoter-primer used
to prime first strand cDNA synthesis comprises a T7 promoter
sequence-dN.sub.9, and the RNA polymerase employed is T7 RNA
polymerase.
[0057] Preferably, the RNA polymerase promoter sequence is located
at or near the terminus of the primer, in an orientation permitting
transcription of the RNA population under study.
[0058] For this transcription step, the presence of the RNA
polymerase promoter region on the double-stranded cDNA is exploited
for the production of sense and/or antisense RNA. To synthesize the
RNA, the double-stranded DNA is contacted with the appropriate RNA
polymerase in the presence of the four ribonucleotides, under
conditions sufficient for RNA transcription to occur.
[0059] In one embodiment, the conditions for RNA transcription are
those described in Section 6, Example 1. Briefly, the transcription
mix and the transcription reaction are as follows. 60 of
Transcription Mix are aliquoted into each sample tube. The
transcription reactions are incubated at 40.degree. C. for 16
hrs.
TABLE-US-00001 Transcription Mix Component Volume (.mu.l)
Nuclease-free water 22.8 5x Transcription Buffer 16 100 mM DTT 6.0
NTPs (25 mM A, G, C, 6.0 mM UTP) 8.0 as UTP (allylamine-derivatized
UTP) 2.0 (75 mM) 200 mM MgCl.sub.2 3.3 RNAGuard .TM., Pharmacia (36
U/.mu.l) 0.5 Inorganic Pyrophosphatase (200 U/ml) 0.6 T7 RNA
polymerase (2500 U/.mu.l) 0.8 Volume of Transcription Mix 60
TABLE-US-00002 Composition of Transcription Reaction Final
concentration or Component amount Double-strand cDNA Approximately
400 ng Tris-HCl, pH 7.5 52 mM MgCl.sub.2 15 mM KCl 19 mM NaCl 10 mM
Spermidine 2 mM DTT 10 mM ATP, GTP, CTP 2.5 mM each UTP 0.6 mM aa
UTP 1.9 mM T7 RNA polymerase 2000 U RNAGuard .TM., Pharmacia 18 U
Inorganic pyrophosphatase 0.12 U Total reaction volume 80 .mu.l
[0060] Other suitable conditions for RNA transcription using RNA
polymerases are known in the art, see e.g., Milligan and Uhlenbeck
(1989), Methods in Enzymol. 180, 51 (which is hereby incorporated
by reference in its entirety).
[0061] In one aspect of the invention, the transcription step is
carried out in the presence of reverse transcriptase that is
present in the reaction mixture from the double-stranded cDNA
synthesis. Thus, the subject methods do not involve a step in which
the double-stranded cDNA is physically separated from the reverse
transcriptase following double-stranded cDNA preparation
facilitating high throughput amplification and analysis. In this
aspect of the invention, the reverse transcriptase that is present
during the transcription step is rendered inactive. Thus, the
transcription step is carried out in the presence of a reverse
transcriptase that is unable to catalyze RNA-dependent DNA
polymerase activity, at least for the duration of the transcription
step. As a result, the RNA products of the transcription reaction
cannot serve as substrates for additional rounds of cDNA synthesis,
and the amplification process cannot proceed exponentially.
[0062] The reverse transcriptase present during the transcription
step may be rendered inactive using any convenient protocol. The
transcriptase may be irreversibly or reversibly rendered inactive.
Where the transcriptase is reversibly rendered inactive, the
transcriptase is physically or chemically altered so as to no
longer be able to catalyze RNA-dependent DNA polymerase activity.
The transcriptase may be irreversibly inactivated by any convenient
means. Thus, the reverse transcriptase may be heat inactivated, in
which the reaction mixture is subjected to heating to a temperature
sufficient to inactivate the reverse transcriptase prior to
commencement of the transcription step. In these embodiments, the
temperature of the reaction mixture and therefore the reverse
transcriptase present therein is typically raised to 55.degree. C.
to 70.degree. C. for 5 to 60 minutes, usually to about 65.degree.
C. for 15 to 20 minutes.
[0063] Alternatively, reverse transcriptase may be irreversibly
inactivated by introducing a reagent into the reaction mixture that
chemically alters the enzyme so that it no longer has RNA-dependent
DNA polymerase activity. In yet other embodiments, the reverse
transcriptase is reversibly inactivated. In these embodiments, the
transcription step may be carried out in the presence of an
inhibitor of RNA-dependent DNA polymerase activity. Any convenient
reverse transcriptase inhibitor may be employed that is capable of
inhibiting RNA-dependent DNA polymerase activity a sufficient
amount to provide for linear amplification. However, these
inhibitors should not adversely affect RNA polymerase activity.
Reverse transcriptase inhibitors of interest include ddNTPs, such
as ddATP, ddCTP, ddGTP or ddTTP, or a combination thereof, the
total concentration of the inhibitor typically ranges from about 50
.mu.M to 200 .mu.M.
[0064] Because of the nature of the subject methods, all of the
necessary polymerization reactions, i.e., first strand cDNA
synthesis, second strand cDNA synthesis and RNA transcription, may
be carried out in the same reaction vessel at the same temperature,
such that temperature cycling is not required. As such, the subject
methods are particularly suited for automation, as the requisite
reagents for each of the above steps need merely be added to the
reaction mixture in the reaction vessel, without any complicated
separation steps being performed, such as phenol/chloroform
extraction. A further feature of the subject invention is that,
despite its simplicity, it yields high amplification extents, where
the amplification extents (mass of RNA product/mass of nucleic acid
target) typically are at least about 50-fold, usually at least
about 200-fold and may be as high as 600-fold or higher.
Furthermore, such amplification extents are achieved with low
variability, e.g., coefficients of variation about the mean
amplification extents that do not exceed about 10%, and usually do
not exceed about 5%.
[0065] The resultant cRNA (particularly antisense RNA) produced by
the subject methods finds use in a variety of applications. RNA
amplified by the methods of the invention may be labeled and
employed to profile gene expression in different populations of
cells. In a preferred embodiment, the amplified RNA is used for
quantitative comparisons of gene expression between different
populations of cells or between populations of cells exposed to
different stimuli. For example, the resultant antisense RNA can be
used in expression profiling analysis on such platforms as DNA
microarrays, for construction of "driver" for subtractive
hybridization assays, for cDNA library construction, and the like.
Especially facilitated by the subject methods are studies of
differential gene expression in mammalian cells or cell
populations. The cells may be from blood (e.g., white cells, such
as T or B cells) or from tissue derived from solid organs, such as
brain, spleen, bone, heart, vascular, lung, kidney, liver,
pituitary, endocrine glands, lymph node, dispersed primary cells,
tumor cells, or the like.
[0066] The RNA amplification technology can also be applied to
improve methods of detecting and isolating nucleic acid sequences
that vary in abundance among different populations using the
technique known as subtractive hybridization. In such assays, two
nucleic acid populations, one sense and the other antisense, are
allowed to mix with one another with one population being present
in molar excess ("driver"). Under appropriate conditions, the
sequences represented in both populations form hybrids, whereas
sequences present in only one population remain single-stranded.
Thereafter, various well known techniques are used to separate the
unhybridized molecules representing differentially expressed
sequences. The amplification technology described herein may be
used to construct large amounts of antisense RNA for use as
"driver" in such experiments.
[0067] 5.3. Oligonucleotides
[0068] A primer may be prepared by any suitable method, such as
phosphotriester and phosphodiester methods of synthesis, or
automated embodiments thereof. It is also possible to use a primer
that has been isolated from a biological source, such as a
restriction endonuclease digest, although a synthetic primer is
preferred.
[0069] An oligonucleotide primer can be DNA, RNA, chimeric mixtures
or derivatives or modified 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.
[0070] For example, an oligonucleotide primer may comprise at least
one modified base moiety which is selected from the group 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, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
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.
[0071] In another embodiment, the oligonucleotide primer comprises
at least one modified sugar moiety selected from the group
including but not limited to arabinose, 2-fluoroarabinose,
xylulose, and hexose.
[0072] In yet another embodiment, the oligonucleotide primer
comprises 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.
[0073] An oligonucleotide primer for use in the methods of the
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 by synthesis
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, etc.) and standard phosphoramidite chemistry.
As examples, 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.
[0074] 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 oligonucleotide that has been separated on
an acrylamide gel, or by measuring the optical density at 260 nm in
a spectrophotometer.
[0075] 5.4. Methods of Labeling of Nucleic Acid Amplification
Products
[0076] Nucleic acid amplification products such as amplified RNA
may be labeled with any art-known detectable marker, including
radioactive labels such as .sup.32P, .sup.35S, .sup.3H, and the
like; fluorophores; chemiluminescers; or enzymatic markers. In a
preferred embodiment, the label is fluorescent. Exemplary suitable
fluorophore moieties that can be selected as labels are listed in
Table 1.
TABLE-US-00003 TABLE 1 Suitable fluorophore moieties that can be
selected as labels
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 Cy5 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)
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) fluorescein
fluorescein isothiocyanate QFITC (XRITC) fluorescamine IR 144
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 .RTM. 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 101 (Texas Red)
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl
rhodamine tetramethyl rhodamine isothiocyanate (TRITC) riboflavin
rosolic acid terbium chelate derivatives
[0077] 5.4.1. Labeling of RNA
[0078] Depending on the particular intended use of the RNA
amplification products, the RNA amplification products may be
labeled. The RNA may be labeled with any art-known detectable
marker, including but not limited to radioactive labels such as
.sup.32P, .sup.35S, .sup.3H, and the like; fluorophores;
chemiluminescers; or enzymatic markers (e.g., as listed in Table
1).
[0079] Labeling of RNA is preferably accomplished by including one
or more labeled NTPs in the in vitro transcription (IVT) reaction
mixture. NTPs may be directly labeled with a radioisotope, such as
.sup.32P, .sup.35S, .sup.3H; 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 Cy5. In one embodiment, biotinylated or
allylamine-derivatized NTPs are incorporated during the IVT
reaction and the resultant cRNAs are thereafter labeled, 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
allylamine. In another embodiment, fluorescently labeled NTPs may
be incorporated during the IVT reaction, which fluorescently labels
the resultant cRNAs directly.
[0080] RNA may be fluorescently labeled with fluorescently tagged
nucleotides (e.g., fluorescently labeled ATP, UTP, GTP or CTP) that
are incorporated into the antisense RNA product during the
transcription step. Fluorescent moieties that may be used to tag
nucleotides for producing labeled antisense RNA include:
fluorescein, the cyanine dyes, such as Cy3, Cy5, Alexa 542, Bodipy
630/650, and the like. Other labels may also be employed as are
known in the art. Exemplary fluorophore moieties that can be used
as labels are listed in Table 1. The preferred label in the subject
methods is a fluorophore, such as fluorescein isothiocyanate,
lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5
particularly preferred.
[0081] Not only fluorophores, but also chemiluminescers and
enzymes, among others, may be used as labels. In yet another
embodiment, the RNA is 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.
[0082] 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.
[0083] 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 (aa UTP) is incorporated during IVT.
Following the IVT reaction, unincorporated nucleotides are removed
and the allylamine-containing RNAs are conjugated to the
N-hydroxysuccinimide (NHS) esters of Cy3 or Cy5.
[0084] In a preferred embodiment, 5-(3-Aminoallyl)uridine
5'-triphosphate is incorporated into the RNA amplification product
during transcription and post-synthetically coupled to Cy-NHS,
either Cy3-NHS or Cy5-NHS.
[0085] In a specific embodiment, a two-step method of preparing
fluorescent-labeled cRNA may be used in two color hybridizations to
DNA microarrays. Such a two-step method is disclosed in U.S. Ser.
No. 09/411,074, filed Oct. 4, 1999, the disclosure of which is
herein incorporated by reference. In one embodiment, 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.RTM. Mini kit (for RNA) or QIAquick PCR
Purification kit (for DNA) (QIAGEN Inc.--USA, 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 are eluted twice with 50 .mu.l volumes of
70.degree. C. H.sub.2O. Alternatively (but less preferably),
samples may be cleaned up by repeated cycles of dilution and
concentration on Microcon-30 filters.
[0086] In a second step of the embodiment, aa-nucleic acids are
derivatized with NHS-esters, preferably Cy3 or Cy5. Preferably, 2-6
.mu.g of aa-labeled nucleic acid are aliquoted into a microfuge
tube, adjusting the total volume to 12 .mu.l with H.sub.2O. The
NHS-ester is dissolved at a concentration of .about.15 mM in
anhydrous DMSO (-200 nmoles in 13 .mu.l). 27 .mu.l of 0.1 M sodium
carbonate buffer, pH 9, are added. 12 .mu.l of the dye mix
(containing .about.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.degree.
C. for 1 hour. The coupling reaction is stopped by adding 5 .mu.l
of a 4M solution of hydroxylamine. Incubation is continued at
23.degree. C. for an additional 0.25 hr. Dye-coupled nucleic acid
is separated from unincorporated dye on an RNeasy.RTM. 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 .mu.l volumes of 70.degree. C.
H.sub.2O.
[0087] 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 l.sub.max. Recovery of
nucleic acid is typically .about.80%. The mole percent of dye
incorporated per nucleotide ranges from 1.5-5% of total
nucleotides.
[0088] Often it is desired to compare gene expression in two
different populations of cells, perhaps derived from different
tissues or perhaps exposed to different stimuli. 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.
[0089] 5.5. Methods of Preparation of Source RNA
[0090] The source RNA may be obtained from a variety of different
sources, typically a biological source. In specific embodiments,
the biological source may be any of a variety of eukaryotic
sources. Biological sources of interest may include sources derived
from single-celled organisms such as yeast and multicellular
organisms, including plants and animals, particularly mammals.
Biological 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 RNA to
be analyzed from its biological source, the source may be subjected
to a number of different processing steps. Such processing steps
might include tissue homogenization, cell isolation followed by
cytoplasm extraction or isolation, nucleic acid extraction and the
like. Such processing steps for isolating RNA from its biological
source are known to those of skill in the art. For example, methods
of isolating RNA from cells, tissues, organs or whole organisms are
described in Sambrook et al. (1989), Molecular Cloning: A
Laboratory Manual 2d Ed. (Cold Spring Harbor Press), incorporated
herein by reference in its entirety. Alternatively, at least some
of the initial steps of the subject methods may be performed in
situ, as described in Eberwine (U.S. Pat. No. 5,514,545, entitled
"Method for characterizing single cells based on RNA amplification
for diagnostics and therapeutics," issued May 7, 1996), the
disclosure of which is herein incorporated by reference.
[0091] Although the amplification methods of the invention can be
adapted to amplify DNA, it is preferred to utilize the methods to
amplify RNA from a population of cells. Total cellular RNA,
cytoplasmic RNA, or poly(A).sup.+ RNA may be used, with
poly(A).sup.+ RNA (mRNA) being preferred. Methods for preparing
total and poly(A).sup.+ 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), incorporated herein by reference in their
entireties. 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, modified cells, and drug-exposed
modified cells.
[0092] 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 CsCl centrifugation to separate the RNA from DNA
(Chirgwin et al., 1979, Biochemistry 18:5294-5299). Poly(A).sup.+
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.
[0093] 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.
[0094] 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 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.TM. (see Ausubel et
al., eds., 1994, Current Protocols in Molecular Biology, vol. 2,
Current Protocols Publishing, New York). Once bound,
poly(A)+[.sup.+?] mRNA is eluted from the affinity column using 2
mM EDTA/0.1% SDS. The sample of RNA can comprise 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 comprise at least 100 different
nucleotide sequences. More preferably, the mRNA molecules of the
RNA sample comprise 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, the mRNA
molecules of the mammalian RNA sample comprising about 20,000 to
30,000 different nucleotide sequences.
[0095] 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, rabbit, bird, yeast, eukaryote,
prokaryote, etc. In one embodiment, the method of the invention is
used with a sample containing total mRNA or total RNA from
1.times.10.sup.6 cells or less.
[0096] 5.6. Methods for Determining Biological Response
Profiles
[0097] This section provides some exemplary methods for measuring
biological responses using cRNA amplified by methods of the
invention. One of skill in the art would appreciate that this
invention is not limited to the following specific methods for
measuring the responses of a biological system, i.e., gene
expression profiles. 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, the use of oligonucleotides tethered to beads as
probes, or the use of polynucleotide microarrays.
[0098] In a specific embodiment of the invention, one or more
labels is introduced into the RNA during the transcription step to
facilitate gene expression profiling. Gene expression can be
profiled in any of several ways, among which the preferred method
is to probe a DNA microarray with the labeled RNA transcripts
generated 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).
[0099] 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.
[0100] Gene expression profiling can be done for purposes of
screening, diagnosis, staging a disease, and monitoring response to
therapy, as well as for identifying genetic targets of drugs and of
pathogens.
[0101] 5.6.1. Transcript Assay Using DNA Arrays
[0102] This invention is particularly useful for the analysis of
gene expression profiles. For expression profiling, DNA microarrays
are typically probed using mRNA, extracted and amplified from the
cells whose gene expression profile it is desired to analyze, using
the random-primed RT-IVT amplification method of the invention. To
facilitate comparison between any two samples of interest, the
polynucleotides representing the mRNA transcripts present in a cell
are typically labeled separately with fluorescent dyes that emit at
different wavelengths. Some embodiments of this invention are based
on measuring the transcriptional rate of genes.
[0103] The transcriptional rate can be measured by techniques of
hybridization to arrays of nucleic acid or nucleic acid mimic
probes, described in the next subsection, or by other gene
expression technologies, such as those described in the subsequent
subsection. 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).
[0104] 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.
[0105] Preferably, measurement of the transcriptional state is made
by hybridization to transcript arrays, which are described in this
subsection. Certain other methods of transcriptional state
measurement are described later in this subsection.
[0106] In a preferred embodiment the present invention makes use of
"transcript arrays" (also called herein "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.
[0107] 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 amplified 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, of which several are described below. However produced,
microarrays share certain preferred characteristics: 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 cm.sup.2, 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 site (hereinafter "site") per
specific mRNA, for the sake of clarity the discussion below will
assume that there is a single site.
[0108] In one embodiment, the microarray is an array of
polynucleotide probes, the array comprising a support with at least
one surface and at least 100 different polynucleotide probes, each
different polynucleotide probe comprising 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.
[0109] In specific embodiments, the array comprises 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.
[0110] 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 comprise expressed polynucleotide
sequences of a cell or organism.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] In specific embodiments, the array has at least 100, at
least 250, at least 1,000, or at least 2,500 probes per 1 cm.sup.2,
preferably all or at least 25% or 50% of which are different from
each other.
[0116] In another embodiment, the array is a positionally
addressable array (in that the sequence of the polynucleotide probe
at each position is known).
[0117] 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.
[0118] When cRNA complementary to the RNA of a cell is made 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.
[0119] In preferred embodiments, cRNAs from two different cells are
hybridized to the binding sites of the microarray. In the case of
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.
[0120] 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 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 site(s) 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.
[0121] 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 by reference in its entirety for all purposes. An
advantage of using eRNA 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 drug-treated
or pathway-perturbed cell and an untreated cell.
[0122] 5.6.2. Preparation of Microarrays
[0123] 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.
[0124] 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 85%, even more often more than about
90%, and most often at least about 99%.
[0125] 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%.
[0126] Preferably, the microarray has binding sites for genes
relevant to the action of a drug 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 messenger RNA 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 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 for
all purposes). In contrast, the human genome is estimated to
contain approximately 10.sup.5 genes.
[0127] 5.6.3. Preparation of Nucleic Acids for Microarrays
[0128] 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 reverse transcription or RT-PCR), or
cloned sequences. Nucleic acid amplification 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 10 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 bp and about 2000 bp, more typically
between about 100 bp and about 1000 bp, and usually between about
300 bp and about 800 bp in length.
Nucleic acid amplification 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 by reference in its entirety
for all purposes. It will be apparent that computer controlled
robotic systems are useful for isolating and amplifying nucleic
acids.
[0129] 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.
[0130] 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.
[0131] 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; see also U.S. Pat. No. 5,539,083, Cook et
al., entitled "Peptide nucleic acid combinatorial libraries and
improved methods of synthesis," issued Jul. 23, 1996).
[0132] 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.
[0133] 5.6.4. Attaching Nucleic Acids to the Solid Surface
[0134] The nucleic acid or analogue are 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.
[0135] 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 tine array (U.S. Pat. No. 5,605,662, Heller et al.,
entitled "Active programmable electronic devices for molecular
biological analysis and diagnostics," issued Feb. 25, 1997). DNA is
typically immobilized at a density of 100 to 10,000
oligonucleotides per cm.sup.2 and preferably at a density of about
1000 oligonucleotides per cm.sup.2.
[0136] 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.)
[0137] 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 are 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 most preferred.
[0138] 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, Trulson et al., entitled "Method and
apparatus for imaging a sample on a device," issued Nov. 26, 1996;
U.S. Pat. No. 5,556,752, Lockhart et al., entitled "Surface-bound,
unimolecular, double-stranded DNA," issued Sep. 17, 1996; and U.S.
Pat. No. 5,510,270, Fodor et al., entitled "Synthesis and screening
of immobilized oligonucleotide arrays," issued Apr. 23, 1996; each
of which is incorporated by reference in its entirety for all
purposes) or other methods for rapid synthesis and deposition of
defined oligonucleotides (Lipshutz et al., 1999, Nat. Genet. 21(1
Suppl):20-4.)
[0139] 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.
[0140] In a particularly preferred embodiment, microarrays of the
invention are manufactured by means of an ink jet printing device
for oligonucleotide synthesis, e.g., using the methods and systems
described by Blanchard in International Patent Publication No. WO
98/41531, published Sep. 24, 1998; Blanchard et al., 1996,
Biosensors and Bioele[c?]tronics 11:687-690; Blanchard, 1998, in
Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow,
Ed., Plenum Press, New York at pages 111-123; U.S. Pat. No.
6,028,189 to Blanchard. Specifically, the oligonucleotide probes in
such microarrays are preferably synthesized in arrays, e.g., on a
glass slide, by serially depositing individual nucleotide bases in
"microdroplets" of a high surface tension solvent such as propylene
carbonate. The microdroplets have small volumes (e.g., 100 .mu.L or
less, more preferably 50 .mu.L or less) and are separated from each
other on the microarray (e.g., by hydrophobic domains) to form
circular surface tension wells which define the locations of the
array elements (i.e., the different probes).
[0141] 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, New York), 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.
[0142] 5.6.5. Hybridization to Microarrays
[0143] 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 (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).
[0144] 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. (1989, 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 5.times.SSC
plus 0.2% SDS at 65.degree. C. for 4 hours followed by washes at
25.degree. C. in low stringency wash buffer (1.times.SSC plus 0.2%
SDS) followed by 10 minutes at 25.degree. C. in high stringency
wash buffer (0.1.times.SSC 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.
[0145] 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.
[0146] 5.6.6. Signal Detection and Data Analysis
[0147] When fluorescently labeled probes are used, the fluorescence
emissions at each site of a transcript array can be, preferably,
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 multi-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.
[0148] 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 bespeckled 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. The 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.
[0149] 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.
[0150] 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.
[0151] 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., co-pending U.S. patent application Ser. No. 09/179,569 filed
on Oct. 27, 1998, by Friend et al.; Ser. No. 09/220,142 filed on
Dec. 23, 1998, by Stoughton et al.; Ser. No. 09/220,275 filed on
Dec. 23, 1998, by Friend et al.; International Publication WO
00/24936, dated May 4, 2000, which are incorporated by reference
herein in their entireties.
[0152] 5.7. Diagnostic Methods
[0153] The random-primed RT-IVT methods of the invention have use
in nucleic acid amplification reactions to generate sufficient
quantities of nucleic acid for detection of a specific nucleic acid
of interest. Accordingly, the methods of the invention can be used
in methods of diagnosis, for example, in amplifying 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 nucleic acid of
interest can be genomic or cDNA or mRNA, or can be 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 nucleic acid of interest 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. By way of example, the mutation can be an insertion,
substitution, and/or deletion of one or more nucleotides, or a
translocation.
[0154] 5.8. Kits for the Amplification and Detection of Selected
Target Nucleotide Sequences
[0155] The present invention also provides kits for the linear
amplification of RNA, and, for example, detection or measurement of
nucleic acid amplification products and for determining the
responses or state of a biological sample. Such a kit may comprise
containers, each with one or more of the various reagents
(typically in concentrated form) utilized in the methods of the
invention, including, for example, buffers, the appropriate
nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP,
GTP and UTP), reverse transcriptase, RNA polymerase specific to the
RNA polymerase promoter, and the random promoter-primers and
primers of the present invention. Optionally also present in the
kit is a reverse transcriptase inhibitor, where, in many
embodiments, the inhibitor is at least ddNTP or a combination of
ddNTPs, e.g., ddATP and/or ddGTP. A set of instructions for use of
kit components in an mRNA amplification method of the present
invention, will also be typically included.
[0156] In a specific embodiment, the kit comprises one or more
primer oligonucleotides of the invention, such as a RNA polymerase
promoter-containing primer, including but not limited to a set of
random RNA polymerase promoter-containing primers and/or a set of
random primers, in one or more containers. The kit can comprise for
example, a random T7-poly dN primer set, a T7-poly dT primer,
and/or a random poly dN primer set. The kit can further comprise
additional components for carrying out the amplification reactions
of the invention, such as reverse transcriptase and RNA polymerase.
Where the target nucleic acid sequence being amplified is one
implicated in disease or disorder, the kit can be used for
diagnosis or prognosis.
[0157] Oligonucleotides in containers can be in any form, e.g.,
lyophilized, or in solution (e.g., a distilled water or buffered
solution), etc. Oligonucleotides ready for use in the same
amplification reaction can be combined in a single container or can
be in separate containers.
[0158] The kit optionally further comprises 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. The kit
optionally further provides means for stimulating and detecting
fluorescent light emissions, e.g., a fluorescence plate reader or a
combination thermocycler-plate-reader to perform the analysis.
[0159] 5.8.1. Analytic Kit Implementation
[0160] In a preferred embodiment, the methods of this invention can
be implemented by use of kits containing oligonucleotide primers of
the invention and microarrays. The microarrays contained in such
kits comprise 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 a 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 that 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 that hybridize to RNA species that are not
increased in response to perturbations to the particular protein
whose activity is determined by the kit.
[0161] In another preferred embodiment, a kit 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 co-pending U.S.
patent application Ser. No. 09/220,276, by Bassett, Jr. et al.,
filed Dec. 23, 1998, which is incorporated herein by reference in
its entirety. Preferably, the expression profile analysis software
contained in a kit of this invention, is essentially identical to
the expression profile analysis software 512 described in U.S.
patent application Ser. No. 09/220,276.
[0162] Alternative kits for implementing the analytic methods of
this invention will be apparent to one 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.
[0163] The following experimental examples are offered by way of
illustration and not by way of limitation.
6. Example 1
cDNA Synthesis and RNA Amplification for the Preparation of Cy3-
and Cy5-Labeled RNA Targets for Gene Expression Monitoring
[0164] This example demonstrates that using the random-primed
RT-IVT method of the invention, linear amplification of mRNA
to[<CUT?] can be used to produce unbiased antisense RNA
profiles. The results of an mRNA amplification produced using the
random-primed RT-IVT method of the invention were compared with
results obtained using the mRNA amplification method disclosed in
Shannon (U.S. Pat. No. 6,132,997, entitled "Method for linear mRNA
amplification," issued Oct. 17, 2000). Using the random primed
RT-IVT method, poly-A.sup.+ RNA was converted to double-stranded
cDNA using degenerate random primers comprising a T7 RNA polymerase
promoter sequence (T7-dN.sub.9) to prime first strand cDNA
synthesis and degenerate random primers (dN.sub.6) to prime second
strand cDNA synthesis to yield a double-stranded cDNA that is
recognized by T7 RNA polymerase. The double-stranded cDNA was then
transcribed into antisense RNA by T7 RNA polymerase in the presence
of a reverse transcriptase that was rendered incapable of
RNA-dependent DNA polymerase activity during this transcription
step by heat inactivation. 5-(3-Aminoallyl)uridine 5'-triphosphate
was incorporated into the antisense RNA during transcription and
post-synthetically labeled with Cy3-NHS or Cy5-NHS. Linear
amplification extents of at least 100-fold and labeling
efficiencies of approximately 3% were achieved using this
method.
[0165] 6.1. Materials and Methods
[0166] Total RNA was isolated from Jurkat and K562 cell lines.
Poly-A.sup.+ RNA was isolated from the total RNA to provide the
initial source mRNA used in the experiment.
[0167] cDNA Synthesis Reagents. [0168] 1. mRNA, 0.2 mg. [0169] 2.
DNA T7-dN.sub.9 (20 .mu.M): (5') AAT TAA TAC GAC TCA CTA TAG GGA
GAT NNN NNN NNN (3') (N=A, T, C or G) (SEQ ID NO.: 1) [0170] 3.
MMLV Reverse Transcriptase (50 U/.mu.l), Epicentre P/N M4425H
[0171] 4. RNAGuard.TM., Pharmacia P/N 27-0815-01 [0172] 5. 5.times.
First Strand Buffer: 250 mM Tris-HCl, pH 8.3, 15 mM MgCl.sub.2, 375
mM KCl, Life Technologies P/N 18057-018 [0173] 6. 100 mM DTT*
(*supplied with MMLV Reverse Transcriptase, Epicentre) [0174] 7.
dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01 [0175] 8.
ultraPURE distilled water, DNAse, RNAse Free, Life Technologies,
Cat #10977-015 [0176] 9. pdN.sub.6 (200 ng/.mu.l), diluted from
Amersham Pharmacia Biotech P/N 27-2166-01
[0177] Transcription Reagents: [0178] 1. T7 RNA Polymerase (2500
units/.mu.l), Epicentre P/N TU950K [0179] 2. RNAGuard.TM.,
Pharmacia P/N 27-0815-01 [0180] 3. Inorganic Pyrophosphatase (200
U/ml), New England Biolabs, #MO296S [0181] 4. 5+ Transcription
Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl, 30 mM MgCl.sub.2, 10 mM
spermidine, Epicentre P/N BP1001 [0182] 5. 100 mM DTT, Epicentre
P/N BP1001 [0183] 6. MgCl.sub.2 (200 mM), diluted from Sigma P/N
M-1028 [0184] 7. NTPs (25 mM ATP, GTP, CTP, 6 mM UTP), diluted from
Pharmacia P/N 27-2025-01 [0185] 8. 5-(3-Aminoallyl)uridine
5'-triphosphate (75 mM), Sigma P/N A-5660 [0186] 9. ultraPURE
distilled water, DNAse, RNAse Free, Life Technologies, P/N
10977-015
[0187] Purification and Labeling Reagents: [0188] 1. RNeasy.RTM.
Mini Kit (250), QIAGEN Inc., P/N 74106 [0189] 2.
Carbonate-Bicarbonate Buffer capsules, Sigma, P/N C-3041 [0190] 3.
Hydrochloric acid, Fisher, P/N A508-500 [0191] 4. Anhydrous MSO
(methyl sulfoxide, also known as DMSO, dimethyl sulfoxide),
Aldrich, P/N 27,685-5 [0192] 5. Cy3-NHS dye pack, Amersham, P/N
PA23001 [0193] 6. Cy5-NHS dye pack, Amersham, P/N PA25001 [0194] 7.
Hydroxylamine ("HA"), Sigma, P/NH-2391
[0195] Other materials: [0196] 1. Pipetman micropipettors, (P-10,
P-20, P-200, P-1000), or equivalent [0197] 2. Sterile,
nuclease-free 1.5 ml microcentrifuge tubes [0198] 3. Sterile,
nuclease-free aerosol-barrier pipette tips [0199] 4. Thermal
Cycler
[0200] Reagent Preparation: [0201] 1. dNTPs (10 mM each) [0202]
Thaw dNTP stocks (100 mM) and place on ice. Add 10111 each dNTP to
60 .mu.l nuclease-free water. Store frozen. [0203] 2. pdN.sub.6
(200 ng/.mu.l) [0204] Add 663 .mu.l nuclease-free water to
lyophilized sample (50 A260 units or approximately 1325 .mu.g) for
2.0 .mu.g/.mu.l. Add 10 .mu.l pdN.sub.6 (2.0 .mu.g/.mu.l) to 90
.mu.l nuclease-free water for 200 ng/.mu.l. Store frozen. [0205] 3.
200 mM MgCl.sub.2 [0206] Add 100 .mu.l of 1 M MgCl.sub.2 to 400
.mu.l nuclease-free water. Store frozen. [0207] 4. NTPs (25 mM ATP,
GTP, CTP, 6.0 mM UTP) [0208] Thaw NTP stocks (100 mM) and place on
ice. Combine 125 .mu.l ATP, 125 .mu.l GTP, 125 .mu.l CTP, 30 .mu.l
UTP and 95 .mu.l nuclease-free water. Store frozen. [0209] 5. aa
UTP (75 mM) Dissolve 5 mg in 125 .mu.l water. [0210] 6. Anhydrous
MSO should be stored with a molecular sieve to absorb water.
[0211] Procedure:
[0212] To prevent contamination of reactions by ribonucleases,
laboratory gloves were worn and dedicated solutions and pipettors
with nuclease-free, aerosol-resistant tips were used.
[0213] Amplified RNA preparations were prepared in batches of no
less than 6 to minimize errors associated with pipetting small
volumes of enzyme solutions. The procedure below specifies reagent
volumes for 1 reaction; for 6 reactions, the specified volumes were
multiplied by 6.5. [0214] 1. Add 0.2 .mu.g of source mRNA to
reaction tube. Add 1.0 .mu.l DNA T7-dN.sub.9 (201 .mu.M) and bring
total sample volume to 10.5 .mu.l in nuclease-free water. [0215] 2.
Incubate at 65.degree. C. for 10 min to denature primer and
template. Move reaction tubes to ice. Store reactions tubes on ice
for 5 rain. [0216] 3. Mix the following components and maintain on
ice.
TABLE-US-00004 [0216] cDNA Mix Component Volume (.mu.l) 5x First
Strand Buffer 4.0 100 mM DTT 2.0 dNTPs (10 mM each) 1.0 pdN.sub.6
(200 ng/.mu.l) 1.0 MMLV-RT (50 U/.mu.l) 1.0 RNAGuard .TM. (36
U/.mu.l) 0.5 Volume of cDNA Mix 9.5
[0217] 4. Aliquot 9.5 .mu.l of cDNA Mix into each sample tube.
Incubate cDNA synthesis reaction at 40.degree. C. for 120 min.
TABLE-US-00005 [0217] Composition of cDNA Synthesis Reaction
Component Final concentration or amount poly-A.sup.+ RNA 200 ng DNA
T7T18VN 1 .mu.M Tris-HCl, pH 8.3 50 mM MgCl.sub.2 3.0 mM KC1 75 mM
DTT 10 mM dNTPs mM each MMLV-RT 50 U RNAGuard .TM. 18 U Total
reaction volume 20 .mu.l
[0218] Incubate reaction tubes at 65.degree. C. for 15 min. This
inactivates the reverse transcriptase activity of MMLV prior to the
WT step. Move reaction tubes to ice. Store reaction tubes on ice
for 5 min. [0219] 5. Immediately before use, mix the following
components in the order indicated at room temperature:
TABLE-US-00006 [0219] Transcription Mix Component Volume (.mu.l)
Nuclease-free water 22.8 5x Transcription Buffer 16 100 mM DTT 6.0
NTPs (25 mM A, G, C, 6.0 mM UTP) 8.0 aa UTP (75 mM) 2.0 200 mM
MgCl.sub.2 3.3 RNAGuard .TM. (36 U/.mu.l) 0.5 Inorganic
Pyrophosphatase (200 U/ml) 0.6 T7 RNA polymerase (2500 U/.mu.l) 0.8
Volume of Transcription Mix 60
[0220] 6. Aliquot 60 .mu.l of Transcription Mix into each sample
tube. Incubate transcription reactions at 40.degree. C. for 16
hrs.
TABLE-US-00007 [0220] Composition of Transcription Reaction
Component Final concentration or amount Double-strand cDNA
Approximately 400 ng Tris-HCl, pH 7.5 52 mM MgCl.sub.2 15 mM KCl 19
mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, CTP 2.5 mM each
UTP 0.6 mM aa UTP 1.9 mM T7 RNA polymerase 2000 U RNAGuard .TM. 18
U Inorganic pyrophosphatase 0.12 U Total reaction volume 80
.mu.l
[0221] 7. RNeasy.RTM. (QIAGEN Inc.) Purification of reactions:
[0222] Add 20 .mu.l water to 80 .mu.l reaction tube. [0223]
Transfer to mixing tube. [0224] Add 350 .mu.l RLT buffer (QIAGEN
Inc.) (plus 2-.beta.-mercaptoethanol), mix well. [0225] Add 250
.mu.l 100% EtOH, mix well. [0226] Transfer to RNeasy.RTM. column.
[0227] Spin 30 seconds in microfuge, 10 K. [0228] Transfer column
to new collection tube. [0229] Add 700 .mu.l 80% EtOH. [0230] Spin
30 seconds in microfuge, 10 K. [0231] Discard flow through. [0232]
Add 700 .mu.l 80% EtOH. [0233] Spin 30 seconds in microfuge, 10 K.
[0234] Transfer column to new collection tube. [0235] Spin 2
minutes, 14 K to dry filter. [0236] Place column in microfuge tube.
[0237] Add 55 .mu.l of nuclease-free water to filter. Let sit 1
minute. [0238] Spin 14 K, 2 minutes. [0239] Add 55 .mu.l of
nuclease-free water to filter. Let sit 1 minute. [0240] Spin 14 K,
2 minutes.
[0241] To quantitate the yield of amplified RNA product, remove a
10.0 .mu.l aliquot of the product and dilute into 90 .mu.l
dH.sub.2O. Add samples to a Costar UV-transparent plate and measure
A260, A280 using a Spectramax (GRM Reader) and template for
whichever lot of plates you are using. Calculate yield using the
relationship A260=1 corresponds to 40 .mu.g/ml. Conversion factor
for Spectramax=3.59 (i.e. multiply A260 bp 3.59 when calculating
yield). [0242] 8. In speed vac, dry down 10 .mu.g per
fluor-reversed pair. [0243] 9. Coupling Reactions: [0244] Resuspend
10 .mu.g IVT product in 7 .mu.l water (or water plus El a) and
divide into two tubes. One tube will be coupled with Cy3 and one
with Cy5.
[0245] Preparation of 3.times. Sodium Bicarbonate Buffer:
[0246] Place the contents of one Carbonate-Bicarbonate Buffer
capsule (Sigma, P/N C-3041) into a 50 ml Falcon tube.
[0247] Add 16.7 ml RNase free water and mix well.
[0248] Add 125 .mu.l 37% HCl and mix.
[0249] pH should be 9-9.5.
[0250] Preparation of Cy-NHS Dyes:
[0251] Spin dye briefly before opening tube.
[0252] Add 10 .mu.l anhydrous MSO to dye.
[0253] Mix by pipetting 20 times.
[0254] Set a pipettman at 3.5 .mu.l.
[0255] Work quickly since the amino esters are unstable in aqueous
environment.
[0256] Add 20 .mu.l 13.times. sodium bicarbonate buffer to dye and
mix well.
[0257] Add 3.5 .mu.l dye to each tube of cRNA. Mix well.
[0258] Incubate in the dark for 1 hour.
[0259] Stop the reaction by adding 3.5 .mu.l 4M HA
(hydroxylamine)
[0260] Incubate 10 minutes. [0261] 10. Repeat RNeasy.RTM. clean-up
as in Step 7, above, except elute in 70.degree. C. nuclease-free
water. [0262] 11. Measure yield and percent incorporation in a
Costar UV plate. Calculate concentration of RNA using 1
OD.sub.260=40 .mu.g/ml RNA. Overall amplification yield is
calculated by multiplying RNA concentration (4/ml) by the sample
volume (0.1 ml) and dividing by the amount of poly-A.sup.+ RNA
initially added to the reaction. Calculate concentration of Cy3-CTP
using .epsilon.(552 nm)=150 (1/mMcm). Calculate concentration of
Cy5-CTP using .epsilon.(650 nm)=250 (1/mMcm).
[0263] Generation of Gene Expression Profile Signatures:
[0264] Source mRNA from Jurkat and K562 cell lines was used to
generate gene expression profile signatures by amplification and
labeling using the Shannon method and using the random-primed
RT-IVT method, followed by hybridization to DNA microarrays.
Approximately 5 .mu.g of Cy-labeled cRNA from each cell line was
hybridized as fluor-reversed pairs to a DNA microarray pattern with
probes tiled (overlapped) across all mRNA sequence for
approximately 33 RefSeq test genes (LocusLink database,
www.ncbi.nlm.nih.gov/locuslink/build.html) known to exhibit 3'
amplification bias when amplified by the Shannon method. Analysis
was performed either on a gene-by-gene basis or with all
oligonucleotides at once. The first goal of the study was to
determine whether the random-primed RT-IVT method produced a
full-length cRNA. The second goal of the study was to determine
whether the random-primed RT-IVT method has less of a 3' bias when
compared with the Shannon method.
[0265] 6.2. Results and Discussion
[0266] FIG. 1 compares the profiles obtained from single-gene
analysis using the mRNA amplification method described in U.S. Pat.
No. 6,132,997 (Shannon, issued Oct. 17, 2000) ("Shannon") and the
random-primed RT-IVT method of the invention. The graphs plot
signal intensity (mlavg) of oligonucleotides in a single gene
(X-axis) as a function of the number of by from the 5' end
(Y-axis). The 3' bias of signal intensity seen when the Shannon
method is used cannot be seen when the random-primed RT-IVT method
is used, indicating that the random-primed RT-IVT method overcomes
the 3' bias of the Shannon method.
[0267] FIG. 2 shows the intensity difference as a function of
distance from the 3' end. The graph shows the intensity of all
oligonucleotides as a function of distance from the 3' end.
[0268] The graph plots mlavg (Shannon method)--mlavg (random-primed
RT-IVT method) (X-axis) versus log.sub.10 of the number of by from
the 3' end (Y-axis). The intensity obtained with the Shannon method
is greater than the intensity obtained with the random-primed
RT-IVT method for probes less than 1000 bp from the 3' end of the
message. The intensity obtained with the Shannon method is less
than the intensity obtained with the random-primed RT-IVT method
for probes greater than 1000 bp from the 3' end of the message.
[0269] At xdev threshold 2.5 (.about.Pvalue 1%), the following
number of signatures were obtained (Table 2):
TABLE-US-00008 TABLE 2 Forward Reverse Total by < 1000 bp >
1000 total by < 1000 bp > 1000 random- 2486 1488 998 2237
1367 870 primed RT-IVT Shannon 2587 1927 660 1218 892 326 method #
of 7416 2965 4451 7413 2962 4451 probes
[0270] FIGS. 3(A-C) shows[show?] the signature differences in the
numbers and percentages of significant data points. The top graph
(A) plots the number of probes (X-axis) versus the [log.sub.10](bp)
(Y-axis). The middle graph (B) plots the number of signatures
(X-axis) versus the [log.sub.10](bp) (Y-axis). The bottom graph (C)
plots the fraction of signatures versus the [log.sub.10] (bp)
(Y-axis). As can be seen in the bottom graph, the random-primed
RT-IVT method outcompetes the Shannon method for probes greater
than 1000 bp from the 3' end. Note the black arrow at approximately
700 bp where random-primed RT-IVT method becomes better than the
Shannon method. Stars: Shannon method. Circles: random-primed
RT-IVT method.
[0271] FIGS. 4(A-C) shows the results obtained when the
amplification methods of the invention were run using a primer
comprising a T7 RNA polymerase promoter site and a poly-dT.sub.18
sequence ("T7-dT.sub.18"), in addition to using random T7-d N.sub.9
and dN.sub.6 primers. The top graph (A) plots the number of probes
(X-axis) versus the log.sub.10 (bp) (Y-axis). The middle graph (B)
plots the number of signatures (X-axis) versus the log.sub.10 (bp)
(Y-axis). The bottom graph (C) plots the fraction of signatures
versus the log.sub.10 (bp) (Y-axis). As can be seen in the bottom
graph, the random-primed RT-IVT method helps improve the fraction
of significant probes at by <1000. Using both the T7-dT.sub.18
and random T7-dN.sub.9 primers for first strand cDNA synthesis
improves the fraction of significant probes more efficiently than
either the Shannon method or the method of the invention in which
just the random T7-d N.sub.9 primer is used. Stars: Shannon method.
Circles: random-primed RT-IVT method.
[0272] These results indicate that the performance of random-primed
RT-IVT is stable. The average yield obtained was 20 .mu.g. The
protocol produced little or no 3' bias and improved the ability to
detect the 5' ends of mRNA. Linear amplification extents of
100-fold and labeling efficiencies of approximately 3% can be
achieved using this method. When poly-dT and random dN primers,
both of which comprise a T7 RNA polymerase promoter sequence, are
used together to prime first strand cDNA synthesis, the fraction of
significant probes is greater than that obtained with either the
Shannon method or the method of the invention in which just a
random T7-dN.sub.9 primer is used.
[0273] The above results and discussion demonstrate that novel and
improved methods of producing linearly amplified amounts of RNA
from an initial RNA source are provided. The methods of the
invention provide an improvement over prior methods of producing
linearly amplified RNA in that the protocol produces little or no
3' bias and improves the ability to detect the 5' ends of mRNA.
Furthermore, linear amplification extents of at least 100-fold can
be achieved using the subject methods. Finally, all of the benefits
of linear amplification are achieved with the subject methods, such
as the production of unbiased antisense RNA libraries from
heterogeneous mRNA mixtures. As such, the subject methods represent
a significant contribution to the art.
[0274] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0275] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0276] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended claims
along with the full scope of equivalents to which such claims are
entitled.
[0277] While illustrative embodiments have been illustrated and
described, it will be appreciated that various changes can be made
therein without departing from the spirit and scope of the
invention.
Sequence CWU 1
1
1136DNAArtificialSynthetic 1aattaatacg actcactata gggagatnnn nnnnnn
36
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References