U.S. patent application number 13/027162 was filed with the patent office on 2012-06-21 for methods for making transcription products.
This patent application is currently assigned to EPICENTRE TECHNOLOGIES. Invention is credited to Gary A. Dahl, Jerome J. Jendrisak.
Application Number | 20120156679 13/027162 |
Document ID | / |
Family ID | 32393336 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156679 |
Kind Code |
A1 |
Dahl; Gary A. ; et
al. |
June 21, 2012 |
METHODS FOR MAKING TRANSCRIPTION PRODUCTS
Abstract
The present invention provides methods, compositions and kits
for using an RNA polymerase for making transcription products
corresponding to a target sequence by obtaining circular
single-stranded DNA transcription substrates using a promoter
primer that encodes one strand of a double-stranded promoter. The
invention has broad applicability for research, diagnostic and
therapeutic applications, such as preparing cDNA corresponding to
mRNA, making sense or anti-sense probes, detecting gene- or
organism-specific sequences, or making RNAi.
Inventors: |
Dahl; Gary A.; (Madison,
WI) ; Jendrisak; Jerome J.; (Madison, WI) |
Assignee: |
EPICENTRE TECHNOLOGIES
Madison
WI
|
Family ID: |
32393336 |
Appl. No.: |
13/027162 |
Filed: |
February 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10719913 |
Nov 21, 2003 |
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13027162 |
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60428013 |
Nov 21, 2002 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12N 15/1096 20130101;
C12Q 1/6853 20130101; C12Q 1/6853 20130101; C12Q 2531/119 20130101;
C12Q 1/6853 20130101; C12Q 2525/125 20130101; C12Q 2521/301
20130101; C12Q 2521/301 20130101; C12Q 2531/119 20130101 |
Class at
Publication: |
435/6.12 ;
435/91.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A method for making a transcription product corresponding to a
target sequence in a target nucleic acid, the method comprising:
(a) obtaining a target nucleic acid; (b) obtaining a sense promoter
primer, the sense promoter primer comprising a 5'-end portion
comprising a sense transcription promoter and a 3'-end portion that
is complementary to the target; (c) annealing the sense promoter
primer with the target nucleic acid so as to form a target nucleic
acid-sense promoter primer complex; (d) contacting the target
nucleic acid-sense promoter primer complex with a DNA polymerase
under polymerization reaction conditions so as to obtain
first-strand cDNA that is complementary to the target sequence; (e)
obtaining first-strand cDNA; (f) ligating the first-strand cDNA
under ligation conditions so as to obtain circular sense
promoter-containing first-strand cDNA; (g) obtaining an anti-sense
promoter oligo; (h) annealing the anti-sense promoter oligo to the
circular sense promoter-containing first-strand cDNA so as to
obtain a circular transcription substrate; (i) obtaining the
circular transcription substrate; and (j) contacting the circular
transcription substrate with an RNA polymerase under transcription
conditions, wherein a transcription product is obtained.
2. The method of claim 1 wherein the anti-sense promoter oligo
comprises an oligo that is immobilized on a solid support.
3. A method for detecting an analyte in or from a sample, the
method comprising: a) obtaining an analyte-binding
substance-oligonucleotide ("ABS-oligo"), wherein the ABS-oligo
comprises an ABS that is joined to a oligonucleotide comprising a
sequence for an anti-sense promoter portion of a double-stranded
promoter for an RNA polymerase that recognizes the promoter; b)
obtaining a Signal Probe, wherein the Signal Probe comprises a
sense promoter that is joined to the 3'-end of a template, wherein
the sense promoter is sufficiently complementary to the anti-sense
promoter of the ABS-oligo to form a complex that can be used for
transcription of the template using an RNA polymerase that binds to
the complex; c) contacting an ABS-oligo with a surface to which an
analyte is bound if present in a sample under analyte-binding
conditions that permit the ABS-oligo to bind the analyte if present
on said surface; d) washing the surface under conditions that
permit removal of unbound ABS-oligo; e) contacting the surface with
a Signal Probe under complexing conditions that permit complexing
of the Signal Probe with the ABS-oligo if present on the surface;
f) optionally, washing the surface under conditions that permit
removal of unbound Signal Probe; g) contacting the surface with an
RNA polymerase under conditions that permit transcription of a
product encoded by the template using the complex between the
ABS-oligo and the Signal Probe; and h) detecting a transcription
product encoded by the template, if present.
4. A method for amplifying the amount of a template-complementary
transcription product, the method comprising: a) obtaining a
transcription product; b) obtaining a sense promoter primer
comprising a 3'-end portion that is complementary to the 3'-end of
the transcription product and optionally, a phosphate group or a
topoisomerase moiety on its 5-end; c) annealing the sense promoter
primer to the transcription product; d) primer-extending the
promoter primer annealed to the transcription product with an
RNA-dependent DNA polymerase under DNA synthesis conditions so as
to obtain first-strand cDNA; e) optionally, removing the RNA that
is annealed to the first-strand cDNA; f) ligating the first-strand
cDNA, wherein the 5'-end is covalently joined to the 3'-end of the
first-strand cDNA so as to obtain circular sense
promoter-containing first-strand cDNA; g) annealing an anti-sense
promoter oligo to the circular sense promoter-containing
first-strand cDNA so as to obtain a circular substrate for
transcription; h) contacting the circular substrate for
transcription with an RNA polymerase under transcription conditions
so as to obtain additional transcription product; and i) obtaining
the additional transcription product.
5. The method of claim 4 wherein the sense promoter primer
comprises a single-stranded sense promoter chosen from the group
consisting of a pseudopromoter or a synthetic promoter that is used
by an RNA polymerase to make additional transcription product and
wherein an anti-sense promoter oligo is not used to obtain
additional transcription product.
6. The method of claim 4 wherein the sense promoter primer
comprises an N4 promoter and the RNA polymerase used for
transcription is chosen from among N4 vRNAP, mini-vRNAP, and a
mutant mini-vRNAP, and wherein an anti-sense promoter oligo is not
used to obtain additional transcription product.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/428,013, filed Nov. 21, 2002. The entire
disclosure of all priority applications is specifically
incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
FIELD OF THE INVENTION
[0003] The present invention relates generally to methods,
compositions and kits for using an RNA polymerase for making
transcription products corresponding to a target sequence by
obtaining circular single-stranded DNA transcription substrates
using a promoter primer that encodes one strand of a
double-stranded promoter. The invention has broad applicability for
research, diagnostic and therapeutic applications, such as
preparing cDNA corresponding to mRNA, making sense or anti-sense
probes, detecting gene- or organism-specific sequences, or making
RNAi.
BACKGROUND OF THE INVENTION
Description of Related Art
[0004] It is well known in the art that a target sequence can be
transcribed in vitro or in vivo if the target sequence is inserted
into a cloning vector downstream of a transcription promoter, such
as, but not limited to a T7 RNA polymerase promoter or another
T7-type RNA polymerase promoter and the resulting clone is
incubated under transcription conditions with an RNA polymerase
that recognizes the respective promoter or the clone is transformed
into a host cell that can express the RNA polymerase (e.g., see
Studier, F W et al., pp. 60-89 in Methods in Enzymology, Vol. 185,
ed. by Goeddel, D V, Academic Press, 1990, incorporated herein by
reference). Under suitable conditions, this system can also be used
to express a protein encoded by a target sequence comprising a gene
in vitro or, in an appropriate host cell, in vivo.
[0005] There are many reasons for which it is beneficial to
transcribe one or more target nucleic acid sequences. For example,
in vitro transcription is frequently used in methods to make probes
corresponding to mRNA sequences in samples in order to profile the
expression of genes in cells of a particular type versus another
type or versus a similar type in response to different conditions
or stimuli. Contemporary gene expression profiling is typically
performed by simultaneously hybridizing labeled probes prepared
from one or more samples to arrays or microarrays having sequences
for up to hundreds or thousands of different genes attached to a
surface. In some cases, the expression or lack of expression of
particular genes may correlate with or be indicative of the
presence or status of a disease state, such as, but not limited to,
a cancer.
[0006] A number of the methods for making probes corresponding to a
target sequence for such purposes are known in the art. Examples of
methods that involve in vitro transcription for making probes for
gene expression profiling are described in: Murakawa et al., DNA
7:287-295, 1988; Phillips and Eberwine, Methods in Enzymol. Suppl.
10:283-288, 1996; Ginsberg et al., Ann. Neurol. 45:174-181, 1999;
Ginsberg et al., Ann. Neurol. 48:77-87, 2000; VanGelder et al.,
Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990; Eberwine et al.,
Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992; U.S. Pat. Nos.
5,021,335; 5,168,038; 5,545,522; 5,514,545; 5,716,785; 5,891,636;
5,958,688; 6,291,170; and PCT Patent Applications WO 00/75356 and
WO 02/065093.
[0007] Still other methods use in vitro transcription as part of a
process for amplifying and detecting one or more target nucleic
acid sequences in order to detect the presence of a pathogen, such
as a viral or microbial pathogen, that is a causative agent for a
disease or to detect a gene sequence that is related to a disease
or the status of a disease for medical purposes. Examples of
methods that use in vitro transcription for this purpose include
U.S. Pat. Nos. 5,130,238; 5,194,370; 5,399,491; 5,409,818;
5,437,990; 5,466,586; 5,554,517; 5,665,545; 6,063,603; 6,090,591;
6,100,024; 6,410,276; Kwoh et al., Proc. Natl. Acad. Sci. USA
86:1173, 1989; Fahy et al, In: PCR Methods and Applications, pp.
25-33, 1991; PCT Patent Application Nos. WO 89/06700 and WO
91/18155; and European Patent Application Nos. 0427073 A2 and
0427074 A2.
[0008] The use of a single-stranded DNA template for transcription
is not common. However, Milligan et al. (Nucleic Acids Res., 15:
8783-8798, 1987) showed that a double-stranded DNA template is not
necessary to synthesize RNA using T7 RNA polymerase, provided that
the single-stranded DNA template contained a double-stranded -17 to
+1 T7 promoter region. Also, a method for synthesizing RNA using a
single-stranded DNA template that is non-covalently immobilized on
a solid support by annealing to a complementary promoter sequence
is described in U.S. Pat. No. 5,700,667. In addition, Japanese
Patent No. JP4304900 of Aono Toshiya et al. disclose synthesis of
RNA using a circular single-stranded template with a
double-stranded T7 RNA polymerase promoter. The circular substrate
for transcription disclosed in JP4304900 is obtained by ligation of
a linear probe having target-complementary 3'- and 5'-end sequences
which are adjacent when the linear probe is annealed to a target
sequence in the sample.
[0009] Nevertheless, all of the methods referenced above that
obtain a probe or a transcription substrate by replication of a
target nucleic acid sequence involve synthesis of linear
double-stranded DNA comprising a double-stranded transcription
promoter that is operably joined to a double-stranded template.
[0010] The reason the methods in the art that obtain a probe or
transcription substrate by replication of a target sequence have
used double-stranded DNA templates is understood when the processes
for these methods are examined in detail. For example, the methods
described by Van Gelder et al. in U.S. Pat. Nos. 5,545,522;
5,716,785; and 5,891,636 use a promoter primer to synthesize a
double-stranded transcription substrate. First, the promoter
primer, which comprises a promoter sequence that is 5'- of a 3'-end
portion, wherein the 3'-end portion is complementary to one or more
mRNA target sequences, is used to reverse transcribe the mRNA
target. Then, second-strand cDNA is synthesized using RNA or a
hairpin from the first-strand cDNA as a primer. Since RNA
polymerase transcribes a template strand in a 5'-to-3' direction,
the only way a promoter sequence can be joined to a target sequence
in the correct orientation using a promoter primer according to
this method is to replicate the promoter sequence of the promoter
primer at the 3'-end of the second-strand cDNA. That is, the
promoter sequence in this promoter primer directs transcription of
RNA using second-strand cDNA as a template. The template strand for
transcription therefore has a Watson-Crick base-pairing sequence
that is identical to an mRNA target sequence in the sample and the
transcription product comprises anti-sense RNA. The single-stranded
promoter sequence used in the promoter primer of Van Gelder et al.,
which is one strand of a functional double-stranded promoter, is
referred to herein as an "anti-sense promoter" and the
corresponding promoter primer is referred to herein as an
"anti-sense promoter primer". Similarly, the promoter sequence of a
double-stranded promoter that is operably joined to the template
strand is referred to as a "sense promoter sequence" herein and a
promoter primer that comprises this sequence is referred to as a
"sense promoter primer."
[0011] The present invention provides, in part, a novel method for
using sense promoter primers in order to obtain transcription
products corresponding to a target sequence. The method makes
transcription substrates comprising a single-stranded DNA template
that encodes the target sequence, which ssDNA template is operably
joined to a functional double-stranded promoter. Additional aspects
of the invention will be understood from the specification
below.
BRIEF SUMMARY OF THE INVENTION
[0012] The methods in the art use anti-sense promoter primers,
whereas some embodiments of the present invention provide methods
that use sense promoter primers. Thus, by way of example, but
without limiting the invention with respect to the promoter
sequence or the respective RNA polymerase used for transcription,
whereas the anti-sense T7 promoter sequence and +1 base used in a
promoter primer in U.S. Pat. Nos. 5,545,522; 5,716,785; and
5,891,636 is:
TABLE-US-00001 (5' TAATACGACTCACTATAG 3');
[0013] a corresponding sense T7 promoter sequence and +1 base that
can be used in a promoter primer in some embodiments of the present
invention is:
TABLE-US-00002 (5' CTATAGTGAGTCGTATTA 3').
[0014] The present invention provides methods, compositions and
kits for making transcription products corresponding to a target
nucleic acid sequence in a sample by transcription of circular
transcription substrates. A first step of the method to obtain
circular transcription substrates, as shown in FIG. 1, is primer
extension of sense promoter primers using a target sequence as a
template. The resulting sense promoter-containing first-strand cDNA
is then ligated using a ligase under ligation conditions to obtain
circular sense promoter-containing first-strand cDNA. A circular
transcription substrate is obtained by annealing of a complementary
anti-sense promoter oligo to the circular sense promoter-containing
first-strand cDNA. The circular transcription substrate comprises a
double-stranded transcription promoter that is operably joined to a
single-stranded template corresponding to a target sequence.
[0015] The invention also comprises methods to obtain linear
transcription substrates. In one embodiment, shown in FIG. 2,
circular sense promoter-containing first-strand cDNA (obtained as
described above) is linearized using methods described herein
below. Then, the resulting linear sense promoter-containing
first-strand cDNA is then complexed with an anti-sense promoter
oligo to obtain a linear transcription substrate. Alternatively,
the circular transcription substrate can be linearized to obtain a
linear transcription substrate comprising a double-stranded
transcription promoter that is operably joined to a single-stranded
template. Both circular and linear transcription substrates can be
used to make transcription products corresponding to target nucleic
acid sequences.
[0016] The invention also includes methods for amplifying the
amount of transcription products obtained from transcription of a
transcription substrate. One embodiment, shown in FIG. 3, uses
transcription products obtained from transcription of a first
transcription substrate to make additional transcription substrates
using a sense promoter primer according to methods of the present
invention described herein.
[0017] The invention also comprises embodiments of the methods that
obtain a transcription substrate by annealing of an anti-sense
promoter oligo to a sense promoter-containing first-strand or
second-strand cDNA which use an oligonucleotide comprising an
anti-sense promoter oligo that is attached or immobilized on a
surface, wherein the sense promoter containing first-strand or
second-strand cDNA is complexed with the immobilized anti-sense
promoter oligo to obtain an immobilized transcription substrate
which is used to obtain transcription products corresponding to the
target nucleic acid. Immobilization of the anti-sense promoter
oligo permits methods and assays using dipsticks, arrays and the
like. Methods for using an immobilized circular or linear
transcription substrates to detect transcription products
corresponding to a target sequence are shown in FIG. 4 and FIG. 5,
respectively.
[0018] If a transcription substrate is obtained by using a sense
promoter primer to prime synthesis of first-strand cDNA and the
target nucleic acid as a template, transcription of the
transcription substrate results in sense transcription
products.
[0019] However, in some embodiments of the invention, anti-sense
transcription products are obtained. One method for obtaining
anti-sense transcription products is shown in FIG. 6. Briefly, this
method uses a primer comprising anti-sense promoter sequence to
prime synthesis of first-strand cDNA. Then, after circularizing the
first-strand cDNA by ligation, a concatemeric second-strand cDNA is
obtained by rolling circle replication using a strand displacement
primer and a strand-displacing DNA polymerase. The second-strand
cDNA comprises sense promoter sequences. Annealing of an anti-sense
promoter oligo to the sense promoter sequences permits
transcription of anti-sense transcription products with respect to
the target sequence of the target nucleic acid.
[0020] In another embodiment (not illustrated), a sense promoter
primer is used according to the invention to obtain transcription
substrates for synthesis of anti-sense transcription products. In
these embodiments of the invention, synthesis of first-strand cDNA
that is complementary to the target nucleic acid comprising the
target sequence is carried out using a primer that lacks a promoter
sequence. If the sequence of the first-strand cDNA is known, a
sense promoter primer that has a sequence at its 3'-end that is
complementary to a specific 3' sequence of the first-strand cDNA
can then be used to prime synthesis of second-strand cDNA.
Alternatively, the first-strand cDNA can be "tailed" and an
additional sequence that is not complementary to the target
sequence can be added to the 3'-end of the first-strand cDNA using
methods, such as those known in the art (e.g., U.S. Pat. No.
5,962,272 and Schmidt, W M and Mueller, M W, Nucleic Acids Res.,
27: e31, 1999 or as described herein. The additional sequence that
is added to the 3'-end of the first-strand cDNA can be used as a
unique priming site for annealing of a sense promoter primer of the
invention. A tail and/or additional sequence for binding of a sense
promoter primer is especially useful if the sequence of the
3'-portion of the first-strand cDNA is not known or if the sense
promoter primer is used to prime synthesis of a multiplicity of
target sequences comprising the target nucleic acid (e.g., to prime
synthesis of first-strand cDNA corresponding to substantially all
mRNA molecules in a sample). Primer extension of the sense promoter
primer using first-strand cDNA as a template (including the tail or
additional sequence, if present) results in synthesis of sense
promoter-containing second-strand cDNA, which can be used to obtain
a circular sense promoter-containing second-strand cDNA by ligation
using a ligase under ligation conditions. Then, a circular
transcription substrate of the invention can be obtained by
annealing an anti-sense promoter oligo to the circular sense
promoter-containing second-strand cDNA. The circular transcription
substrate can be linearized using methods described herein to
obtain a linear transcription substrate. Alternatively, the
circular sense promoter-containing second-strand cDNA can be
linearized and then annealed to an anti-sense promoter oligo to
obtain a linear transcription substrate. Transcription of the
resulting circular or linear transcription substrates yield
transcription products that are anti-sense transcription products
with respect to the starting target nucleic acid.
[0021] Yet another aspect of the invention is a signaling system
for detecting and/or quantifying analytes of any type, including,
without limitation, protein, carbohydrate, nucleic acid or other
analytes. Embodiments of this aspect of the invention use Signal
Probes, including RCT Signal Probes or LINT Signal Probes, which
comprise a sense promoter that is joined to the 3'-end of a signal
template in order to detect and/or quantify an ABS-oligo comprising
an analyte-binding substance that is joined to an oligonucleotide
comprising an anti-sense promoter sequence. Transcription of the
Signal Probe-ABS-oligo indicates the presence and/or quantity of
the analyte in the sample. In this aspect of the invention, the
template sequence is not obtained by copying a target sequence in a
sample. Rather, the template that is transcribed by an RNA
polymerase to detect and/or quantify an analyte serves as a
"signal" for the analyte-binding substance and for the analyte.
Embodiments of the invention that use Signal Probes enable simple
assays and methods, such as, but not limited to those illustrated
in FIGS. 7 and 8.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0023] FIG. 1--Schematic of an embodiment of the invention that
uses a sense promoter primer to obtain a circular transcription
substrate.
[0024] FIG. 2--Schematic of an embodiment of the invention for
obtaining a linear transcription substrate by linearization of a
circular sense promoter-containing first-strand cDNA and complexing
with an anti-sense promoter oligo.
[0025] FIG. 3--Schematic of an embodiment of a method or assay that
uses a sense promoter primer having a transcription termination
sequence in order to obtain a circular transcription substrate that
is used to obtain additional transcription product. The assay can
be performed in a stepwise or in a continuous manner.
[0026] FIG. 4--Schematic of an assay that is used to detect a
transcription product corresponding to a target sequence in a
target nucleic acid in a sample. In this example, the sense
promoter-containing first-strand cDNA is annealed to an anti-sense
promoter sequence that is immobilized on a solid support, such as a
dipstick or a bead. The resulting immobilized circular
transcription substrate is transcribed by rolling circle
transcription and the transcription product is detected using a
molecular beacon.
[0027] FIG. 5--Schematic of an assay that uses a linear
transcription substrate made by linearizing a circular sense
promoter-containing first-strand cDNA and complexing with an
anti-sense promoter oligo that is immobilized on a solid support to
make a transcription product corresponding to a target sequence in
a target nucleic. The transcription product can be detected by any
of a variety of methods known in the art.
[0028] FIG. 6--Embodiment that uses an anti-sense promoter primer.
Primer extension of the promoter primer and ligation of the
first-strand cDNA makes a circular anti-sense-promoter-containing
first-strand cDNA that serves as a template for rolling circle
replication, the product of which is a linear sense
promoter-containing second-strand cDNA. Annealing of an anti-sense
promoter oligo makes a concatemeric linear transcription substrate
that is used to make a transcription product.
[0029] FIG. 7--Schematic of an assay that uses an RCT Signal Probe
to detect an analyte using an analyte-binding substance comprising
a second antibody to which a polynucleotide comprising an
anti-sense promoter sequence is joined. The anti-sense promoter
sequence complexes the sense promoter of the RCT Signal Probe and
thereby generates a substrate for transcription by an RNA
polymerase that recognizes the double-stranded promoter and
initiates rolling circle transcription therefrom under
transcription conditions. In this example, a molecular beacon is
used to detect the transcription product, which indicates the
presence of the analyte. If performed with controls of known
quantities of analyte, the level of analyte can also be
determined.
[0030] FIG. 8--Embodiment of a method that uses a LINT Signal Probe
to detect, and under suitable conditions, to quantify an analyte in
a sample using an analyte-binding substance comprising a second
antibody to which a polynucleotide comprising an anti-sense
promoter sequence is joined. In this embodiment The anti-sense
promoter sequence complexes the sense promoter of a LINT Signal
Probe and thereby generates a substrate for transcription by an RNA
polymerase that recognizes the double-stranded promoter and
initiates linear run-off transcription therefrom under
transcription conditions. The transcription product can be detected
by any of a variety of methods known in the art.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention relates to novel methods,
compositions, and kits for synthesizing RNA corresponding to one or
multiple target nucleic acid sequences in or from a sample. The
target sequence can comprise at least a portion of one or more
target nucleic acids comprising either RNA or DNA from any source.
By way of example, but not of limitation, the target nucleic acid
sequences that are transcribed using a method of the invention can
comprise first-strand cDNA corresponding to from one to
substantially all mRNA molecules in a sample. Alternatively, the
target nucleic acid sequence that is transcribed can comprise a
specific sequence in genomic DNA of a particular organism. The
present invention overcomes deficiencies in the art by providing
methods to obtain circular DNA transcription substrates that
comprise a double-stranded promoter that is operably joined to a
single-stranded template for transcription and provides a unique
system to synthesize RNAs of a desired sequence. The circular
transcription substrates can be used to synthesize RNA for use as
probes for expression studies using arrays or microarrays, for
RNase protection studies, for in situ hybridization studies, for
Southern and Northern blot analysis, for the synthesis of defined
RNA:DNA hybrids, for RNA interference, for in vitro translation,
for microinjection, or for nucleic acid amplification. The present
invention also allows for the synthesis of derivatized RNA for
these or other applications.
[0032] In general, it is envisioned that the RNA or transcription
product probes described herein will be useful both as reagents in
solution hybridization for detection of expression of corresponding
genes, as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the test DNA (or RNA) is
adsorbed or otherwise affixed to a selected matrix or surface. This
fixed, single-stranded nucleic acid is then subjected to
hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic
acid, size of hybridization probe, etc.). Following washing of the
hybridized surface to remove non-specifically bound probe
molecules, hybridization is detected, or even quantified, by means
of the label.
Methods for Making Transcription Products Corresponding to a Target
Nucleic Acid Sequence
[0033] One aspect of the invention comprises a method for making a
transcription product corresponding to a target sequence in a
target nucleic acid, the method comprising:
[0034] (a) obtaining a target nucleic acid;
[0035] (b) obtaining a sense promoter primer, the sense promoter
primer comprising a 5'-end portion comprising a sense transcription
promoter and a 3'-end portion that is complementary to the
target;
[0036] (c) annealing the sense promoter primer with the target
nucleic acid so as to form a target nucleic acid-sense promoter
primer complex;
[0037] (d) contacting the target nucleic acid-sense promoter primer
complex with a DNA polymerase under polymerization reaction
conditions so as to obtain first-strand cDNA that is complementary
to the target sequence;
[0038] (e) obtaining first-strand cDNA;
[0039] (f) ligating the first-strand cDNA under ligation conditions
so as to obtain circular sense promoter-containing first-strand
cDNA;
[0040] (g) obtaining an anti-sense promoter oligo;
[0041] (h) annealing the anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate;
[0042] (i) obtaining the circular transcription substrate; and
[0043] (j) contacting the circular transcription substrate with an
RNA polymerase under transcription conditions, wherein a
transcription product is obtained.
[0044] The invention comprises use of any polymerase that
synthesizes a transcription product using a circular transcription
substrate that is obtained by circularization of a primer extension
product obtained by primer extension on a target sequence of a
sense promoter primer, the sense promoter primer comprising a
sequence that encodes a sense strand of a transcription promoter in
its 5'-end portion and a target-complementary sequence in its
3'-end portion, and wherein the sense promoter is operably or
functionally joined or linked to the primer extension product
comprising the target sequence by ligating the sense promoter
sequence to the 3'-end of the primer extension product and
complexing the resulting circular ssDNA with an oligo comprising an
anti-sense promoter sequence that is complementary to the sense
promoter sequence therein (unless a single-stranded sense
pseudopromoter is used in the promoter primer in place of a sense
strand for a double-stranded promoter). Thus, the methods of the
invention can use any RNA polymerase for which a suitable sense
promoter sequence is known or can be obtained.
[0045] Preferred promoters of the invention comprise promoters for
T7-type RNA polymerases. By "T7-type RNAPs" we mean T7, T3, phi,
phalli, W31, gh1, Y, A1122, SP6 and mitochondrial RNAPs, as well as
mutant forms of these RNAPs (Sousa et al., U.S. Pat. No. 5,849,546;
Padilla, R and Sousa, R, Nucleic Acids Res., 15: e138, 2002; Sousa,
R and Mukherjee, S, Prog Nucleic Acid Res Mol. Biol., 73: 1-41,
2003), such as, but not limited to T7 RNAP Y639F mutant enzyme, T3
RNAP Y573F mutant enzyme, SP6 RNAP Y631F mutant enzyme, T7 RNAP
having altered amino acids at both positions 639 and 784, T3 RNAP
having altered amino acids at both positions 573 and 785, or SP6
RNAP having altered amino acids at both positions 631 and 779, or
other mutant forms of an RNAP that functions in a method of the
invention.
[0046] Sense promoters of the invention also comprise
single-stranded pseudopromoters or synthetic promoters that are
recognized by an RNA polymerase (RNAP) so as to function in a
method of the invention. A "pseudopromoter" or "synthetic promoter"
of the present invention can be any single-stranded sequence that
is identified and/or selected to be functional as a promoter for in
vitro transcription by an RNA polymerase that binds the promoter
with specificity and functions as a promoter for the RNA polymerase
in a transcription reaction. That is, a single-stranded
pseudopromoter or synthetic promoter that is recognized by the RNA
polymerase is not suitable for aspects of the invention that used
Signal Probe, which is incorporated herein by reference.
Single-stranded promoters for phage N4 vRNAP can also be used in
some embodiments, in which case a wild-type or mutant form of N4
mini-vRNAP is used in the method or kit, both of which are
discussed elsewhere herein.
[0047] If a pseudopromoter or synthetic promoter is used as a sense
promoter in a method or assay of the invention, then the
corresponding RNA polymerase for which the pseudopromoter or
synthetic promoter was identified and/or selected is used in the
method. By way of example, but not of limitation, a sense promoter
comprising a ssDNA pseudopromoter can be obtained as described by
Ohmichi et al. (Proc. Natl. Acad. Sci. USA 99:54-59, 2002,
incorporated herein by reference) and used in a sense promoter
primer of a method or assay of the invention that uses E. coli RNAP
or a T7-type phage RNAP. If a single-stranded pseudopromoter or
synthetic promoter is used in a sense promoter primer of the
invention, a circular sense promoter-containing single-stranded
cDNA obtained using a method of the invention comprises a circular
transcription substrate without annealing an anti-sense promoter
oligo. Therefore, no anti-sense promoter oligo is needed in these
embodiments. Single-stranded promoters for phage N4 vRNAP can also
be used in some embodiments, in which case a wild-type or mutant
form of N4 mini-vRNAP is used in the method or kit, both of which
are discussed elsewhere herein, and no anti-sense promoter oligo is
needed in those embodiments.
[0048] A. Definitions and General Methods
[0049] 1. Transcription Product
[0050] The term "transcription product" as used herein can comprise
RNA or, in view of the ability of certain polymerases of the
invention, including, without limitation, a T7 RNAP Y639F mutant
enzyme or a T7 RNAP mutant enzyme having altered amino acids at
both positions 639 and 784 (Sousa et al., U.S. Pat. No. 5,849,546;
Padilla, R and Sousa, R, Nucleic Acids Res., 15: e138, 2002; Sousa,
R and Mukherjee, S, Prog Nucleic Acid Res Mol. Biol., 73: 1-41,
2003), to use base-substituted ribonucleotides, such as
5-allyamino-UTP, or non-canonical nucleotide substrates such as
dNTPs or 2'-substituted 2'-deoxyribonucleotides such as, but not
limited to 2'-fluoro-, 2'-amino-, 2'-methoxy-, or
2'-azido-substituted 2'-deoxyribonucleotides, a transcription
product can comprise, in addition to RNA, DNA or modified DNA, or
modified RNA, or a mixture thereof. A transcription product does
not necessarily have perfect sequence complementarity or identity
to the target sequence. For example, a transcription product can
include nucleotide analogs such as deoxyinosine or deoxyuridine,
intentional sequence alterations, such as sequence alterations
introduced through a primer comprising a sequence that is
hybridizable, but not complementary, to the target sequence, and/or
sequence errors that occur during transcription.
[0051] Also, for a variety of reasons, a nucleic acid or
polynucleotide of the invention may comprise one or more modified
nucleic acid bases, sugar moieties, or internucleoside linkages. By
way of example, some reasons for using nucleic acids or
polynucleotides that contain modified bases, sugar moieties, or
internucleoside linkages include, but are not limited to: (1)
modification of the T.sub.m; (2) changing the susceptibility of the
polynucleotide to one or more nucleases; (3) providing a moiety for
attachment of a label; (4) providing a label or a quencher for a
label; or (5) providing a moiety, such as biotin, for attaching to
another molecule which is in solution or bound to a surface.
[0052] 2. Sample/Target/Target Nucleic Acid/Target Sequence
[0053] A "sample" or a "biological sample" according to the present
invention is used in its broadest sense. A sample is any specimen
that is collected from or is associated with a biological or
environmental source, or which comprises or contains biological
material, whether in whole or in part, and whether living or
dead.
[0054] Biological samples may be plant or animal, including human,
fluid (e.g., blood or blood fractions, urine, saliva, sputum,
cerebral spinal fluid, pleural fluid, milk, lymph, or semen), swabs
(e.g., buccal or cervical swabs), solid (e.g., stool), microbial
cultures (e.g., plate or liquid cultures of bacteria, fungi,
parasites, protozoans, or viruses), or cells or tissue (e.g., fresh
or paraffin-embedded tissue sections, hair follicles, mouse tail
snips, leaves, or parts of human, animal, plant, microbial, viral,
or other cells, tissues, organs or whole organisms, including
subcellular fractions or cell extracts), as well as liquid and
solid food and feed products and ingredients such as dairy items,
vegetables, meat and meat by-products, and waste. Biological
samples may be obtained from all of the various families of
domestic plants or animals, as well as wild animals or plants.
[0055] Environmental samples include environmental material such as
surface matter, soil, water, air, or industrial samples, as well as
samples obtained from food and dairy processing instruments,
apparatus, equipment, utensils, disposable and non-disposable
items. These examples are not to be construed as limiting the
sample types applicable to the present invention.
[0056] In short, a sample comprises a specimen from any source that
contains or may contain a naturally occurring target nucleic
acid.
[0057] A sample on which the assay method of the invention is
carried out can be a raw specimen of biological material, such as
serum or other body fluid, tissue culture medium or food material.
More typically, the method is carried out on a sample that is a
processed specimen, derived from a raw specimen by various
treatments to remove materials that would interfere with detection
of a target nucleic acid or an amplification product thereof.
Methods for processing raw samples to obtain a sample more suitable
for the assay methods of the invention are well known in the
art.
[0058] An "analyte" means a substance whose presence, concentration
or amount in a sample is being determined in an assay. An analyte
is sometimes referred to as a "target substance" or a "target
molecule" or a "target analyte" of an assay. An analyte may also be
referred to more specifically. Embodiments of the present invention
pertain to analytes that are naturally occurring nucleic acids, and
the analyte may be referred to as a "target nucleic acid" or a
"target polynucleotide" or a "target oligonucleotide" or a "target
sequence," depending on the particular case. A composition, kit, or
method of the invention can be used for an "analyte-specific
reagent" to detect a target nucleic acid analyte or another analyte
or analyte-binding substance in a sample.
[0059] With respect to the present invention, an analyte is often
associated with a biological entity that is present in a sample if
and only if the analyte is present. Such biological entities
include viroids (analyte is, e.g., a nucleic acid or a segment
thereof); viruses (analyte is, e.g., a viral genome, or a segment
of viral genome); other microorganisms (analyte is, e.g., a segment
of the genome or the RNA of the microorganism); abnormal cells,
such as cancer cells (analyte is, e.g., an oncogene); or an
abnormal gene (analyte is, e.g., a gene segment which includes the
altered bases which render the gene abnormal, or a messenger RNA
segment which includes altered bases as a result of having been
transcribed from the abnormal gene).
[0060] From the description of analyte, it is apparent that the
present invention has widespread applicability, including in
applications in which nucleic acid probe hybridization assays are
often employed. Thus, among other applications, the invention is
useful in diagnosing diseases in plants and animals, including
humans; and in testing products, such as food, blood, and tissue
cultures, for contaminants.
[0061] A "target" of the present invention is a biological organism
or material that is the reason or basis for which a biological
assay or a diagnostic assay is performed. By way of example, but
not of limitation, an assay of the present invention may be
performed to detect a target that is a virus which is indicative of
a present disease or a risk of future disease (e.g., HIV which is
believed to result in AIDS), or a target that is a gene which is
indicative of antibiotic resistance (e.g., an antibiotic resistance
gene in an infectious pathogenic bacterium), or a target that is a
gene which, if absent, may be indicative of disease (e.g., a
deletion in an essential gene). In developing assays according to
the present invention, it is important to identify target analytes
that yield assay results that are sufficiently specific, accurate,
and sensitive to be meaningful related to the presence or condition
of the target.
[0062] A target analyte that is a "target polynucleotide" or a
"target nucleic acid" comprises at least one nucleic acid molecule
or portion of at least one nucleic acid molecule, whether the
molecule or molecules is or are DNA, RNA, or both DNA and RNA, and
wherein each the molecule has, at least in part, a defined
nucleotide sequence. The target polynucleotide may also have at
least partial complementarity with other molecules used in an
assay, such as, but not limited to, primers, splice template
oligos, ligation splint oligos, capture probes or detection probes.
The target polynucleotide may be single- or double-stranded. A
target polynucleotide of the present invention may be of any
length. However, it must comprise a polynucleotide sequence of
sufficient sequence specificity and length so as to be useful for
its intended purpose. By way of example, but not of limitation, a
target nucleic acid that is to be detected using a
sequence-complementary detection probe must have a sequence of
sufficient sequence specificity and length so as remain hybridized
by the detection probe under assay hybridization conditions wherein
sequences that are not target polynucleotides are not hybridized. A
target polynucleotide having sufficient sequence specificity and
length for an assay of the present invention may be identified,
using methods known to those skilled in the art, by comparison and
analysis of nucleic acid sequences known for a target and for other
sequences which may be present in the sample. For example,
sequences for nucleic acids of many viruses, bacteria, humans
(e.g., for genes and messenger RNA), and many other biological
organisms can be searched using public or private databases, and
sequence comparisons, folded structures, and hybridization melting
temperatures (i.e., T.sub.m's) may be obtained using computer
software known to those knowledgeable in the art.
[0063] The terms "source of target nucleic acid" or "source of
target polynucleotide" refers to any sample that contains a
naturally occurring target nucleic acid, RNA or DNA.
[0064] Thus, a method of the present invention can be carried out
on nucleic acid from a variety of sources, including unpurified
nucleic acids, or nucleic acids purified using any appropriate
method in the art, such as, but not limited to, various "spin"
columns, cationic membranes and filters, or salt precipitation
techniques, for which a wide variety of products are commercially
available (e.g., MasterPure.TM. DNA & RNA Purification Kits
from Epicentre Technologies, Madison, Wis., USA). Methods of the
present invention can also be carried out on nucleic acids isolated
from viroids, viruses or cells of a specimen and deposited onto
solid supports as described by Gillespie and Spiegelman (J. Mol.
Biol. 12:829-842, 1965), including solid supports on dipsticks and
the inside walls of microtiter plate wells. The method can also be
carried out with nucleic acid isolated from specimens and deposited
on solid support by "dot" blotting (Kafatos, et al., Nucl. Acids
Res. 7:1541-1552, 1979); White, and Bancroft, J. Biol. Chem.
257:8569-8572, 1982); Southern blotting (Southern, E., J. Mol.
Biol. 98:503-517, 1975); "northern" blotting (Thomas, Proc. Natl.
Acad. Sci. USA 77:5201-5205, 1980); and electroblotting (Stellwag,
and Dahlberg, Nucl. Acids Res. 8:299-317, 1980). The method can
also be carried out for nucleic acids spotted on membranes, on
slides, or on chips as arrays or microarrays, or the method can be
carried out to prepare probes for detecting or quantifying nucleic
acids present in a sample based on hybridization to nucleic acids
spotted or synthesized on one of these surfaces. Nucleic acid of
specimens can also be assayed by the method of the present
invention applied to water phase hybridization (Britten, and Kohne,
Science 161:527-540, 1968) and water/organic interphase
hybridizations (Kohne, et al., Biochemistry 16:5329-5341, 1977).
Water/organic interphase hybridizations have the advantage of
proceeding with very rapid kinetics but are not suitable when an
organic phase-soluble linking moiety, such as biotin, is joined to
the nucleic acid affinity molecule.
[0065] The methods of the present invention can also be carried out
on amplification products obtained by amplification of a naturally
occurring target nucleic acid, provided that the target sequence in
the target nucleic acid is amplified by the method used only if the
target nucleic acid is present in the sample. Suitable
amplification methods include, but are not limited to, PCR, RT-PCR,
NASBA, TMA, 3SR, LCR, LLA, SDA (e.g., Walker et al., Nucleic Acids
Res. 20:1691-1696, 1992), RCA, Multiple Displacement Amplification
(Molecular Staging), ICAN.TM. or UCAN.TM. (TAKARA), Loop-AMP
(EIKEN), and SPIA.TM. or Ribo-SPIA.TM. (NuGEN Technologies). There
are various reasons for using a nucleic acid that is a product of
another amplification method as a target nucleic acid for an assay
of the present invention, such as, but not limited to, for
obtaining more sensitive detection of targets, greater specificity,
or to decrease the time required to obtain an assay result.
[0066] The methods of the invention can also be carried out on
nucleic acids isolated from specimens and deposited onto solid
supports by dot-blotting, or by adsorption onto walls of microtiter
plate wells or solid support materials on dipsticks, on membranes,
on slides, or on chips as arrays or microarrays.
[0067] Still further, the methods of the invention are applicable
to detecting cellular nucleic acids in whole cells from a specimen,
such as a fixed or paraffin-embedded section, or from
microorganisms immobilized on a solid support, such as
replica-plated bacteria or yeast. In some embodiments, the methods
of the invention can be used to make a transcription product
corresponding to a target sequence to detect target nucleic acids
in living cells.
[0068] A target nucleic acid can be a nucleic acid from any source
in purified or unpurified form. For example, a target nucleic acid
comprising DNA, can be dsDNA or ssDNA such as mitochondrial DNA,
chloroplast DNA, chromosomes, plasmids or other episomes, the
genomes of bacteria, yeasts, viruses, viroids, mycoplasma, molds,
or other microorganisms, or genomes of fungi, plants, animals, or
humans. Target nucleic acids comprising RNA can be tRNA, mRNA,
rRNA, mitochondrial RNA, chloroplast RNA, micro RNA, or other RNA
molecules, without limit. Target nucleic acids can also be mixtures
of DNA and RNA, including, but not limited to, mixtures of the
above nucleic acids or fragments thereof, or DNA-RNA hybrids. The
target nucleic acid can be only a minor fraction of a complex
mixture such as a biological sample and can be obtained from
various biological materials by procedures known in the art.
Numerous methods for purification of a particular target nucleic
are known in the art, if further purification is necessary.
[0069] The term "target nucleic acid sequence" or "target sequence"
refers to the particular nucleotide sequence of the target nucleic
acid(s) that is/are to be transcribed to make a transcription
product. A "target sequence" comprises one or more sequences within
one or more target nucleic acids. A target sequence can also have
"complexing sequences" which are added during processes of some
embodiments of the invention to facilitate joining of a target
sequence to another polynucleotide for a particular purpose. For
example, a complexing sequence can provide a complementary sequence
to which an oligonucleotide (e.g., a primer and/or splice template)
used in a method of the invention can anneal or complex. A
complexing sequence usually comprises a "tail" sequence that is
added by means such as those discussed herein, including, but not
limited to, non-templated addition of dCMP residues to first-strand
cDNA by reverse transcriptase pausing at cap structures of mRNA (in
the presence or absence of manganese cations) and/or controlled
ribonucleotide tailing using TdT. If a complexing sequence is added
to a target sequence during a process of a method of the invention,
it is desirable that a complexing sequence is chosen that does not
to affect the specificity of the transcription products made using
the resulting transcription substrate comprising the target
sequence.
[0070] A target nucleic acid can be either single-stranded or
double-stranded RNA, DNA, or mixed RNA and DNA. A target nucleic
acid is sometimes referred to more specifically by the type of
nucleic acid. By way of example, but not of limitation, a target
nucleic acid can be a "target RNA" or an "RNA target," or a "target
mRNA," or a "target DNA" or a "DNA target." Similarly, the target
sequence can be referred to as "a target RNA sequence" or a "RNA
target sequence", or as a "target mRNA sequence" or a "target DNA
sequence," or the like. In some embodiments, the target sequence
comprises one or more entire target nucleic acids, such as, one or
all full-length mRNA molecules in a particular sample. In other
embodiments, the target sequence comprises only a portion of one or
more target nucleic acid molecules. When the target nucleic acid is
originally single-stranded, the term "target sequence" is also
meant to refer to the sequence complementary to the "target
sequence." When the "target nucleic acid" is originally
double-stranded, the term "target sequence" may refer to both the
sense strand of the sequence or its complement, or both, depending
on the intended purpose of the method. The target sequence may be
known or not known, in terms of its actual sequence. In some
instances, the terms "target sequence," "target nucleic acid,"
"target polynucleotide," and variations thereof are used
interchangeably.
[0071] 3. cDNA/First-Strand cDNA/Second-Strand cDNA/Reverse
Transcriptase/RNaseH
[0072] In some important embodiments of the invention, the target
sequence of a transcription substrate comprises cDNA. In general,
"cDNA" refers to "complementary DNA" that is synthesized by primer
extension using a DNA polymerase, including, but not limited to, an
RNA-dependent DNA polymerase or reverse transcriptase, using at
least a portion of a target nucleic acid as a template, and which
cDNA is "homologous to" or "base pairs with" at least a portion of
the target nucleic acid template. In some embodiments of the
invention, which are preferred embodiments, cDNA is obtained by
reverse transcription primer extension using a reverse
transcriptase and a target nucleic acid comprising messenger RNA
(mRNA) obtained from a biological sample as a template, and which
cDNA is homologous to the mRNA. Methods in the art related to
making cDNA from mRNA involve synthesis of double-stranded cDNA
comprising first-strand cDNA and second-strand cDNA, which usually
are synthesized sequentially using different methods. However
herein, we often refer to "first-strand cDNA" even when a method of
the invention results in synthesis of only one strand of DNA that
is complementary to the mRNA (i.e., the term "first-strand cDNA" is
not intended to imply that there is also a second-strand cDNA). In
some embodiments, the terms "first-strand cDNA" or "cDNA" refer to
a single-stranded DNA molecule obtained by reverse transcription of
any RNA molecule, even if it is not mRNA. In other embodiments, the
terms "first-strand cDNA" or "cDNA" refer to a single-stranded DNA
molecule obtained by primer extension using a target nucleic acid
comprising either a single-stranded DNA or one strand of a
double-stranded DNA as a template for a DNA polymerization
reaction.
[0073] If a promoter primer of the invention is used for primer
extension of a target sequence, the promoter sequence is at the
5'-end of the resulting first-strand cDNA. Even if the promoter
sequence of the promoter primer is a sense promoter sequence, it is
not operably joined to the 3'-end of the target sequence as
required to obtain a functional double-stranded promoter by
complexing with an anti-sense promoter oligo. Therefore, a product
obtained by primer extension of a promoter primer is referred to
herein simply as "first-strand cDNA" or as a "first-strand cDNA
primer extension product" or as a "primer extension product."
However, once the promoter sequence is joined to the 3'-end of the
target sequence obtained by primer extension, the resulting
circular molecule is referred to herein as "circular sense
promoter-containing first-strand cDNA." Similarly, the molecule
resulting from linearization of circular sense promoter-containing
first-strand cDNA to obtain a linear moleculewith a sense promoter
on the 3'-end of the target sequence is referred to herein as
"linear sense promoter-containing first-strand cDNA." These terms,
which designate the presence of a sense promoter at the 3'-end of
the target sequence in the respective circular or linear
first-strand cDNA, are used so that the reader will understand that
a circular or linear transcription substrate, respectively, can be
obtained by complexing (or annealing) an anti-sense promoter oligo
to the respective circular or linear sense promoter-containing
first-strand cDNA.
[0074] Based on a reading of the present description, the reader
will understand that a transcription substrate cannot be obtained
by annealing a sense promoter oligo to an "anti-sense promoter
containing first-strand cDNA" (because the sense promoter sequence
must be joined to the 3'-end of a target sequence on the template
strand). That is, a functional double-stranded transcription
promoter is only obtained by complexing an anti-sense promoter
oligo to a sense promoter on the template strand. Therefore, the
reader will understand that circularization of anti-sense
promoter-containing first-strand cDNA will not yield a
transcription substrate by complexing with a sense promoter oligo.
However, rolling circle DNA replication of circular anti-sense
promoter-containing first-strand cDNA yields concatemeric "linear
sense promoter-containing first-strand cDNA," which can be used to
obtain a concatemeric linear transcription substrate by complexing
with an anti-sense promoter oligo. The transcription products are
anti-sense transcription products with respect to the target
nucleic acid.
[0075] An "RNA-dependent DNA polymerase" or "reverse transcriptase"
is an enzyme that can synthesize a complementary DNA copy ("cDNA")
from an RNA template. All known reverse transcriptases also have
the ability to make a complementary DNA copy from a DNA template;
thus, they are both RNA- and DNA-dependent DNA polymerases.
[0076] A "template" is the nucleic acid molecule that is copied by
a nucleic acid polymerase. If the nucleic acid comprises two
strands (i.e., is "double-stranded"), and sometimes even if the
nucleic acid comprises only one strand (i.e., is
"single-stranded"), the strand that is copied is the "template" or
"the template strand." The synthesized copy is complementary to the
template. Both RNA and DNA are always synthesized in the 5'-to-3'
direction and the two strands of a nucleic acid duplex always are
aligned so that the 5' ends of the two strands are at opposite ends
of the duplex (and, by necessity, so then are the 3' ends). A
primer is required for both RNA and DNA templates to initiate
synthesis by a DNA polymerase. Examples of reverse transcriptases
that can be used in methods of the present invention include, but
are not limited to, AMV reverse transcriptase, MMLV reverse
transcriptase, Tth DNA polymerase, rBst DNA polymerase large
fragment, also called IsoTherm.TM. DNA Polymerase (Epicentre
Technologies, Madison, Wis., USA), and BcaBEST.TM. DNA polymerase
(Takara Shuzo Co, Kyoto, Japan). In some cases, a mutant form of a
reverse transcriptase, such as, an MMLV reverse transcriptase that
lacks RNase H activity is used. In still other embodiments,
IsoTherm.TM. DNA polymerase is most suitable. In other embodiments,
a wild-type enzyme is preferred. In general, the invention is not
limited with respect to the reverse transcriptase used so long as
it functions for its intended purpose.
[0077] In embodiments of the invention that obtain a transcription
substrate in which a ssDNA target sequence comprising first-strand
cDNA is synthesized using a reverse transcriptase and a primer that
is complementary to and anneals to the 3'-end of a target nucleic
acid comprising mRNA, the primer can comprise oligo(dT).sub.n or
modified oligo(dT).sub.n, or it can comprise an oligo d(T).sub.nX
anchor primer, wherein "X" comprises either a specific base for a
specific mRNA or a randomized nucleotide (i.e., synthesized with a
mixture of all four nucleotides) for priming all mRNA molecules in
a sample, or the primer can comprise an oligonucleotide having a
specific sequence that is complementary to the sequence of a
specific mRNA molecule, or in some cases, it can comprise an
oligonucleotide having a random sequence (i.e., synthesized using a
mixture of all four nucleotides for every position of the
primer).
[0078] If a target nucleic acid is RNA, such as mRNA, and it is
desirable to remove the RNA that is annealed to first-strand cDNA
following reverse transcription using a sense promoter primer, this
can be accomplished by one of several means. By way of example, but
not of limitation, the RNA can be removed by treatment with RNase
H, by treatment with a base, such as, but not limited to sodium or
potassium hydroxide, or the RNA can be removed from the hybrid by
heat denaturation. In preferred embodiments for some applications,
the RNA is removed by an RNase H activity of a reverse
transcriptase that is used for reverse transcription (or primer
extension), such as, but not limited to, MMLV reverse
transcriptase. Alternatively, in some embodiments, the RNA is
dissociated from the first-strand cDNA by incubating the hybrid or
performing the reverse transcription in the presence of a
single-strand binding (SSB) protein of the invention, such as, but
not limited to E. coli SSB (EcoSSB).
[0079] In some embodiments of the invention, especially in
embodiments for obtaining additional rounds of transcription
products, a separate RNase H enzyme is also used, whether or not
the reverse transcriptase has RNase H activity. If RNase H activity
is desirable in an embodiment for obtaining multiple rounds of
transcription, but a separate RNase H enzyme is not added, MMLV
reverse transcriptase (wild-type RNase H-positive) can be used. AMV
reverse transcriptase can be used in some embodiments for obtaining
multiple rounds of transcription in which a separate RNase H enzyme
is also added. Kacian et al. (U.S. Pat. No. 5,399,491) disclose
information related to the effects of adding different amounts of a
separate RNase H enzyme to transcription-mediated amplification
assays that use either MMLV or AMV reverse transcriptase, which
reference and information is incorporated herein by reference and
made a part of the present disclosure.
[0080] "Ribonuclease H" or "RNase H" is an enzyme that degrades the
RNA portion of an RNA:DNA duplex. An RNase H can be an endonuclease
or an exonuclease. Most wild-type reverse transcriptase enzymes
have an RNase H activity in addition to their polymerase activity.
However, other sources of the RNase H are available without an
associated polymerase activity. The degradation may result in
separation of RNA from an RNA:DNA complex. Alternatively, the RNase
H may simply cut the RNA at various locations such that portions of
the RNA melt off or permit enzymes to unwind portions of the RNA.
When used in an embodiment of the invention, RNaseH enzymes that
can be used include, but are not limited to, E. coli RNase H,
Thermus thermophilus RNase H, and Thermus flavus RNase H (U.S. Pat.
Nos. 5,268,289; 5,459,055; and 5,500,370, incorporated herein by
reference). The latter two enzymes, which are thermostable and,
therefore, maintain more consistent activity in reactions and are
more easily stored and shipped, are preferred in most embodiments
in which a separate RNase H enzyme is used. Other RNase H enzymes
that can be used are those that are described by Sagawa et al. in
PCT Patent Publication No. WO 02/16639; and in PCT Patent
Publications Nos. WO 00/56877 and AU 00/29742, all of which are
incorporated herein by reference. In other embodiments, it is
desirable to use a less thermally stable enzyme, such as E. coli
RNase H, because it is easier to inactivate the enzyme in a
reaction mixture.
[0081] Kacian et al. disclosed in U.S. Pat. No. 5,399,491,
incorporated herein by reference, that the number, distribution,
and position of putative RNase H cut sites determine, in part, the
usefulness of a given primer and that amplification can be improved
by inclusion of intentional mismatches or insertion of sequences in
order to affect the number, distribution, and position of putative
RNase H cut sites. Thus, in preferred processes of the invention
for removing RNA from RNA:DNA hybrids following reverse
transcription to make first-strand cDNA, the RNA target sequence is
determined and then analyzed to determine where RNase H degradation
will cause cuts or removal of sections of RNA from the duplex upon
synthesis of first-strand cDNA. The processes of the invention
include conducting experiments to determine the effect on
amplification of the target sequence of the degradation of the RNA
target sequence by RNase H present in the reverse transcriptase
and/or separate RNase H enzyme(s) used, including, but not limited
to, AMV reverse transcriptase, and both RNase H-plus and RNase
H-minus MMLV reverse transcriptase, and E. coli RNase H or
thermostable RNase H enzymes that are stable for more than 10
minutes at 70.degree. C. (U.S. Pat. Nos. 5,268,289; 5,459,055; and
5,500,370, incorporated herein by reference), such as, but not
limited to, Hybridase.TM. thermostable RNase H (Epicentre
Technologies, Madison, Wis., USA), Tth RNase H, and Tfl RNase H, or
by different combinations of a reverse transcriptase and a separate
RNase H.
[0082] In selecting a primer, including a promoter primer of the
invention, for use in reverse transcription of an RNA target
sequence to make first-strand cDNA, it is preferable that the
primer be selected so that it will hybridize to a section of RNA
which is substantially nondegraded by the RNase H present in the
reaction mixture. If there is substantial degradation, the cuts in
the RNA strand in the region of the primer may stop or inhibit DNA
synthesis and prevent extension of the primer. Thus, it is
desirable to select a primer that will hybridize with a sequence of
the RNA target, located so that when the RNA is subjected to RNase
H, there is no substantial degradation that would prevent formation
of the primer extension product.
[0083] 4. Transcription Substrate
[0084] As used herein, a "transcription substrate" according to the
present invention comprises a target sequence that is operably
joined to a transcription promoter, wherein an RNA polymerase can
bind to the transcription promoter with specificity and synthesize
a transcription product corresponding to the target sequence under
suitable transcription conditions. In order to be operably joined
to the target sequence, a sense transcription promoter is 3'- of
the target sequence and, if the respective promoter for the RNA
polymerase must be double-stranded to be functional, then an oligo
comprising an anti-sense promoter sequence is annealed to the sense
promoter sequence that is joined to the target sequence. If the
sense transcription promoter comprises a single-stranded
pseudopromoter or synthetic promoter that has been selected or
identified, as described elsewhere herein, then the transcription
substrate can comprise single-stranded DNA (i.e., without annealing
an anti-sense promoter oligo).
[0085] A transcription substrate of the invention can also have
additional nucleic acid sequences that are 5'- of and/or 3'- of the
transcription promoter sequence, but a transcription substrate is
not required to have such additional other sequences. By way of
example, but not of limitation, a transcription substrate can have
a transcription initiation site 5'- of the promoter sequence. Also,
in some embodiments of the invention, a transcription substrate can
have one or more transcription termination sequences, one or more
sites for DNA cleavage to permit controlled linearization of a
circular first-strand cDNA that can be used to obtain a linear
transcription substrate as described elsewhere herein, one or more
origins ("ori's") of replication (preferably an ori for a
single-stranded replicon, such as, but not limited to, a phage M13
replicon), a selectable or screenable marker, such as, but not
limited to an antibiotic-resistance gene or a beta-galactosidase
gene, respectively, or one or more transposon recognition
sequences, such as outer end ("OE") or mosaic end ("ME") sequences
for a Tn5-type transposon, that, in double-stranded form, can be
recognized and used by a transposase for in vitro or in vivo
transposition, or one or more sites that are recognized by a
recombinase (such as, but not limited to, the cre-lox system),
and/or other sequences and genetic elements for a particular
purpose, including, but not limited to, sequences that are
transcribed by the RNA polymerase so as to provide additional
regions of complementarity in the RNA transcription products for
annealing of primers for reverse transcription in order to make
cDNA for additional rounds of transcription. In most embodiments,
the target sequence or template portion of a transcription
substrate of the present invention is single-stranded, whereas
double-stranded DNA templates are used in other methods in the art
for obtaining transcription products corresponding to a target
sequence.
[0086] Since, except for the promoter sequence, a transcription
substrate of the present invention is single-stranded, the terms
"3'- of" and "5'- of" are used herein with respect to the present
invention to refer to the position or orientation of a particular
nucleic acid sequence or genetic element, such as, but not limited
to, a transcription promoter, relative to other sequences or
genetic elements within the DNA template strand comprising the
transcription substrate. Thus, although the synthesis of RNA in a
5'-to-3' direction during transcription is thought of as proceeding
in a "downstream" direction, the sense transcription promoter
sequence on the transcription substrate is referred to herein as
being 3'- of the target sequence. Those with knowledge in the art
will understand these terms in the context of nucleic acid
chemistry and structure, particularly related to the 3'- and
5'-positions of sugar moieties of canonical nucleic acid
nucleotides. By way of example, a sense transcription promoter that
is "3'- of the target sequence" on a linear transcription substrate
refers to a sense promoter sequence that is at or closer to the
3'-end of the transcription substrate relative to the target
sequence on the same strand. If a first nucleic acid sequence is
3'- of a second sequence on one strand, the complement of the first
sequence will be 5'- of the complement of the second sequence on
the complementary strand. The description of the invention will be
understood with respect to the relative 5' or 3' position and
orientation of a sequence or genetic element within a particular
nucleic acid strand, unless explicitly stated to the contrary.
[0087] 5. Nucleic Acids, Polynucleotides and Analogs Thereof
[0088] A "nucleic acid" or "polynucleotide" of the invention is a
polymer molecule comprising a series of "mononucleotides," also
referred to as "nucleosides," in which the 3'-position of the
pentose sugar of one nucleoside is linked by an internucleoside
linkage, such as, but not limited to, a phosphodiester bond, to the
5'-position of the pentose sugar of the next nucleoside. A
nucleoside linked to a phosphate group is referred to as a
"nucleotide." The nucleotide that is linked to the 5'-position of
the next nucleotide in the series is referred to as "5' of" or the
"5' nucleotide" and the nucleotide that is linked to the
3'-position of the 5' nucleotide is referred to as "3' of or the
"3' nucleotide." The pentose sugar of the nucleic acid can be
ribose, in which case, the nucleic acid or polynucleotide is
referred to as "RNA," or it can be 2'-deoxyribose, in which case,
the nucleic acid or polynucleotide is referred to as "DNA."
Alternatively, especially if the nucleic acid is synthesized
chemically, the nucleic acid can be composed of both DNA and RNA
mononucleotides. In both RNA and DNA, each pentose sugar is
covalently linked to one of four common or "canonical" nucleic acid
bases (each also referred to as a "base"). Three of the predominant
naturally-occurring bases that are linked to the sugars (adenine,
cytidine and guanine) are common for both DNA and RNA, while one
base is different; DNA has the additional base thymine, while RNA
has the additional base uridine. Those in the art commonly think of
a small polynucleotide as an "oligonucleotide." The term
"oligonucleotide" as used herein is defined as a molecule comprised
of two or more deoxyribonucleotides or ribonucleotides, preferably
about 10 to 200 nucleotides, but there is no defined limit to the
length of an oligonucleotide. The exact size will depend on many
factors, which in turn depends on the ultimate function or use of
the oligonucleotide.
[0089] In order to accomplish the goals of the invention, there is
no limit to the composition of the nucleic acids or polynucleotides
of the invention including any splice template oligos, primers,
including promoter primers, ligation splint oligos, detection
probes, such as, but not limited to molecular beacons (U.S. Pat.
Nos. 5,925,517 and 6,103,476 of Tyagi et al. and 6,461,817 of
Alland et al., which are incorporated herein by reference), capture
probes, oligonucleotides, or other nucleic acids used or detected
in the assays or methods, so long as each nucleic acid functions
for its intended use. By way of example, but not of limitation, the
nucleic acid bases in the mononucleotides may comprise guanine,
adenine, uracil, thymine, or cytidine, or alternatively, one or
more of the nucleic acid bases may comprise xanthine,
allyamino-uracil, hypoxanthine, 2-aminoadenine, 6-methyl and other
alkyl adenines, 2-propyl and other alkyl adenines, 5-halouracil,
5-halo cytosine, 5-propynyl uracil, 5-propynyl cytosine,
7-deazaadenine, 7-deazaguanine, 7-deaza-7-methyl-adenine,
7-deaza-7-methyl-guanine, 7-deaza-7-propynyl-adenine,
7-deaza-7-propynyl-guanine and other 7-deaza-7-alkyl or 7-aryl
purines, N2-alkyl-guanine, N2-alkyl-2-amino-adenine, purine 6-aza
uracil, 6-aza cytosine and 6-aza thymine, 5-uracil (pseudo uracil),
4-thiouracil, 8-halo adenine, 8-amino-adenine, 8-thiol adenine,
8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted
adenines and 8-halo guanines, 8-amino-guanine, 8-thiol guanine,
8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted
guanines, other aza and deaza uracils, other aza and deaza
thymidines, other aza and deaza cytosine, aza and deaza adenines,
aza and deaza guanines or 5-trifluoromethyl uracil and
5-trifluorocytosine. Still further, they may comprise a nucleic
acid base that is derivatized with a biotin moiety, a digoxigenin
moiety, a fluorescent or chemiluminescent moiety, a quenching
moiety or some other moiety. The invention is not limited to the
nucleic acid bases listed; this list is given to show the broad
range of bases which may be used for a particular purpose in a
method.
[0090] In some embodiments of the invention, a molecule comprising
a "peptide nucleic acid" (PNA) or a molecule comprising both a
nucleic acid and a PNA, as described in U.S. Pat. Nos. 5,539,082;
5,641,625; 5,700,922; 5,705,333; 5,714,331; 5,719,262; 5,736,336;
5,773,571; 5,786,461; 5,817,811; 5,977,296; 5,986,053; 6,015,887;
and 6,020,126 (and references therein), can also be used. In
general, a PNA molecule is a nucleic acid analog consisting of a
backbone comprising, for example, N-(2-aminoethyl)glycine units, to
each of which a nucleic acid base is linked through a suitable
linker, such as, but not limited to an aza, amido, ureido, or
methylene carbonyl linker. The nucleic acid bases in PNA molecules
bind complementary single-stranded DNA or RNA according to
Watson-Crick base-pairing rules. However, the T.sub.m 's for
PNA/DNA or PNA/RNA duplexes or hybrids are higher than the
T.sub.m's for DNA/DNA, DNA/RNA, or RNA/RNA duplexes. PNA provides
tighter binding and greater binding stability than a nucleic acid
of similar base sequence (e.g., see U.S. Pat. No. 5,985,563). Also,
since PNA is not naturally occurring, PNA molecules and highly
resistant to protease and nuclease activity. PNA can be prepared
according to methods know in the art, such as, but not limited to,
methods described in the above-mentioned patents, and references
therein.
[0091] When a molecule comprising both a nucleic acid and a peptide
nucleic acid (PNA) is used in the invention, modified bases can be
used in one or both parts. For example, binding affinity can be
increased by the use of certain modified bases in both the
nucleotide subunits that make up the 2'-deoxyoligonucleotides of
the invention and in the peptide nucleic acid subunits. Such
modified bases may include 5-propynylpyrimidines, 6-azapyrimidines,
and N2, N-6 and O-6 substituted purines including
2-aminopropyl-adenine. Other modified pyrimidine and purine base
are also expected to increase the binding affinity of
macromolecules to a complementary strand of nucleic acid.
[0092] With respect to nucleic acids or polynucleotides of the
invention, one or more of the sugar moieties can comprise ribose or
2'-deoxyribose, or alternatively, one or more of the sugar moieties
can be some other sugar moiety, such as, but not limited to,
2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose, which provide
resistance to some nucleases, or 2'-amino-2'-deoxyribose or
2'-azido-2'-deoxyribose, which can be used to label transcription
products by reacting them with visible, fluorescent, infrared
fluorescent or other detectable dyes or chemicals having an
electrophilic, photoreactive or other reactive chemical moiety.
[0093] The internucleoside linkages of nucleic acids or
polynucleotides of the invention can be phosphodiester linkages, or
alternatively, one or more of the internucleoside linkages can
comprise modified linkages, such as, but not limited to,
phosphorothioate, phosphorodithioate, phosphoroselenate, or
phosphorodiselenate linkages, which are resistant to some
nucleases.
[0094] A variety of methods are known in the art for making nucleic
acids having a particular sequence or that contain particular
nucleic acid bases, sugars, internucleoside linkages, chemical
moieties, and other compositions and characteristics. Any one or
any combination of these methods can be used to make a nucleic
acid, polynucleotide, or oligonucleotide for the present invention.
The methods include, but are not limited to:
[0095] (1) chemical synthesis (usually, but not always, using a
nucleic acid synthesizer instrument);
[0096] (2) post-synthesis chemical modification or
derivatization;
[0097] (3) cloning of a naturally occurring or synthetic nucleic
acid in a nucleic acid cloning vector (e.g., see Sambrook, et al.,
Molecular Cloning: A Laboratory Approach Second Edition, 1989, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and
Sambrook and Russell, Molecular Cloning, A Laboratory Manual, Third
Edition, 2001, Cold Spring Harbor Laboratory Press), such as, but
not limited to a plasmid, bacteriophage (e.g., M13 or lamba),
phagemid, cosmid, fosmid, YAC, or BAC cloning vector, including
vectors for producing single-stranded DNA;
[0098] (4) primer extension using an enzyme with DNA
template-dependent DNA polymerase activity, such as, but not
limited to, Klenow, T4, T7, rBst, Taq, Tfl, or Tth DNA polymerases,
including mutated, truncated (e.g., exo-minus), or
chemically-modified forms of such enzymes;
[0099] (5) PCR (e.g., see Dieffenbach, C. W., and Dveksler, eds.,
PCR Primer: A Laboratory Manual, 1995, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.);
[0100] (6) reverse transcription (including both isothermal
synthesis and RT-PCR) using an enzyme with reverse transcriptase
activity, such as, but not limited to, reverse transcriptases
derived from avian myeloblasosis virus (AMV), Maloney murine
leukemia virus (MMLV), Bacillus stearothermophilus (rBst), or
Thermus thermophilus (Tth);
[0101] (7) in vitro transcription using an enzyme with RNA
polymerase activity, such as, but not limited to, SP6, T3, or T7
RNA polymerase, Tth RNA polymerase, E. coli RNA polymerase, or SP6
or T7 R&DNA.TM. Polymerase (Epicentre Technologies, Madison,
Wis., USA), or another enzyme;
[0102] (8) use of restriction enzymes and/or modifying enzymes,
including, but not limited to exo- or endonucleases, kinases,
ligases, phosphatases, methylases, glycosylases, terminal
transferases, including kits containing such modifying enzymes and
other reagents for making particular modifications in nucleic
acids;
[0103] (9) use of polynucleotide phosphorylases to make new
randomized nucleic acids;
[0104] (10) other compositions, such as, but not limited to, a
ribozyme ligase to join RNA molecules; and/or
[0105] (11) any combination of any of the above or other techniques
known in the art.
[0106] Oligonucleotides and polynucleotides, including chimeric
(i.e., composite) molecules and oligonucleotides with modified
bases, sugars, or internucleoside linkages are commercially
available (e.g., TriLink Biotechnologies, San Diego, Calif., USA or
Integrated DNA Technologies, Coralville, Iowa).
[0107] A "portion" or "region," used interchangeably herein, of a
polynucleotide or oligonucleotide is a contiguous sequence of 2 or
more bases. In other embodiments, a region or portion is at least
about any of 3, 5, 10, 15, 20, 25 contiguous nucleotides.
[0108] 6. Ligation/Ligase
[0109] "Ligation" refers to the joining of a 5'-phosphorylated end
of one nucleic acid molecule to a 3'-hydroxyl end of the same or
another nucleic acid molecule by an enzyme called a "ligase."
Alternatively, in some embodiments of the invention, ligation is
effected by a type I topoisomerase moiety attached to one end of a
nucleic acid (see U.S. Pat. No. 5,766,891, incorporated herein by
reference). The terms "ligating," "ligation," and "ligase" are
often used in a general sense herein and are meant to comprise any
suitable method and composition for joining a 5'-end of one nucleic
acid to a 3'-end of the same or another nucleic acid. Different
ligases are preferred in different embodiments of the invention, as
discussed elsewhere herein.
[0110] In general, if a nucleic acid to be ligated comprises RNA, a
ligase such as, but not limited to, T4 RNA ligase, a ribozyme
ligase, Tsc RNA Ligase (Prokaria Ltd., Reykjavik, Iceland), or
another ligase can be used for non-homologous joining of the ends.
T4 DNA ligase can also be used to ligate RNA molecules when a
5'-phosphoryl end is adjacent to a 3'-hydroxyl end annealed to a
complementary sequence (e.g., see U.S. Pat. No. 5,807,674 of Tyagi,
incorporated herein by reference).
[0111] If the nucleic acids to be joined comprise DNA and the
5'-phosphorylated and the 3'-hydroxyl ends are ligated when the
ends are annealed to a complementary DNA so that the ends are
adjacent (such as, when a "ligation splint" or "ligation splint
oligo" is used), then enzymes such as, but not limited to, T4 DNA
ligase, Ampligase.RTM. DNA Ligase (Epicentre Technologies, Madison,
WI, USA), Tth DNA ligase, Tfl DNA ligase, or Tsc DNA Ligase
(Prokaria Ltd., Reykjavik, Iceland) can be used. However, the
invention is not limited to the use of a particular ligase and any
suitable ligase can be used. Still further, Faruqui discloses in
U.S. Pat. No. 6,368,801 that T4 RNA ligase can efficiently ligate
DNA ends of nucleic acids that are adjacent to each other when
hybridized to an RNA strand. Thus, T4 RNA ligase is a suitable
ligase of the invention in embodiments in which DNA ends are
ligated on a ligation splint oligo comprising RNA or modified RNA,
such as, but not limited to modified RNA that contains 2'-F-dCTP
and 2'-F-dUTP made using the DuraScribe.TM. T7 Transcription Kit
(Epicentre Technologies, Madison, Wis., USA). With respect to
ligation on an homologous ligation template, especially ligation
using a "ligation splint" or a "ligation splint oligo" (as
discussed elsewhere herein), a region, portion, or sequence that is
"adjacent" to another sequence directly abuts that region, portion,
or sequence.
[0112] In other embodiments comprising intramolecular ligation of
linear ssDNA, ligation can be effected in the absence of a ligation
splint using a ligase that can catalyze non-homologous ligation of
ssDNA, such as, but not limited to, ThermoPhage.TM. RNA Ligase II
(Prokaria Ltd., Reykjavik, Iceland), which is derived from phage
TS2126 that infects Thermus scotoductus.
[0113] 7. DNA Polymerases/Strand-Displacing DNA Polymerases/Strand
Displacement/Rolling Circle Replication
[0114] A "DNA-dependent DNA polymerase" is an enzyme that
synthesizes a complementary DNA ("cDNA") copy from a DNA template.
Examples are DNA polymerase I from E. coli and bacteriophage T7 DNA
polymerase. All known DNA-dependent DNA polymerases require a
complementary primer to initiate synthesis. It is known that under
suitable conditions a DNA-dependent DNA polymerase may synthesize
(i.e., "reverse transcribe") a complementary DNA copy from an RNA
template, a process that is also referred to as "reverse
transcription," for which application the DNA polymerase can also
be referred to as a "reverse transcriptase."
[0115] Some DNA polymerases are able to displace the strand
complementary to the template strand as a new DNA strand is
synthesized by the polymerase. This process is called "strand
displacement" and the DNA polymerases that have this activity are
referred to herein as "strand-displacing DNA polymerases." The
template for strand displacement DNA synthesis using a method of
the invention can be a linear or circular ssDNA. If the DNA
template is a single-stranded circle, primed DNA synthesis proceeds
around and around the circle, with continual displacement of the
strand ahead of the replicating strand, a process called "rolling
circle replication." Rolling circle replication results in
synthesis of tandem copies of the circular template. The
suitability of a DNA polymerase for use in an embodiment of the
invention that comprises strand displacement on linear templates or
rolling circle replication can be readily determined by assessing
its ability to carry out rolling circle replication. By way of
example, but not of limitation, the ability of a polymerase to
carry out rolling circle replication can be determined by using the
polymerase in a rolling circle replication assay such as those
described by Fire and Xu (Proc. Natl. Acad. Sci. USA 92:4641-4645,
1995), incorporated herein by reference. It is preferred that a DNA
polymerase be a strand displacing DNA polymerase and lack a
5'-to-3' exonuclease activity for strand displacement
polymerization reactions using both linear or circular templates
since a 5'-to-3' exonuclease activity, if present, might result in
the destruction of the synthesized strand. It is also preferred
that DNA polymerases for use in the disclosed strand displacement
synthesis methods are highly processive. The ability of a DNA
polymerase to strand-displace can vary with reaction conditions, in
addition to the particular enzyme used. Strand displacement and DNA
polymerase processivity can also be assayed using methods described
in Kong et al. (J. Biol. Chem. 268:1965-1975, 1993 and references
cited therein, all of which are incorporated herein by
reference).
[0116] Preferred strand displacing DNA polymerases of the invention
are rBst DNA polymerase large fragment (also called IsoTherm.TM.
DNA Polymerase (EPICENTRE Technologies, Madison, Wis., USA),
BcaBEST.TM. DNA polymerase (Takara Shuzo Co., Kyoto, Japan),
RepliPHI.TM. DNA Polymerase (EPICENTRE Technologies, Madison, Wis.,
USA), .PHI.29 DNA polymerase (U.S. Pat. Nos. 5,576,204 and
5,001,050), SequiTherm.TM. DNA polymerase (Epicentre Technologies,
Madison, Wis., USA), and MMLV reverse transcriptase. Other
strand-displacing DNA polymerases which can be used include, but
are not limited to, phage M2 DNA polymerase (Matsumoto et al., Gene
84:247, 1989), phage .PHI. PRD1 DNA polymerase (Jung et al., Proc.
Natl. Acad. Sci. USA 84: 8287, 1987), VENT.RTM. DNA polymerase
(Kong et al., J. Biol. Chem. 268:1965-1975, 1993), Klenow fragment
of DNA polymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627,
1974), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19, 1991),
PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta 1219:
267-276, 1994), modified T7 DNA polymerase (Tabor and Richardson,
J. Biol. Chem. 262:15,330-15,333, 1987); Tabor and Richardson, J.
Biol. Chem. 264:6447-6458, 1989); Sequenase.TM. (U.S. Biochemicals,
Cleveland, Ohio, USA), and T4 DNA polymerase holoenzyme (Kaboord
and Benkovic, Curr. Biol. 5:149-157, 1995), all of which
references, are incorporated herein by reference. Strand displacing
DNA polymerases are useful in some embodiments of the invention for
strand-displacing rolling circle replication of circular
first-strand cDNA or circular second-strand cDNA. IsoTherm.TM. DNA
polymerase (rBst DNA polymerase large fragment; Epicentre) is most
preferred because, in addition to having strand-displacing DNA
polymerase activity, it can also be used as a reverse transcriptase
for synthesis of first-strand cDNA from RNA target nucleic acids
(e.g., U.S. Pat. No. 6,030,814 of Jendrisak et al). BcaBEST.TM. DNA
polymerase (Takara Shuzo Co., Kyoto, Japan) can also be used as a
reverse transcriptase as well as a strand-displacing DNA
polymerase.
[0117] Strand displacement can be facilitated through the use of a
strand displacement factor, such as helicase. It is considered that
any DNA polymerase that can perform rolling circle replication in
the presence of a strand displacement factor is suitable for use in
embodiments of the invention that comprise strand displacement or
rolling circle replication, even if the DNA polymerase does not
perform rolling circle replication in the absence of such a factor.
Strand displacement factors useful in rolling circle replication
include, but are not limited to, BMRF1 polymerase accessory subunit
(Tsurumi et al., J. Virology 67:7648-7653, 1993), adenovirus
DNA-binding protein (Zijderveld and van der Vliet, J. Virology
68:1158-1164, 1994), herpes simplex viral protein ICP8 (Boehmer and
Lehman, J. Virology 67:711-715, 1993); Skaliter and Lehman, Proc.
Natl. Acad. Sci. USA 91:10,665-10,669, 1994), single-stranded DNA
binding proteins (SSB; Rigler and Romano, J. Biol. Chem.
270:8910-8919, 1995), and calf thymus helicase (Siegel et al., J.
Biol. Chem. 267:13,629-13,635, 1992).
[0118] 8. Hybridize/Hybridization
[0119] The terms "hybridize" and "hybridization" refer to the
formation of complexes between nucleotide sequences that are
sufficiently complementary to form complexes via Watson-Crick base
pairing. With respect to the present invention, nucleic acid
sequences that "hybridize" or "anneal" with each other should form
"hybrids" or "complexes" that are sufficiently stable to serve the
intended purpose. By way of example, but not of limitation, where a
primer or splice template oligo hybridizes or anneals with a target
nucleic acid in a sample or with a "tailed" target sequence,
respectively, each respective complex or hybrid should be
sufficiently stable to serve the respective priming functions
required for a DNA polymerase to copy the target sequence by primer
extension of the annealed primer or to extend the 3'-end of the
target sequence using the annealed splice template oligo as a
template, respectively.
[0120] 9. RNA Polymerase Promoters
[0121] In certain embodiments of the invention, promoter sequences
may be used that that are recognized specifically by a
DNA-dependent RNA polymerase, such as, but not limited to, those
described by Chamberlin and Ryan, In: The Enzymes. San Diego,
Calif., Academic Press, 15:87-108, 1982, and by Jorgensen et al.,
J. Biol. Chem. 266:645-655, 1991. Several RNA polymerase promoter
sequences are especially useful, including, but not limited to,
promoters derived from SP6 (e.g., Zhou and Doetsch, Proc. Nat.
Acad. Sci. USA 90:6601-6605, 1993), T7 (e.g., Martin, and Coleman,
Biochemistry 26:2690-2696, 1987) and T3 (e.g., McGraw et al., Nucl.
Acid. Res. 13:6753-6766, 1985). An RNA polymerase promoter sequence
derived from Thermus thermophilus can also be used (see, e.g.,
Wendt et al., Eur. J. Biochem. 191:467-472, 1990; Faraldo et al.,
J. Bact. 174:7458-7462, 1992; Hartmann et al., Biochem.
69:1097-1104, 1987; Hartmann et al., Nucl. Acids Res. 19:5957-5964,
1991). The length of the promoter sequence will vary depending upon
the promoter chosen. For example, the T7 RNA polymerase promoter
can be only about 25 bases in length and act as a functional
promoter, while other promoter sequences require 50 or more bases
to provide a functional promoter.
[0122] In other embodiments of the invention, a promoter is used
that is recognized by an RNA polymerase from a T7-like
bacteriophage. The genetic organization of all T7-like phages that
have been examined has been found to be essentially the same as
that of T7. Examples of T7-like phages according to the invention
include, but are not limited to Escherichia coli phages T3, .PHI.I,
.PHI.I, W31, H, Y, A1, 122, cro, C21, C22, and C23; Pseudomonas
putida phage gh-1; Salmonella typhimurium phage SP6; Serratia
marcescens phages IV; Citrobacter phage ViIII; and Klebsiella phage
No. 11 (Hausmann, Current Topics in Microbiology and Immunology
75:77-109, 1976; Korsten et al., J. Gen. Virol. 43:57-73, 1975;
Dunn, et al., Nature New Biology 230:94-96, 1971; Towle, et al., J.
Biol. Chem. 250:1723-1733, 1975; Butler and Chamberlin, J. Biol.
Chem. 257:5772-5778, 1982).
[0123] Some embodiments of the invention also comprises use of the
coliphage N4 RNA polymerase (N4 vRNAP) (Rothman-Denes, L. B., and
Schito, G. C., Virology, 60: 65-72, 1974; Falco, S. C. et al.,
Proc. Natl. Acad. Sci. USA, 74: 520-523, 1977; Falco, S. C. et al.,
J. Biol. Chem., 255: 4339-4347, 1980; Kazmierczak, K. M., et al.,
EMBO J., 21: 5815-5823, 2002, all of which are incorporated herein
by reference) and the promoter sequences of said ssDNA oligos
comprise a conserved promoter sequence recognized by the
Escherichia coli phage N4 vRNAP, wherein said promoter sequence
comprises a 5-basepair stem and 3-base loop hairpin structure
(Glucksmann, M. A. et al., Cell, 70: 491-500, 1992; Haynes, L. L.
and Rothman-Denes, L. B., Cell, 41: 597-605, 1985), which
references and which promoters are incorporated herein as part of
the invention by reference. By way of example, but not of
limitation, promoters comprising the following sequences can be
used:
TABLE-US-00003 P1: 3'-CAACGAAGCGTTGAATACC T-5'; or P2:
3'-TTCTTCGAGGCGAAGAAAACCT-5'; or P3:
3'-CGACGAGGCGTCGAAAACCA-5'.
[0124] In contrast to other known RNA polymerases, the N4 vRNAP
transcribes single-stranded, promoter-containing templates in vitro
with in vivo specificity (Falco, S. C et al., Proc. Natl. Acad.
Sci. USA, 75: 3220-3224, 1978; Haynes, L. L. and Rothman-Denes, L.
B., Cell, 41: 597-605, 1985, all of which are incorporated herein
by reference). In most preferred embodiments of the invention, the
RNA polymerase comprises a transcriptionally active 1,106-amino
acid domain of the N4 vRNAP (herein designated "mini-vRNAP"), which
corresponds to amino acids 998-2103 of N4 vRNAP (Krystyna M.
Kazmierczak, Ph.D. Dissertation, University of Chicago, 2001;
Kazmierczak, K. M., et al., EMBO J., 21: 5815-5823, 2002,
incorporated herein by reference), and reaction conditions are as
described therein.
[0125] 10. Reaction Conditions
[0126] Appropriate reaction media and conditions for carrying out
the methods of the present invention are those that permit nucleic
acid transcription and other reactions according to the methods of
the present invention. With respect to transcription reactions of
the invention with a wild-type or mutant T7 RNAP enzymes, the
reaction conditions for in vitro transcription are those provided
with the AmpliScribe.TM. T7-Flash.TM. Transcription Kit, or the
AmpliScribe.TM. T7 High Yield Transcription Kit, or the
DuraScribe.TM. T7 Transcription Kit or, for incorporation of
2'-substituted deoxyribonucleotides other than
2'-fluorine-substituted deoxyribonucleotides, with the T7
R&DNA.TM. Polymerase, in each case according to the
instructions of the manufacturer EPICENTRE Technologies, Madison,
Wis.). If a T3 or SP6 RNAP is used for transcription using a method
of the invention, the reaction conditions for in vitro
transcription are those provided with the AmpliScribe.TM. T3 High
Yield Transcription Kit or with the AmpliScribe.TM. T3-Flash.TM.
High Yield Transcription Kit, or with the AmpliScribe.TM. SP6 High
Yield Transcription Kit, in each case according to the instructions
of the manufacturer EPICENTRE Technologies, Madison, Wis.).
[0127] If mini-vRNAP or mini-vRNAP Y678F enzymes are used in vitro
transcription of a single-stranded DNA template having a
single-stranded promoter, such as, but not limited to uses for
obtaining additional amplification of transcription products
resulting from an RCT Signal Probe, the following in vitro
transcription reaction is prepared by setting up a reaction mixture
containing the following final concentrations of components, added
in the order given: 0.1 micromolar of a N4 vRNAP
promoter-containing DNA oligo; 1.0 micromolar EcoSSB Protein;
1.times. transcription buffer comprising 40 mM Tris-HCl (pH 7.5), 6
mM MgCl.sub.2., 2 mM spermidine, and 10 mM NaCl; 1 mM DTT; 0.5 mM
of each NTP (ATP, CTP, GTP and UTP); deionized RNase-free water so
the final volume will be 50 microliters after addition of an RNAP;
and 0.1 micromolar of mini-vRNAP or mini-vRNAP Y678F enzyme. In
some embodiments of the invention, 2'-F-dUTP and 2'-F-dCTP are used
at a final concentration of 0.5 mM each in place of UTP and CTP in
order to obtain synthesis of modified RNA which is resistant to
ribonuclease A-type enzymes. Other modified nucleoside
triphosphates can also be used in place of or in addition to the
canonical NTPs for specific applications. The reaction mixture is
then incubated at 37.degree. C. to permit synthesis of RNA from the
template. The reaction can be followed by gel electrophoresis on a
PAGE gel.
[0128] Reaction conditions for in vitro transcription using other
RNA polymerases are well known in the art and can be obtained from
the public literature.
[0129] The invention is not limited to these reaction conditions or
concentrations of reactants. Those with skill in the art will know
that other suitable reaction conditions under which an RNA
polymerase of the invention can be used can be found by simple
experimentation, and any of these reaction conditions are also
included within the scope of the invention. Such media and
conditions are known to persons of skill in the art, and are
described in various publications, such as U.S. Pat. No. 5,679,512
and PCT Pub. No. WO99/42618, incorporated herein by reference. For
example, a buffer can be Tris buffer, although other buffers can
also be used as long as the buffer components are non-inhibitory to
enzyme components of the methods of the invention. The pH is
preferably from about 5 to about 11, more preferably from about 6
to about 10, even more preferably from about 7 to about 9, and most
preferably from about 7.5 to about 8.5. The reaction medium can
also include bivalent metal ions such as Mg.sup.+2 or Mn.sup.+2, at
a final concentration of free ions that is within the range of from
about 0.01 to about 10 mM, and most preferably from about 1 to 6
mM. The reaction medium can also include other salts, such as KCl,
that contribute to the total ionic strength of the medium. For
example, the range of a salt such as KCl is preferably from about 0
to about 100 mM, more preferably from about 0 to about 75 mM, and
most preferably from about 0 to about 50 mM. The reaction medium
can further include additives that could affect performance of the
reactions, but that are not integral to the activity of the enzyme
components of the methods. Such additives include proteins such as
BSA, and non-ionic detergents such as NP40 or Triton. Reagents,
such as DTT, that are capable of maintaining activities enzyme with
sulfhydryl groups can also be included. Such reagents are known in
the art. Where appropriate, an RNase inhibitor, such as, but not
limited to a placental ribonuclease inhibitor (e.g., RNasin.RTM.,
Promega Corporation, Madison, Wis., USA) or an antibody RNase
inhibitor, that does not inhibit the activity of an RNase employed
in the method can also be included. Any aspect of the methods of
the present invention can occur at the same or varying
temperatures. Preferably, the reactions are performed isothermally,
which avoids the cumbersome thermocycling process. The reactions
are carried out at a temperature that permits hybridization of the
oligonucleotides of the present invention to the target sequence
and/or first-strand cDNA of a method of the invention and that does
not substantially inhibit the activity of the enzymes employed. The
temperature can be in the range of preferably about 25.degree. C.
to about 85.degree. C., more preferably about 30.degree. C. to
about 75.degree. C., and most preferably about 37.degree. C. to
about 70.degree. C. In the processes that include RNA
transcription, the temperature for the transcription steps is lower
than the temperature(s) for the preceding steps. In these
processes, the temperature of the transcription steps can be in the
range of preferably about 25.degree. C. to about 85.degree. C.,
more preferably about 30.degree. C. to about 75.degree. C., and
most preferably about 37.degree. C. to about 55.degree. C.
[0130] As disclosed in U.S. Pat. Nos. 6,048,696 and 6,030,814, as
well as in German Patent No. DE4411588C1, all of which are
incorporated herein by reference and made part of the present
invention, it is preferred in many embodiments to use a final
concentration of about 0.25 M, about 0.5 M, about 1.0 M, about 1.5
M, about 2.0 M, about 2.5 M or between about 0.25 M and 2.5 M
betaine (trimethylglycine) in DNA polymerase or reverse
transcriptase reactions in order to decrease DNA polymerase stops
and increase the specificity of reactions which use a DNA
polymerase.
[0131] Nucleotide and/or nucleotide analogs, such as
deoxyribonucleoside triphosphates, that can be employed for
synthesis of reverse transcription or primer extension products in
the methods of the invention are provided in an amount that is
determined to be optimal or useful for a particular intended
use.
[0132] The oligonucleotide components of reactions of the invention
are generally in excess of the number of target nucleic acid
sequence to be amplified. They can be provided at about or at least
about any of the following: 10, 10.sup.2, 10.sup.4, 10.sup.6,
10.sup.8, 10.sup.10, 10.sup.12 times the amount of target nucleic
acid. Promoter primers, splice templates, ligation splint oligos,
blocker sequence oligos, strand-displacement primers, and the like,
can each be provided at about or at least about any of the
following concentrations: 50 nM, 100 nM, 500 nM, 1000 nM, 2500 nM,
5000 nM, or 10,000 nM, but higher or lower concentrations can also
be used. By way of example, but not of limitation, a concentration
of one or more oligonucleotides may be desirable for production of
one or more target nucleic acid sequences that are used in another
application or process. The invention is not limited to a
particular concentration of an oligonucleotide, so long as the
concentration is effective in a particular method of the
invention.
[0133] In some embodiments, the foregoing components are added
simultaneously at the initiation of the process. In other
embodiments, components are added in any order prior to or after
appropriate time points during the process, as required and/or
permitted by the reaction. Such time points can readily be
identified by a person of skill in the art. The enzymes used for
nucleic acid reactions according to the methods of the present
invention are generally added to the reaction mixture following a
step for denaturation of a double-stranded target nucleic acid in
or from a sample, and/or following hybridization of primers and/or
oligos of a reaction to a denatured double-stranded or
single-stranded target nucleic acid, as determined by their thermal
stability and/or other considerations known to the person of skill
in the art.
[0134] The reactions can be stopped at various time points, and
resumed at a later time. The time points can readily be identified
by a person of skill in the art. Methods for stopping the reactions
are known in the art, including, for example, cooling the reaction
mixture to a temperature that inhibits enzyme activity. Methods for
resuming the reactions are also known in the art, including, for
example, raising the temperature of the reaction mixture to a
temperature that permits enzyme activity. In some embodiments, one
or more of the components of the reactions is replenished prior to,
at, or following the resumption of the reactions. Alternatively,
the reaction can be allowed to proceed (i.e., from start to finish)
without interruption.
[0135] 11. Detection and Identification of Transcription Products
or of Compositions Obtained or Derived Therefrom
[0136] In some embodiments, the detection of the product is
indicative of the presence of the target sequence. Quantitative
analysis, including analysis in real time, can also be performed in
some embodiments. Direct and indirect detection methods (including
quantification) are well known in the art. For example, by
comparing the amount of product amplified from a test sample
containing an unknown amount of a polynucleotide containing a
target sequence to the product of a reference sample that has a
known quantity of a polynucleotide that contains the target
sequence, the amount of target sequence in the test sample can be
determined. The methods of the present invention can also be
extended to analysis of sequence alterations and sequencing of the
target nucleic acid. The amplified nucleic acid can be sequenced
using any suitable procedure. Many such procedures are known.
Preferred forms of sequencing for use with amplified sequences
produced from some embodiments are nanosequencing methods described
by Jalanko et al., Clinical Chemistry 38:39-43, 1992; Nikiforov et
al., Nucleic Acids Research 22:4167-4175, 1994; and Kobayashi et
al., Molecular and Cellular Probes 9:175-182, 1995, and primer
extension sequencing, as described in PCT Application WO 97/20948,
all of which references are included herein by reference. Further,
detection could be effected by, for example, examination of
translation products from RNA products.
[0137] B. Methods for Obtaining a ssDNA Comprising a Target
Sequence
[0138] 1. General Aspects and Methods for Obtaining a Target
Sequence
[0139] An initial step in obtaining a target sequence is rendering
the target nucleic acid single-stranded. If the target nucleic acid
is a double-stranded DNA (dsDNA), the initial step is target
denaturation. The denaturation step may be thermal denaturation or
any other method known in the art, such as alkali treatment.
[0140] In some embodiments of the invention in which the target
nucleic acid in a sample is DNA, the ssDNA target sequence
comprises either ssDNA that is present in a biological sample or
ssDNA that is obtained by denaturation of dsDNA in the sample.
[0141] In other embodiments, the ssDNA target sequence comprises
ssDNA that is obtained as a result of a "primer extension
reaction," meaning an in vitro or in vivo DNA polymerization
reaction using either ssDNA or denatured dsDNA that is present in
the sample as a template and an oligonucleotide as a primer under
DNA polymerization reaction conditions. A "primer" is an
oligonucleotide (oligo), generally with a free 3'-OH group, for
which at least the 3'-portion of the oligo is complementary to a
portion of the template and which oligo "binds" (or "complexes" or
"anneals" or "hybridizes"), by hydrogen bonding and other molecular
forces, to the template to give a primer/template complex for
initiation of synthesis by a DNA polymerase, and which is extended
(i.e., "primer extended") by the addition of covalently bonded
bases linked at its 3'-end which are complementary to the template
in the process of DNA synthesis. The result is a primer extension
product. Virtually all DNA polymerases (including reverse
transcriptases) that are known require complexing of an
oligonucleotide to a single-stranded template ("priming") to
initiate DNA synthesis, whereas RNA replication and transcription
(copying of RNA from DNA) generally do not require a primer.
[0142] In some embodiments, the target nucleic acid in the sample
or the primer extension product, or both, are made into smaller DNA
fragments by methods known in the art in order to generate a DNA
target sequence. In some embodiments using samples containing DNA
target nucleic acids, a ssDNA target sequence is obtained using a
strand displacement method, such as but not limited to, a methods
described in PCT Patent Publication Nos. WO 02/16639; WO 00/56877;
and AU 00/29742; of Takara Shuzo Company, Kyoto, Japan; U.S. Pat.
Nos. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211;
5,733,752; 5,744,311; 5,756,702; and 5,916,779 of Becton Dickinson
and Company; U.S. Pat. Nos. 6,238,868; 6,309,833; and 6,326,173 of
Nanogen/Becton Dickinson Partnership; U.S. Pat. Nos. 5,849,547;
5,874,260; and 6,218,151 of Bio Merieux; U.S. Pat. Nos. 5,786,183;
6,087,133; and 6,214,587 of Gen-Probe, Inc.; U.S. Pat. No.
6,063,604 of Wick et al.; U.S. Pat. No. 6,251,639 of Kurn; U.S.
Pat. No. 6,410,278; and PCT Publication No. WO 00/28082 of Eiken
Kagaku Kabushiki Kaishi, Tokyo, Japan; U.S. Pat. Nos. 5,591,609;
5,614,389; 5,773,733; 5,834,202; and 6,448,017 of Auerbach; and
U.S. Pat. Nos. 6,124,120; and 6,280,949 of Lizardi, all of which
are incorporated herein by reference. In still other embodiments,
the ssDNA target sequence is obtained from a rolling circle
replication reaction. The 3'-end of the DNA target sequence can be
defined, if it need be defined, by using any suitable method known
in the art, such as, but not limited to, a method discussed in the
section herein entitled "Methods for Defining the 5'- and 3'-Ends
of Target Sequences That Comprise Only a Portion of a Larger RNA or
DNA Target Nucleic Acid."
[0143] If the target nucleic acid is RNA, the initial step for
obtaining a target sequence comprises synthesis of a
single-stranded first-strand cDNA by reverse transcription of the
RNA target, meaning an in vitro reaction that utilizes an RNA
present in a sample as a template and a nucleic acid
oligonucleotide that is complementary to at least a portion of a
sequence of the RNA template as a primer in order to synthesize
ssDNA using an RNA-dependent DNA polymerase (i.e., reverse
transcriptase) under reaction conditions. Techniques for the
synthesis of cDNA from RNA are known in the art.
[0144] In some embodiments, a first-strand cDNA for use in methods
of the invention is synthesized in situ in cells or tissue in a
tissue section using methods similar to those described in U.S.
Pat. Nos. 5,168,038; 5,021,335; and 5,514,545, which are
incorporated herein by reference. Thus, the first-strand cDNA is
synthesized by contacting the cells or tissue in the tissue section
under hybridizing conditions with a primer, wherein the primer
hybridizes to one or more target sequences in the cell or
tissue.
[0145] The present invention comprises a method for making a
transcription substrate comprising a circular sense
promoter-containing first-strand cDNA that is complementary to a
target sequence comprising a target nucleic acid in cells or tissue
in a tissue section, the method comprising:
[0146] (a) contacting the cells or tissue in the tissue section
under hybridizing conditions with a sense promoter primer, the
sense promoter primer comprising (i) a 5'-phosphorylated portion
comprising a sense transcription promoter for an RNA polymerase
that can synthesize RNA using this promoter in a transcription
substrate, and (ii) a 3'-end that is complementary to the target
sequence comprising the target nucleic acid;
[0147] (b) contacting the cells or tissue containing the sense
promoter primer in the tissue section with a reverse transcriptase
under reverse transcription conditions so as to obtain a
first-strand cDNA that is complementary to the target sequence;
[0148] (c) obtaining linear sense promoter-containing first-strand
cDNA;
[0149] (d) ligating the linear sense promoter-containing
first-strand cDNA so as to obtain circular sense
promoter-containing first-strand cDNA;
[0150] (e) annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate.
[0151] The circular transcription substrate in the cells or tissues
can be incubated with an RNA polymerase that uses the promoter
under transcription conditions, wherein a transcription product is
obtained.
[0152] A primer can have a sequence that is complementary to a
specific known sequence in the RNA target in a sample, or a primer
can have a sequence comprising a mixture of all possible or many
possible sequences, such as, but not limited to, a primer
comprising a random hexamer priming sequence. Random primer
sequences can be made using an oligonucleotide synthesizer by
including nucleotide reagents that are complementary to each of the
four canonical bases (i.e., all four nucleotides) during the
chemical synthesis of each nucleotide position of the
oligonucleotide that is complementary to the target sequence. In
embodiments of the invention using samples containing mRNA targets,
the ssDNA target sequence comprises first-strand cDNA that is made
by reverse transcription of the mRNA using an oligonucleotide
primer comprising either a specific sequence which is complementary
to a known sequence of a specific mRNA or, if the mRNA has a
poly(A) tail at its 3'-end, an oligo(dT) primer or an oligo(dT)
anchor primer. In other embodiments of the invention, a sense
promoter primer is used, which serves both to prime synthesis of
the first-strand cDNA target sequence and to join a sense
transcription promoter to the target sequence.
[0153] 2. Methods for Defining the 5'- and 3'-Ends of Target
Sequences That Comprise Only a Portion of a Larger RNA or DNA
Target Nucleic Acid
[0154] When a method of the invention is used to obtain a
transcription product corresponding to the complete sequence(s) of
one or a multitude of nucleic acid molecules, such as, but not
limited to the complete sequences (excluding the cap structure) of
substantially all polyadenylated mRNA molecules in a sample, it is
not necessary to devise methods to define the 5'- and 3'-ends of
the sequences. However, if a method of the invention is used to
obtain a transcription product corresponding to a target sequence
that comprises only a portion of a larger RNA or DNA nucleic acid
in a sample, then methods are needed to delimit the target sequence
that becomes the transcription template.
[0155] There are two general approaches for delimiting the ends of
the target sequence that becomes the transcription template
sequence. In the first direct approach, methods are used to
determine the size and end sequences of a target nucleic acid
molecule or molecules present in the sample itself. In the second
indirect approach, instead of changing the size and end sequences
of the target nucleic acid molecules present in a sample, methods
are used to determine the size and end sequences of one or more
first-strand cDNA molecules that is/are synthesesized by reverse
transcription or primer extension, respectively, of RNA or of at
least one strand of DNA in a sample.
[0156] With respect to the direct approach, a number of methods are
known in the art for cleaving a nucleic acid molecule at or near a
specific sequence, and any of the methods which delimit the size
and end sequences of a target nucleic acid for an application of
the present invention can be used. By way of example, but not of
limitation, a DNA in a sample comprising a dsDNA molecule or a
ssDNA molecule to which an appropriate complementary DNA oligo is
annealed can be digested with a restriction endonuclease, provided
a restriction site that would provide a suitable 5'-end and/or
3'-end sequence is present. Alternatively, one or more DNA
oligonucleotides having a double-stranded segment that contains a
FokI restriction enzyme site and a single-stranded segment that
binds to the desired cleavage site on a first-strand cDNA can be
used. As is well known in the art, this type of oligonucleotide can
be used with the restriction enzyme FokI to cut a single-stranded
DNA at almost any desired sequence (Szybalski, W., Gene 40:169-173,
1985; Podhajska A. J. and Szybalski W., Gene 40:175, 1985,
incorporated herein by reference).
[0157] By way of further example, but not of limitation, a ssRNA
target nucleic acid present in a sample can be cleaved using a
ribonuclease H in regions to which complementary oligonucleotides
comprising at least three-to-four deoxynucleotides are annealed.
Alternatively, a linear DNA oligonucleotide can be annealed to an
RNA in a sample at a location that encodes a recognition site of a
restriction enzyme that can cut RNA:DNA heteroduplexes. Cutting the
target RNA:DNA oligo with the enzyme will then generate a defined
end. Alternatively, an RNA or DNA oligo or polynucleotide with a
sequence complementary to the region of an RNA target sequence that
is intended to become a transcription substrate can be annealed to
the RNA and the sequences of the RNA to which the oligo or
polynucleotide is not annealed can be digested using a
single-strand-specific ribonuclease, such as RNase A or RNase T1.
Still further, either RNA or DNA nucleic acids of known sequence
can be cleaved at specific sites using a 5'-nuclease or
Cleavase.TM. enzyme and specific oligonucleotides, as described by
Kwiatkowski, et al., (Molecular Diagnosis 4:353-364, 1999) and in
U.S. Pat. No. 6,001,567 and related patents assigned to Third Wave
Technologies (Madison, Wis., USA), which are incorporated herein by
reference.
[0158] In general, with respect to the second indirect approach,
the 5'-end of the primer that is used for reverse transcription of
RNA in a sample or for primer extension of at least one strand of
DNA in a sample defines the 5'-end of the first-strand cDNA target
sequence. Thus, a sample target nucleic acid that is reverse
transcribed or primer extended to make a first-strand cDNA target
sequence need not have a defined 3'-end.
[0159] In order to generate a defined 3'-end on a first-strand cDNA
(i.e., corresponding to the 5'-end of the target sequence), a
number of methods can be used to obtain a target sequence of the
present invention. By way of example, but not of limitation, if a
specific sequence is present in the first-strand cDNA that
corresponds to a restriction endonuclease site that would provide a
suitable 3'-end sequence, a complementary DNA oligo can be annealed
to this sequence and the site can be cleaved with the restriction
enzyme. The complementary DNA oligo used to provide the
double-stranded restriction site can optionally have a
2',3'-dideoxynucleotide or another terminator nucleotide at its
3'-end so that it cannot be extended by a DNA polymerase.
Alternatively, the 3'-end of the target sequence can be defined
using a DNA oligonucleotide having a double-stranded segment that
contains a Fold restriction enzyme site and a single-stranded
segment that binds to the desired cleavage site on a first-strand
cDNA (Szybalski, W., Gene 40:169-173, 1985; Podhajska A. J. and
Szybalski W., Gene 40:175, 1985). Still further, a 5'-nuclease can
be used to cleave a first-strand cDNA at a defined 3'-end as
discussed above.
[0160] In addition to the above methods, the 3'-end of a
first-strand cDNA can also be limited by other methods. A preferred
method of the invention is to use a "blocking oligo" or a "blocker
sequence," as disclosed by Laney, et al. in U.S. Pat. No.
5,679,512, and by Kurn in U.S. Pat. No. 6,251,639, both of which
are incorporated herein by reference. The "blocker sequence" or
"blocker oligo" is a polynucleotide, which is usually a synthetic
polynucleotide that is single-stranded and comprises a sequence
that is hybridizable, and preferably complementary, to a segment of
target nucleic acid, wherein the blocking oligo anneals to the
target nucleic acid so as to block further primer extension of the
3'-end of first-strand cDNA at a desired position. Some embodiments
of strand displacement methods of the present invention for
obtaining a ssDNA target sequence comprise use of a blocking oligo.
The blocking oligo comprises nucleotides that bind to the target
nucleic acid with an affinity, preferably a high affinity, such
that the blocker sequence resists displacement by DNA polymerase in
the course of primer extension, in preferably more than about 30%,
more preferably more than about 50%, even more preferably more than
about 75%, and most preferably more than about 90%, of primer
extension events. The length and composition of the blocker
polynucleotide should be such that excessive random non-specific
hybridization is avoided under the conditions of the methods of the
present invention. The length of the blocker polynucleotide is
preferably from about 3 to about 30 nucleotides, more preferably
from about 5 to about 25 nucleotides, even more preferably from
about 8 to about 20 nucleotides, and most preferably from about 10
to about 15 nucleotides. In other embodiments, the blocker
polynucleotide is at least about any of the following: 3, 5, 8, 10,
15; and less than about any of the following: 20, 25, 30, 35. It is
understood that the length can be greater or less as appropriate
under the reaction conditions of the methods of this invention. The
complementarity of the blocker polynucleotide is preferably at
least about 25%, more preferably at least about 50%, even more
preferably at least about 75%, and most preferably at least about
90%, to its intended binding sequence on the target nucleic acid.
In some embodiments, the blocker sequence that hybridizes to a DNA
target nucleic acid is attached to the DNA such that displacement
of the blocker sequence by the polymerase that affects primer
extension is substantially, or at least sufficiently, inhibited.
Suitable methods for achieving such attachment include techniques
known in the art, such as using a cytosine analog that contains a
G-clamp heterocycle modification as described by Flanagan et al.,
(Proc. Natl. Acad. Sci. USA 96:3513-3518, 1999); and locked nucleic
acids as described, e.g., by Kumar et al., (Bioorg. Med. Chem.
Lett. 8:2219-2222, 1998; and by Wahlestedt et al. (Proc. Natl.
Acad. Sci. USA 97:5633-5638, 2000), all of which are incorporated
herein by reference. Other suitable methods include using, where
appropriate, sequences with a high GC content and/or cross-linking.
Any of these methods for obtaining enhanced attachment may be used
alone or in combination. Alternatively, a molecule comprising a
peptide nucleic acid ("PNA") can be used.
[0161] Still further, another method that can be used to limit the
3'-end of a first-strand cDNA is to use a thermocycler with short
DNA synthesis elongation cycles during reverse transcription or
primer extension to synthesize first-strand cDNA. The length of the
primer extension product can be somewhat controlled by the length
of the DNA synthesis cycle. Conditions can be determined to define
an approximate chain length of first-strand cDNA by controlling the
temperature and time interval of DNA synthesis before denaturing
the growing first-strand cDNA from the template by raising the
temperature.
[0162] Further, the 3'-end of a first-strand cDNA that is to become
the template sequence for a transcription reaction can be defined
by first amplifying the target nucleic acid sequence using any
suitable amplification method that delimits the end sequence. By
way of example, but not of limitation, it can be prepared using
PCR, RT-PCR, NASBA, TMA, 3SR, Ligation Chain Reaction (LCR), Linked
Linear Amplification (BioRad), SDA, RCA, ICAN.TM. (Takara: Sagawa
et al. in PCT Patent Publication No. WO 02/16639; and in PCT Patent
Publications Nos. WO 00/56877 and AU 00/29742; or a
strand-displacement method of Kurn (U.S. Pat. No. 6,251,639), all
of which are incorporated herein by reference.
[0163] If a 3'-end of a target sequence need not be at an exact
location, and can be random or imprecise, which is the case in some
embodiments of the invention, there are a number of other methods
that can be used for making smaller fragments of a DNA molecule,
whether for a target nucleic acid, a target sequence, or otherwise.
By way of example, but not of limitation, a target nucleic acid can
be fragmented by physical means, such as by movement in and out of
a syringe needle or other orifice or by sonication, preferably with
subsequent end repair, such as using a T4 DNA polymerase or a kit,
such as the End-It.TM. DNA End Repair Kit (Epicentre Technologies,
Madison, Wis., USA). Still another method that can be used is to
incorporate dUMP randomly into the first-strand cDNA during reverse
transcription or primer extension by using dUTP in place of a
portion of the TTP in the reaction. The dUMP will be incorporated
randomly in place of TMP at a frequency based on the ratio of dUTP
to TTP. Then, the first-strand cDNA can be cleaved at sites of dUMP
incorporation by treatment (e.g., see U.S. Pat. No. 6,048,696,
incorporated herein by reference) with uracil-N-glycosylase (UNG)
and endonuclease IV (endo N), which are available from Epicentre
Technologies (Madison, Wis., USA). UNG hydrolyzes the N-glycosidic
bond between the deoxyribose sugar and uracil in single- and
double-stranded DNA that contains uracil in place of thymidine. It
has no activity on uracil residues in RNA or on dUTP. Endo IV
cleaves the phosphodiester linkage at the abasic site. It may be
useful to use a thermolabile UNG (e.g., HK.TM.-UNG from Epicentre
Technologies, Madison, Wis., USA) for some applications. (Also,
incorporation of dUMP at specific sites within a synthetic
oligonucleotide or, for example, within a promoter primer of the
invention between the 3'-target-sequence-complementary portion and
the promoter sequence, introduces specific cleavage sites which can
be used at any time to cleave a resulting nucleic acid which
contains the site by treatment with UNG and endo N.) Still further,
in some cases, the 3'-end of a first-strand cDNA can be defined by
treatment with exonuclease III (Henikoff, S., Gene 28:351, 1984).
In still other cases, the 3'-end of a first-strand cDNA that is
annealed to a DNA target nucleic acid can be incubated with T4 DNA
polymerase or unmodified T7 DNA polymerase (Epicentre Technologies,
Madison, Wis.) in the absence or the presence of dNTPs in the
reaction; these enzymes have the 3'-to-5' exonuclease activity in
the absence of dNTPs, but the polymerase activity predominates in
the presence of dNTPs. These are only some of the methods that can
be used to define the 3'-ends of a first-strand cDNA, and the
invention is not limited to these methods, which are presented only
as examples.
[0164] C. Methods for Obtaining a Transcription Substrate
[0165] 1. Introduction
[0166] In a first general embodiment, first-strand cDNA is obtained
by reverse transcription or primer extension using a sense promoter
primer comprising a sense transcription promoter in its 5'-portion
and a sequence complementary to the target sequence at its 3'-end
as a primer and a target nucleic acid sequence as a template. A
circular transcription substrate is obtained by ligating the
first-strand cDNA to obtain circular sense promoter-containing
first-strand cDNA and then annealing an anti-sense promoter oligo
to the sense promoter sequence. In another embodiment, a linear
transcription substrate having the sense promoter 3'- of the target
sequence is obtained by linearizing circular sense
promoter-containing first-strand cDNA (e.g., without limiting the
invention, using uracil-N-glycosylase and endonuclease IV) and then
a circular transcription substrate is obtained by annealing of the
anti-sense promoter oligo.
[0167] In still another embodiment, a transcription substrate
comprising second-strand cDNA is obtained by:
[0168] (a) synthesizing linear first-strand cDNA using a reverse
transcriptase or a DNA polymerase and an anti-sense promoter primer
comprising an anti-sense transcription promoter in its 5'-portion
and a sequence complementary to the target sequence at its
3'-end;
[0169] (b) ligating the resulting linear first-strand cDNA to
obtain a circular first-strand cDNA having an anti-sense
transcription promoter;
[0170] (c) obtaining a linear sense-promoter-containing
second-strand cDNA by DNA synthesis, preferably rolling circle
replication DNA synthesis, using the circular first-strand cDNA as
a template, a primer with a 3'-end complementary to a sequence on
first-strand cDNA and a DNA polymerase, preferably a
strand-displacing DNA polymerase; and
[0171] (d) annealing an anti-sense promoter oligo to obtain a
linear transcription substrate. If the second-strand cDNA is
obtained by rolling circle replication the linear transcription
substrate obtained is a concatemeric linear transcription
substrate. In this embodiment, the transcription product of the
transcription substrate comprises an anti-sense transcription
product.
[0172] The embodiments of processes for obtaining a transcription
substrate for methods of the invention described above are provided
only as examples, and are not intended to limit the present
invention. The description above and herein will reveal and make
evident to those with knowledge in the art numerous other
embodiments of methods and processes for obtaining a transcription
substrate for use in making a transcription product corresponding
to a target nucleic acid sequence in a method of the invention, and
the invention includes all of those methods and processes for
obtaining a transcription substrate for use in the methods.
[0173] In embodiments in which the target sequences comprise mRNA,
whether of a single species of mRNA or all of the mRNA in a
particular sample, the transcription products can subsequently be
used for a variety of applications. By way of example, but not of
limitation, transcription products can be used for in vitro or in
vivo translation, for use as RNAi to silence one or more genes in
vivo, for spotting on a surface to make expression arrays or
microarrays, or for making hybridization probes for arrays or
microarrays for gene expression profiling or other uses. In still
other embodiments, methods of the invention can be used to make
first-strand cDNA from mRNA, which in turn can be used for
techniques such as random amplification of cDNA ends (RACE) or to
make hybridization probes.
[0174] A "ligation splint" or a "ligation splint oligo" is an oligo
that is used to provide an annealing site or a "ligation template"
for joining two ends of one nucleic acid (i.e., "intramolecular
joining") or two ends of two nucleic acids (i.e., "intermolecular
joining") using a ligase or another enzyme with ligase activity.
The ligation splint holds the ends adjacent to each other and
"creates a ligation junction" between the 5'-phosphorylated and a
3'-hydroxylated ends that are to be ligated. For example, when a
ligation splint oligo is used to join a sense promoter ligation
oligo to the 3'-end of a first-strand cDNA comprising a target
sequence, the ligation splint oligo has a sequence complementary to
the 3'-end of the target sequence, including a "tailed" target
sequence, if any, and a second adjacent sequence that is
complementary to the 5'-end of a the 5'-phosphorylated promoter
ligation oligo. Ligases that can be used to ligate suitable ends
that are annealed to a ligation splint comprising DNA include, but
are not limited to, Ampligase.RTM. DNA Ligase (EPICENTRE
Technologies, Madison, Wis.), Tth DNA ligase, Tfl DNA ligase, Tsc
DNA ligase (Prokaria, Ltd., Reykjavik, Iceland), or T4 DNA ligase.
These ligases can be used for both intermolecular and
intramolecular ligations when a ligation splint comprising DNA is
used to bring the respect ends adjacent. If a ligation splint
comprising RNA is used, T4 DNA ligase can be used to join the ends
that are annealed to the ligation splint. In some embodiments, a
ligase that catalyzes non-homologous intramolecular ligation of a
5'-phosphorylated end with a 3'-hydroxyl end can be used in methods
of the invention for circularization of single-stranded DNA without
a ligation splint. By way of example, but not of limitation, a
ligation splint is not required for ligation of a ssDNA using
ThermoPhage.TM. RNA Ligase II (Prokaria, Ltd., Reykjavik,
Iceland).
[0175] In some embodiments, remaining linear nucleic acids, such
as, but not limited to, ligation splint oligos, are removed during
the reaction using the gene 6 exonuclease of phage T7. This
exonuclease digests DNA starting from the 5'-end of a
double-stranded structure. It has been used successfully for the
generation of single-stranded DNA after PCR amplification (Holloway
et al., Nucleic Acids Res. 21:3905-3906, 1993; Nikiforov et al.,
PCR Methods and Applications 3:285-291, 1994, incorporated herein
by reference). The gene 6 exonuclease of phage T7 can be added
after ligation, together with the rolling circle DNA polymerase to
remove unligated oligos. To protect the product of a strand
displacement reaction from degradation, a strand displacement
primer for rolling circle replication can contain 3 or 4
phosphorothioate linkages at the 5'-end, to make this molecule
resistant to the exonuclease (Nikiforov et al., PCR Methods and
Applications 3:285-291, 1994). The exonuclease degrades unprotected
linear molecules as they become associated with the rolling circle
DNA product. Based on this description, those with knowledge in the
art will understand and know other embodiments of the invention in
which this process of the invention for removing single-stranded
DNA oligos can be used to advantage, and the invention comprises
all such embodiments.
[0176] A ligation splint or a ligation splint oligo provides an
annealing site or a "ligation template" on which a
5'-phosphorylated end and a 3'-hydroxyl end of one or two different
nucleic acids, such as, but not limited to, a promoter ligation
oligo and a first-strand cDNA target nucleic acid sequence, can
hybridize so as to bring the two ends adjacent to one another in a
ligation reaction for joining by a ligase (or a topoisomerase or
other enzyme with ligase activity). A ligation splint comprises a
sufficient number and composition of nucleotides, and is present in
sufficient concentration, so that the complementary sequences of
both the 5'- and 3'-ends remain annealed to the ligation splint
under ligation conditions so to permit ligation of the ends to
occur. Thus, in most embodiments, the ligation splint comprises at
least about 4 nucleotides up to about 20 nucleotides, but the
invention is not limited to a specific number of nucleotides. An
appropriate sequence (and T.sub.n), size, and concentration for a
ligation splint can be determined empirically by those with
knowledge in the art. In most embodiments of the present invention,
it is preferable that the 3'-terminal nucleotide of a ligation
splint is a dideoxynucleotide or another termination nucleotide, so
that the ligation splint oligo cannot serve as a primer for
polymerases in the reaction. In most embodiments in which there are
multiple rounds of transcription of a target nucleic acid sequence,
the other nucleotides in a ligation splint oligo comprise
deoxynucleotides. However, in some embodiments, the other
nucleotides can comprise ribonucleotides and/or purine
ribonucleotides and 2'-fluoro-pyrimidine nucleotides, which confer
resistance to RNase A-type nucleases. The composition of a ligation
splint oligo will also depend on the ligase and ligation conditions
used. By way of example, Ampligase.RTM. Thermostable Ligase
(Epicentre Technologies, Madison, Wis., USA) will only ligate the
5'-phosphoryl and 3'-hydroxyl ends of DNA that is annealed to a DNA
ligation splint and will not ligate DNA ends annealed to RNA.
However, T4 RNA ligase has been reported to efficiently ligate DNA
ends that are annealed to an RNA ligaton splint (Faruqui et al.,
U.S. Pat. No. 6,368,801, incorporated herein by reference).
Ligation splint oligos can be synthesized on an oligo synthesizer,
which is usually preferred, or enzymatically, using methods
discussed elsewhere herein.
[0177] 2. Obtaining Transcription Substrates Using a Promoter
Primer
[0178] a. Methods for Using Promoter Primers and Anti-Sense
Promoter Oligos
[0179] A "promoter primer" is a primer, generally with a free 3'-OH
group, that comprises a sequence that is complementary to a target
sequence at its 3'-end and which encodes a transcription promoter
in its 5'-portion. The transcription promoter in the 5'-end portion
can be either a "sense" promoter" or an "anti-sense promoter." As
defined herein, the promoter sequence of a double-stranded promoter
that is operably joined to the 3'-end of the template strand
sequence that is transcribed is a "sense promoter sequence" and a
promoter primer that comprises this sequence is "a sense promoter
primer." The sequence of a double-stranded promoter that is
complementary to the sense promoter is defined herein as "an
anti-sense promoter" and a promoter primer that comprises this
sequence is "an anti-sense promoter primer."
[0180] A sense promoter primer is used in most embodiments of the
present invention. If a sense promoter primer is used, a "circular
transcription substrate" that comprises a functional
double-stranded promoter can be obtained by annealing "an
anti-sense promoter oligo" to a molecule that is obtained by primer
extension of the sense promoter primer on a template (which is
usually a target nucleic acid) and then ligating the resulting
first-strand cDNA to obtain a "circular sense promoter-containing
first-strand cDNA." That is, annealing of the anti-sense promoter
oligo to the circular sense promoter-containing first-strand cDNA
makes a circular transcription substrate. If the circular sense
promoter-containing first-strand cDNA is linearized 3'- of the
sense promoter sequence therein using methods described elsewhere
in the description of the invention, a "linear sense
promoter-containing first-strand cDNA" is obtained. A "linear
transcription substrate" is obtained by annealing an anti-sense
promoter oligo to the linear sense promoter-containing first-strand
cDNA.
[0181] An anti-sense promoter oligo is also used in other
embodiments of the invention for obtaining a transcription
substrate, such as in embodiments wherein the anti-sense promoter
oligo is complexed with a linear sense promoter-containing
second-strand cDNA to obtain a transcription substrate, as
described elsewhere herein.
[0182] An "antisense promoter oligo" as defined herein comprises an
oligonucleotide that comprises a sequence for an anti-sense
transcription promoter, wherein an RNA polymerase can use the
double-stranded complex between the anti-sense promoter oligo and a
sense promoter that is joined to the 3'-end of a template sequence
to make a transcription product that is complementary to the
template under transcription conditions.
[0183] A promoter primer can have a sequence at its 3'-end that is
complementary to a specific known sequence in a target nucleic
acid., in which case it is referred to as a "specific-sequence
promoter primer." However, other embodiments of promoter primers
can also be used in methods of the invention. An "oligo(dT)
promoter primer" has an oligo(dT) sequence at its 3'-end, and is
used mainly in embodiments of the invention which pertain to mRNA
molecules having polyadenylated [i.e., poly(A) tails], although an
oligo(dT) promoter primer can also be used in embodiments in which
another target nucleic acid is tailed with poly(A) or poly(dA). An
"anchored oligo d(T) promoter primer," in addition to having an
oligo(dT) sequence in its 3'-portion, also has one (or a small
number) of nucleotides 3'- of the oligo(dT) sequence, called
"anchor nucleotides," which anneal to the 3'-portion of the mRNA
target sequence just prior to the poly(A) sequence. Thus, the
anchor nucleotides serve to "anchor" the mRNA-complementary portion
of the anchored oligo(dT) promoter primer to the beginning of the
protein-coding sequence of the mRNA target sequence. The anchor
nucleotides can comprise either a specific base for a specific mRNA
or a randomized nucleotide (i.e., synthesized with a mixture of all
four nucleotides) for priming all mRNA molecules in a sample. A
"random-sequence promoter primer" has a random sequence, such as,
but not limited to a random hexamer sequence or a random octamer
sequence, at its 3'-end. In most cases, a random-sequence promoter
primer comprises a mixture of primers with all possible sequences
(e.g., all possible hexamers) in its target sequence-complementary
portion. Random-sequence promoter primers can be made by including
all four canonical nucleotide reagents during the chemical
synthesis of each of the nucleotide positions of the random
sequence (e.g., the hexamer sequence) of the target
sequence-complementary portion of the primer. A random-sequence
promoter primer can be used in those embodiments of the invention
in which it is desired to amplify all target nucleic acid sequences
in a sample, or to amplify all target nucleic acid sequences in a
random manner, such as for making a library of all target nucleic
acid sequences. However, although all sequences may be amplified,
use of a random-sequence promoter primer does not necessarily
generate only full-length copies of target nucleic acids (e.g.,
full-length cDNA copies of mRNA molecules from a cell). Thus,
embodiments of the invention which use random-sequence promoter
primers are usually used when full-length copies of a target
nucleic acid sequence are not required, such as, for obtaining
hybridization probes for some applications.
[0184] In embodiments of the invention in which a transcription
substrate of the invention comprises first-strand cDNA obtained by
reverse transcription or primer extension of a promoter primer
using a target nucleic acid as a template, the transcription
promoter in the promoter primer comprises a sense promoter sequence
that is located in the 5'-portion of the promoter primer. Thus, the
transcription promoter in the linear first-strand cDNA obtained by
reverse transcriptase- or DNA polymerase-catalyzed extension of the
promoter primer using the target nucleic acid as a template is not
operable as a promoter for transcription of the target sequence
since the promoter is not operably joined to the 3'-end of the
target sequence. A method of the present invention solves this
problem by operably joining the single-stranded sense transcription
promoter in the 5'-portion of the linear first-strand cDNA to the
3'-end of the target sequence using a ligase or another joining
means, thereby forming a "circular sense promoter-containing
first-strand cDNA" that can be used to obtain a circular
transcription substrate by annealing of an anti-sense promoter
oligo to the sense promoter sequence. The circular transcription
substrate can be used to make a transcription product corresponding
to the target sequence by transcription using an RNA polymerase
that can bind the single-stranded promoter and transcribe the
target sequence joined thereto.
[0185] Thus, in one embodiment of the invention, a promoter primer
having a sequence complementary to a target sequence at its 3'-end
and a transcription promoter in its 5'-portion is used to obtain a
circular transcription substrate of the invention (FIG. 1). After
annealing to a target nucleic acid, the promoter primer is used to
prime first-strand cDNA synthesis using a DNA polymerase or reverse
transcriptase under suitable reaction conditions in order to obtain
linear first-strand cDNA.
[0186] The first-strand cDNA is then ligated using a ligase under
suitable ligation conditions, or using another joining method, such
as, but not limited to a topoisomerase (e.g., see U.S. Pat. No.
5,766,891, incorporated herein by reference), under suitable
joining conditions, so as to obtain a circular promoter-containing
first-strand cDNA. By ligation of the phosphorylated 5'-end of
linear first-strand cDNA to its 3'-end, the transcription promoter
is joined to the target sequence so that in vitro transcription
using an RNA polymerase of the invention under transcription
conditions will synthesize transcription products corresponding to
the target sequence.
[0187] In the example in FIG. 1, the target sequence can comprise
target nucleic acid comprising all mRNA molecules in a sample and
the transcription products that are made by transcription of the
transcription substrate comprise RNA that has essentially the same
sequence as sense mRNA. If there is no sequence in the circular
transcription substrate that results in termination of
transcription (i.e., a transcription terminator sequence),
transcription continues around and around the circular
transcription substrate multiple times and generates concatemers of
sense transcription products (i.e., comprising tandem copies of the
same nucleic acid sequence as an mRNA target nucleic acid sequence
in the sample), which concatemers are useful for certain
applications of the invention.
[0188] In some embodiments of the invention, one or more
transcription termination sequences is/are incorporated into the
promoter primer between the target sequence-complementary 3'-region
and the transcription promoter 5'-portion in order to permit
synthesis of single-copy rather than concatemeric sense
transcription products. For example, if a transcription termination
sequence is present in the promoter primer, the transcription
product can correspond in length to a single copy of an mRNA target
nucleic acid sequence following in vitro transcription of the
circular transcription substrate using an RNA polymerase of the
invention. Transcription termination sequences are known in the art
and those with knowledge in the art will know how to find
information about the sequences, as well as experimental methods
for identifying additional termination sequences that can be used.
By way of example, but not of limitation, information about
transcription termination sequences can be found in a book entitled
"RNA Polymerases and the Regulation of Transcription," edited by
Reznikoff, W. S., et al., (Elsevier Science Publishing Co., Inc.,
New York, 1987) and in Section 17 of a book by Miller, J. H.
entitled "A Short Course in Bacterial Genetics. A Laboratory
Handbook for Escherichia coli and Related Bacteria" (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1992), both
incorporated herein by reference.
[0189] In still other embodiments, the circular sense
promoter-containing first-strand cDNA obtained by ligation of the
first-strand cDNA is linearized prior to use for in vitro
transcription in order to form a "linear sense promoter-containing
first-strand cDNA," which is annealed to an anti-sense promoter
oligo to obtain a linear transcription substrate of the invention
(FIG. 2).
[0190] By way of example, but not of limitation, the circular sense
promoter-containing first-strand cDNA can be linearized by
treatment with uracil-N-gycosylase ("UNG") and endonuclease IV
("endo IV") (e.g., see methods in U.S. Pat. No. 6,048,696, which is
incorporated herein by reference) if the promoter primer is
synthesized to have a dUMP nucleotide between the target
sequence-complementary 3'-region and the transcription promoter in
its 5'-portion. However, the use of a promoter primer comprising a
dUMP nucleotide can only be used in embodiments of the invention
which are performed in a stepwise manner, because the presence of
UNG in a continuous reaction would cleave the transcription
promoter portion of a promoter primer from the target-complementary
portion of the promoter primer, thus destroying the ability of the
promoter primer to generate more transcription substrates. There
are a number of other methods known in the art for linearizing a
circular DNA molecule, which can be used in embodiments of the
invention, and those with knowledge in the art will know or know
how to find suitable methods for use in the invention. By way of
example, but not of limitation, a number of such methods which can
be used are described herein in the section entitled "Methods for
Defining the 5'- and 3'-Ends of Target Sequences That Comprise Only
a Portion of a Larger RNA or DNA Target Nucleic Acid."
[0191] Other embodiments of the invention comprise use of a
promoter primer for synthesis of a second-strand cDNA as a
transcription substrate of the invention. In those embodiments, a
transcription promoter can be incorporated into the second-strand
cDNA by, either (a) synthesizing first-strand cDNA using a promoter
primer that has a sequence in its 5'-portion comprising a sequence
that is complementary to a sense transcription promoter (i.e., it
comprises an anti-sense promoter primer) or, (b) using a promoter
primer comprising a sense transcription promoter for synthesis of
second-strand cDNA. The promoter sequence used in a promoter primer
can be determined based on knowledge of sequences of sense
promoters for RNA polymerases of the invention and of the rules of
nucleic acid base complementarity and the directionality of DNA and
RNA synthesis by DNA and RNA polymerases. By way of example, if a
sense transcription promoter is desired in a second-strand cDNA
that is made by primer extension of first-strand cDNA, which is in
turn made by reverse transcription of mRNA, one can work backwards
from having a sense transcription promoter of known sequence in a
transcription substrate at the 3'-end of a target nucleic acid
sequence that is to be amplified by an RNA polymerase of the
invention, in order to determine the appropriate sense or
anti-sense sequence (and position of the sequence with respect to
the target sequence) that is needed, respectively, for promoter
primers that are used for first-strand cDNA synthesis, or for
second-strand cDNA synthesis.
[0192] In some embodiments of the invention, more than one promoter
can be present on the promoter primer. By way of example, but not
of limitation, a promoter primer can encode two promoter sequences,
both of which encode sense promoters on first-strand cDNA (e.g.,
for two different RNA polymerases of the invention). Alternatively,
a first promoter sequence of the promoter primer and the resulting
first-strand cDNA can comprise a sense promoter and a second
promoter sequence can comprise an anti-sense promoter, in which
case, the first promoter sequence will be anti-sense and the second
promoter sequence will be sense in second-strand cDNA. In
embodiments that include additional rounds of transcription by
using RNA from the first round to obtain a second transcription
substrate for transcription, it is necessary to take into account
the fate of the promoter sequences through the subsequent rounds of
transcription when designing the reaction.
[0193] In addition to the transcription promoter sequence and the
target-complementary sequence, a promoter primer of the invention
can also have additional nucleic acid sequences that are 5'- of
and/or 3'- of the transcription promoter sequence, but a promoter
primer is not required to have such additional other sequences. By
way of example, but not of limitation, a promoter primer can have a
transcription initiation site 5'- of the promoter sequence. In some
embodiments of the invention, a promoter primer can have one or
more transcription termination sequences, one or more sites for DNA
cleavage, (such as, but not limited to, a dUMP residue that can be
cleaved using uracil-N-glycosylase and endonuclease IV, or other
cleavage methods discussed elsewhere herein) to permit controlled
linearization of a circular first-strand cDNA that is a
transcription substrate, one or more origins ("ori's") of
replication (preferably an ori for a single-stranded replicon, such
as, but not limited to, a phage M13 replicon), a selectable or
screenable marker, such as, but not limited to an
antibiotic-resistance gene or a beta-galactosidase gene,
respectively, or one or more transposon recognition sequences
(e.g., OE or ME sequences) that can be recognized and used by a
transposase for in vitro or in vivo transposition, or one or more
sites that are recognized by a recombinase (such as, but not
limited to, the cre-lox system), and/or other sequences or genetic
elements for a particular purpose. After reading the specification
of the present invention, those with knowledge in the art will know
that a sequence that is 5'- of a functional promoter will be
transcribed by an RNA polymerase of the invention, and will
therefore know where to position particular additional sequences or
genetic elements relative to the promoter sequence in a promoter
primer. In some embodiments of the invention, a promoter primer has
a 5'-phosphate or is phosphorylated at its 5'-end using an enzyme,
such as, but not limited to, a polynucleotide kinase (e.g., T4
PNK), during the processes of a method of the invention. A primary
reason for providing a 5'-phosphate group on a promoter primer is
to permit ligation of linear first-strand cDNA following reverse
transcription or primer extension of a promoter primer on a target
sequence in order to obtain a circular promoter-containing
first-strand cDNA.
[0194] A number of examples of embodiments that use promoter
primers of the invention are described below. The invention
comprises all methods for using a sense promoter primer wherein the
transcription promoter, in double-stranded form, can be used by an
RNA polymerase that can synthesize RNA using a transcription
substrate and is not limited to only the example embodiments
presented.
[0195] With respect to methods that use a promoter primer, one
embodiment of the invention comprises a method for using a sense
promoter primer for making a circular transcription substrate for
making transcription product corresponding to a target sequence in
a target nucleic acid, the method comprising:
[0196] a. obtaining a sense promoter primer for synthesis of a
first-strand cDNA, the promoter primer comprising a sequence at its
3'-end that is complementary to the 3'-end of the target sequence
that is to be transcribed, and a 5'-end portion comprising a
sequence for a sense transcription promoter, and optionally, a
phosphate group or a topoisomerase moiety on its 5'-end;
[0197] b. annealing the promoter primer to the target nucleic
acid;
[0198] c. primer-extending or the promoter primer annealed to the
target nucleic acid with a DNA polymerase under DNA synthesis
conditions so as to obtain first-strand cDNA that is complementary
to the target sequence or sequences to which the promoter primer
was annealed;
[0199] d. optionally, removing the target nucleic acid that is
annealed to the first-strand cDNA;
[0200] e. ligating the first-strand cDNA, wherein the 5'-end of the
first-strand cDNA is covalently joined to the 3'-end of the
first-strand cDNA so as to obtain circular first-strand cDNA,
wherein the circular first-strand cDNA comprises circular sense
promoter-containing first-strand cDNA; and
[0201] f. annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate.
[0202] Another embodiment of the invention comprises a method for
obtaining a linear transcription substrate for making a
transcription product corresponding to a target sequence in a
target nucleic acid, the method comprising:
[0203] a. obtaining a circular sense promoter-containing
first-strand cDNA circular transcription substrate by carrying out
steps (a) through (e) of the method immediately above;
[0204] b. linearizing the circular sense promoter-containing
first-strand cDNA at a site 3'- of the transcription promoter and
5'- of the target-complementary sequence of the sense promoter
primer portion of said circular sense promoter-containing
first-strand cDNA, wherein a linear sense promoter-containing
first-strand is obtained; and
[0205] c. annealing an anti-sense promoter oligo to the linear
sense promoter-containing first-strand cDNA so as to obtain a
linear transcription substrate.
[0206] A general embodiment of the invention comprises a method for
making a transcription product corresponding to a target sequence
in a target nucleic acid, the method comprising:
[0207] a. obtaining a transcription substrate, chosen from among a
circular transcription substrate and a linear transcription
substrate;
[0208] b. contacting the transcription substrate under
transcription conditions with an RNA polymerase that recognizes the
promoter in the transcription substrate and makes a transcription
product therefrom; so as to obtain transcription product; and
[0209] c. obtaining the transcription product.
[0210] The target nucleic acid can be DNA or RNA. By way of
example, but not of limitation, a target sequence can comprise a
target nucleic acid comprising a single species of mRNA or a target
sequence can comprise a target nucleic acid, which can comprise all
of the mRNA in a sample.
[0211] Thus, one embodiment of the invention is a method for using
a sense promoter primer for making a transcription product
corresponding to a target sequence comprising a target nucleic acid
comprising mRNA, the method comprising:
[0212] a. obtaining a target nucleic acid comprising mRNA;
[0213] b. obtaining a sense promoter primer comprising a a
target-complementary portion at its 3'-end that is complementary to
the 3'-end of the target sequence, wherein the target complementary
sequence is chosen from among an oligo(dT) sequence, an anchored
oligo(dT)X sequence, a target-specific sequence, and a random
sequence, and the 5'-end optionally comprises a phosphate or
topoisomerase moiety;
[0214] c. annealing the promoter primer to the target nucleic
acid;
[0215] d. primer-extending the promoter primer annealed to the
target nucleic acid with a DNA polymerase under DNA synthesis
conditions so as to obtain first-strand cDNA that is complementary
to the target sequence or sequences to which the promoter primer
was annealed;
[0216] e. optionally, removing the RNA that is annealed to the
first-strand cDNA;
[0217] f. ligating the first-strand cDNA, wherein the 5'-end is
covalently joined to the 3'-end of the first-strand cDNA so as to
obtain circular sense promoter-containing first-strand cDNA;
[0218] g. annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate
[0219] h. contacting the circular transcription substrate with an
RNA polymerase under transcription conditions so as to obtain
transcription product; and
[0220] i. obtaining the transcription product.
[0221] In another embodiment, the circular sense-promoter
first-strand cDNA is linearized to obtain linear sense-promoter
first-strand cDNA, and then an anti-sense promoter primer is
annealed to the sense promoter sequence therein to obtain a linear
transcription substrate. Then, the linear transcription substrate
is contacted with an RNA polymerase under transcription conditions
so as to obtain transcription product, and transcription product is
obtained.
[0222] The methods for obtaining a transcription substrate and for
making a transcription product corresponding to a target sequence
can be performed in a stepwise manner, or, under suitable reaction
conditions, they can be performed continuously in a single reaction
mixture.
[0223] Thus, one embodiment of the invention comprises a method for
obtaining additional rounds of transcription of a target sequence
in a target nucleic acid (FIG. 3), the method comprising:
[0224] a. obtaining a transcription product;
[0225] b. obtaining a sense promoter primer comprising a
target-complementary portion at its 3'-end that is complementary to
the 3'-end of the target sequence and optionally, a phosphate group
or a topoisomerase moiety on its 5-end;
[0226] c. annealing the promoter primer to the transcription
product;
[0227] d. primer-extending the promoter primer annealed to the
transcription product with a DNA polymerase under DNA synthesis
conditions so as to obtain first-strand cDNA;
[0228] e. optionally, removing the RNA that is annealed to the
first-strand cDNA;
[0229] f. ligating the first-strand cDNA, wherein the 5'-end is
covalently joined to the 3'-end of the first-strand cDNA so as to
obtain circular sense promoter-containing first-strand cDNA;
[0230] g. annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate
[0231] h. contacting the circular transcription substrate with an
RNA polymerase under transcription conditions so as to obtain
additional transcription product; and
[0232] i. obtaining the additional transcription product.
[0233] In another embodiment, the circular sense-promoter
first-strand cDNA is linearized to obtain linear sense-promoter
first-strand cDNA, and then an anti-sense promoter primer is
annealed to the sense promoter sequence therein to obtain a linear
transcription substrate. Then, the linear transcription substrate
is contacted with an RNA polymerase under transcription conditions
so as to obtain transcription product, and transcription product is
obtained.
[0234] Different embodiments of methods of the invention can also
be used to make a transcription product corresponding to target
sequences that are internal to a target nucleic acid sequence. By
way of example, some embodiments can be used to make a
transcription product corresponding to sequences, such as, but not
limited to, wild-type or mutated sequence in genomic DNA. In these
embodiments, one or more processes, such as, but not limited to,
annealing a blocking oligo to the target nucleic acid, are required
to limit the 3'-end of the target sequence that is transcribed, as
is discussed elsewhere herein. In general, the target nucleic acid
must be single-stranded for use in a method of the invention. Thus,
a double-stranded nucleic acid must be denatured.
[0235] Thus, one embodiment of the invention for using a promoter
primer is a method for making a transcription product corresponding
to a target sequence that comprises only a portion of a target
nucleic acid comprising single-stranded DNA or RNA, the method
comprising:
[0236] a. obtaining a target nucleic acid comprising
single-stranded DNA or RNA;
[0237] b. obtaining a sense promoter primer comprising a
target-complementary portion at its 3'-end that is complementary to
the 3'-end of the target sequence and optionally, a phosphate group
or a topoisomerase moiety on its 5-end;
[0238] c. obtaining a blocking oligo, the blocking oligo comprising
a sequence that anneals tightly to a sequence on the target nucleic
acid so as to delimit the 3'-end of a primer extension product of
the promoter primer using the target nucleic acid as a
template,
[0239] wherein the blocking oligo is not displaced by the primer
extension product, and wherein the blocking oligo is not itself
capable of being primer extended by a DNA polymerase;
[0240] d. annealing the promoter primer and the blocking oligo to
the target nucleic acid;
[0241] d. primer-extending the promoter primer annealed to the
target nucleic acid with a DNA polymerase under DNA synthesis
conditions so as to obtain first-strand cDNA;
[0242] f. optionally, removing the target nucleic acid that is
annealed to the first-strand cDNA;
[0243] g. ligating the first-strand cDNA, wherein the 5'-end is
covalently joined to the 3'-end of the first-strand cDNA so as to
obtain circular sense promoter-containing first-strand cDNA;
[0244] h. optionally, linearizing the circular sense
promoter-containing first-strand cDNA at a site that is 3'- of the
promoter sequence and 5'- of the target-complementary portion of
the promoter primer in circular sense promoter-containing
first-strand cDNA so as to obtain linear sense promoter-containing
first-strand cDNA;
[0245] g. annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular transcription substrate; or annealing an anti-sense
promoter oligo to the linear sense promoter-containing first-strand
cDNA so as to obtain a linear transcription substrate;
[0246] h. contacting the circular transcription substrate or the
linear transcription substrate with an RNA polymerase under
transcription conditions so as to obtain transcription product;
and
[0247] i. obtaining the transcription product.
[0248] k. optionally, repeating steps b through k to obtain
additional rounds of transcription to obtain transcription
products.
[0249] In still other embodiments of the invention, the circular
first-strand cDNA comprising an anti-sense promoter is used as a
template for DNA synthesis using a strand-displacing DNA polymerase
and at least one strand displacement primer, and in some
embodiments, multiple strand displacement primers. A "strand
displacement primer," as used herein, is an oligonucleotide or
polynucleotide that can be primer extended by a strand-displacing
DNA polymerase of the invention, wherein strand displacement DNA
synthesis occurs. In general, strand displacement is more a
property of the DNA polymerase and the reaction conditions used
than of the primer. Thus, the composition and properties of a
strand displacement primer can vary greatly. For example, in some
embodiments of the invention, a strand displacement primer can be
an oligonucleotide that is hybridizable to a circular DNA template,
wherein the DNA synthesis product resulting from rolling circle
replication by a strand displacing DNA polymerase results in
displacement of the strand displacement primer extension product,
resulting in tandem complementary ssDNA copies of circular
template. In other embodiments, it is preferred that the strand
displacement primer has a 3'-portion that is complementary to a
sequence in a circular DNA template and a 5'-portion that is
non-complementary, and therefore has a "flap;" the protruding flap
appears to facilitate displacement of the primer in some cases.
When strand displacement is carried out on linear DNA templates,
strand displacement primers can be designed to have particular
nucleotide compositions and/or structures, and additional methods
and reaction components can be used in order to facilitate strand
displacement by liberating the strand displacement primer from the
template.
[0250] A strand-displacing DNA polymerase of the invention can be
any DNA polymerase that results in strand displacement. Preferred
strand-displacing DNA polymerases of the present invention lack
5'-exonuclease activity, including structure-dependent 5'-nuclease
activity. Preferred strand-displacing DNA polymerases comprise rBst
DNA polymerase large fragment, also called "IsoTherm.TM. DNA
Polymerase" (Epicentre Technologies, Madison, Wis., USA), Bca DNA
Polymerase (TAKARA Shuzo Company, Kyoto, Japan), RepliPHI.TM. DNA
Polymerase (Epicentre Technologies, Madison, Wis., USA), .PHI.29
DNA polymerase (U.S. Pat. Nos. 5,198,543 and 5,001,050,
incorporated herein by reference), SequiTherm.TM. DNA Polymerase
(Epicentre Technologies, Madison, Wis., USA), MMLV reverse
transcriptase, and Sequenase.RTM. DNA Polymerase (USB, Cleveland,
Ohio, USA). In these embodiments, a strand displacement primer is
used to prime second-strand DNA synthesis using circular
first-strand cDNA as a template. Once DNA synthesis has proceeded
completely around the circular first-strand cDNA template, the
second-strand cDNA is displaced so that the displaced
single-stranded second DNA strand is released into the reaction
medium. Since a transcription promoter is present at least once in
every round of DNA synthesis of the circular first-strand cDNA
template, this released single-stranded "sense-promoter-containing
second-strand cDNA" can be used to obtain a linear transcription
substrate after annealing of an anti-sense promoter oligo. As the
second-strand cDNA continues to grow, longer and longer concatemers
of the anti-sense RNA are formed. The linear transcription
substrate obtained can be used by an RNA polymerase of the
invention under transcription conditions to make an anti-sense
transcription product with respect to the target nucleic acid
sequence. Thus, this differs from previous embodiments that used
sense promoter primers to make sense transcription products using
first-strand cDNA as a template. Anti-sense RNA can be used for
many applications, such as, but not limited to, for use as probes
for nucleic acid arrays or microarrays. As the second-strand cDNA
continues to grow, longer and longer concatemers of the anti-sense
RNA are formed.
[0251] As discussed above, some embodiments obtain a transcription
template for making a transcription product corresponding to a
target sequence in a target nucleic acid by first obtaining a
circular first-strand cDNA comprising an anti-sense transcription
promoter, wherein the circular first-strand cDNA is then used as a
template for strand displacement DNA synthesis of the linear sense
promoter-containing second-strand cDNA by a strand-displacing DNA
polymerase. A linear transcription substrate is obtained by
annealing an anti-sense promoter oligo to the linear sense
promoter-containing second-strand cDNA.
[0252] Thus, one embodiment of the invention comprises a method for
making a transcription product corresponding to a target sequence
in a target nucleic acid (FIG. 6), the method comprising:
[0253] a. obtaining a target nucleic acid comprising
single-stranded DNA or RNA;
[0254] b. obtaining an anti-sense promoter primer comprising a
3'-end portion that is complementary to a target sequence and
optionally, a phosphate group or a topoisomerase moiety on its
5-end;
[0255] c. optionally, obtaining a blocking oligo, the blocking
oligo comprising a sequence that anneals tightly to a sequence on
the target nucleic acid so as to delimit the 3'-end of a primer
extension product of the promoter primer using the target nucleic
acid as a template, wherein the blocking oligo is not displaced by
the primer extension product, and wherein the blocking oligo is not
itself capable of being primer extended by a DNA polymerase;
[0256] d. annealing the promoter primer and, optionally, the
blocking oligo to the target nucleic acid;
[0257] e. primer-extending or the promoter primer annealed to the
target nucleic acid with a DNA polymerase under DNA synthesis
conditions so as to obtain first-strand cDNA;
[0258] f. optionally, removing the target nucleic acid that is
annealed to the first-strand cDNA;
[0259] g. ligating the linear first-strand cDNA, wherein the 5'-end
is covalently attached to the 3'-end of the first-strand cDNA so as
to obtain circular first-strand cDNA;
[0260] h. obtaining a strand-displacement primer;
[0261] i. annealing the strand displacement primer to the circular
first-strand cDNA;
[0262] j. obtaining a strand-displacing DNA polymerase;
[0263] k. contacting the circular first-strand cDNA to which the
strand displacement primer is annealed with a strand-displacing DNA
polymerase under DNA synthesis conditions so as to obtain linear
sense promoter-containing second-strand cDNA;
[0264] l. obtaining the linear sense promoter-containing
second-strand cDNA;
[0265] m. annealing an anti-sense promoter oligo to the linear
sense promoter-containing second-strand cDNA and obtaining a linear
second-strand transcription substrate;
[0266] n. obtaining an RNA polymerase that transcribes the linear
second-strand transcription substrate using the promoter under
transcription conditions;
[0267] o. contacting the linear second-strand transcription
template with the RNA polymerase under transcription conditions so
as to obtain anti-sense transcription products; and
[0268] p. obtaining the anti-sense transcription products; and
[0269] q. optionally, repeating steps b through q to obtain
additional rounds of transcription of anti-sense transcription
products.
[0270] b. Composition of Promoter Primers and Anti-Sense Promoter
Oligos of the Invention
[0271] In preferred embodiments of the invention, a promoter primer
comprises DNA nucleotides. A promoter primer can also comprise one
or more modified nucleotides for a particular purpose. By way of
example, but not of limitation, a promoter primer can comprise one
or more dUMP nucleotides 3'- of a sense transcription promoter and
5'- of the sequence that is complementary to a target nucleic acid
sequence, which provides a site for linearizing a sense
promoter-containing first-strand cDNA (prior to annealing an
anti-sense promoter oligo to obtain a circular transcription
substrate), or in some cases, for linearizing a circular
transcription substrate, using UNG and endo IV as discussed
elsewhere herein. However, the invention is not limited to promoter
primers comprising DNA nucleotides or modified DNA nucleotides, and
in some cases, a promoter primer can comprise RNA or modified RNA
nucleotides or both DNA and RNA nucleotides or modified
nucleotides.
[0272] The nucleic acid target-complementary portion of a promoter
primer can be complementary to a specific known sequence in the RNA
target in a sample, or it can comprise a mixture of all possible or
many possible sequences, such as, but not limited to, random
hexamer sequences. Random primer sequences can be made by including
nucleotide reagents which are complementary to all four canonical
bases during the chemical synthesis of each nucleotide position of
the mRNA-complementary portion of the promoter primer. In
embodiments of the invention using samples containing mRNA target
nucleic acids, which are preferred embodiments, the 3'-end of a
promoter primer comprises either a specific sequence that is
complementary to a known sequence of a specific mRNA or, if the
mRNA has a poly(A) tail at its 3'-end, the 3'-end of the promoter
primer can comprise an oligo(dT) sequence. In still other
embodiments of the invention for mRNA target nucleic acids, the
3'-end of a promoter primer can comprise a random sequence, such
as, but not limited to a random hexamer sequence.
[0273] A promoter primer of the invention comprises a transcription
promoter in its 5'-portion. In most embodiments, the transcription
promoter comprises a sense promoter sequence that is capable of
binding an RNA polymerase of the invention. However, those with
knowledge in the art will understand that, due to the fact that the
transcription promoter is 5'- of the linear first-strand cDNA that
is synthesized by reverse transcription of the RNA target nucleic
acid using the promoter primer, an RNA polymerase of the invention
cannot use the transcription promoter to synthesize RNA
complementary to first-strand cDNA that is complementary to the RNA
target nucleic acid. However, if the 5'-end of the linear
first-strand cDNA is ligated to its 3'-end so as to form circular
first-strand cDNA, then an RNA polymerase of the invention can use
the transcription promoter to synthesize RNA complementary to
first-strand cDNA that is complementary to the RNA target nucleic
acid; thus, a circular first-strand cDNA of this embodiment
comprises a transcription substrate of the invention. Thus, a
preferred promoter primer of these embodiments of the invention is
phosphorylated at its 5'-end in order to facilitate ligation of
linear first-strand cDNA by a ligase of the invention under
ligation conditions. By means of example, but not of limitation,
the 5'-end of the promoter primer can be phosphorylated using T4
polynucleotide kinase and ATP under suitable reaction conditions
known in the art. In other embodiments, the promoter primer has a
type 1 topoisomerase moiety at its 5'-end in order to facilitate
topoisomerase-mediated ligation of linear first-strand cDNA under
ligation conditions (e.g., U.S. Pat. No. 5,766,891, incorporated
herein by reference).
[0274] In general, an anti-sense promoter oligo comprises
deoxyribonucleotides. Modified nucleotides or modified linkages
should be used in an anti-sense promoter oligo only after carefully
determining that they do not substantially affect the ability of
the anti-sense promoter oligo to complex with a sense promoter
sequence or to bind the RNA polymerase or to affect the ability of
the RNA polymerase to initiate transcription using the template
strand. However, modified nucleotides can be used for a particular
purpose. Similarly, modified linkages, such as, but not limited to
alpha-thiophosphate sugar linkages that are resistant to certain
nucleases can be used for a particular purpose. An anti-sense
promoter oligo can be of any length so long as it has sufficient
length to comprise an anti-sense promoter sequence that, when
annealed to a sense promoter, makes a functional double-stranded
promoter that can be used by an RNA polymerase under transcription
conditions to make a transcription product. The oligo comprising
the anti-sense promoter can comprise additional nucleotides that
are 3'- of or 5'- of the anti-sense promoter sequence so long as
the additional nucleotides do not bind the intended target sequence
or another component of a method of the invention in a manner that
is independent of complexing of the anti-sense promoter sequence
with the sense promoter sequence of a sense promoter primer, or
otherwise negatively affect the results of the method. If modified
nucleotides are used in anti-sense promoter oligo, for a purpose,
such as, but not limited to for attaching a labeling moiety, it is
preferred that the modified nucleotide is in a nucleotide that does
not comprise the anti-sense promoter sequence if possible. If an
anti-sense promoter oligo is present in a reaction when steps such
as primer extension with a DNA polymerase or ligation with a ligase
are performed, the anti-sense promoter oligo is designed so that it
cannot participate in these reactions. This is accomplished, for
example, by synthesizing an anti-sense promoter oligo that has a
dideoxynucleotide or another termination nucleotide on its 3'-end
so that it can't be primer-extended and that does not have a
phosphate group on its 5'-end (which could participate in a
ligation reaction).
[0275] D. Ligases and Ligation Methods for Circularizing Linear
ssDNA
[0276] It will be clear from the above descriptions of methods for
obtaining transcription substrates that it is useful in various
embodiments to ligate linear ssDNA to obtain circular ssDNA. The
invention is not limited to a specific ligase for circularizing a
linear ssDNA molecule and different ligases and ligation methods
can be used in different embodiments in order to accomplish a
particular purpose. In embodiments that use a ligase, the 5'-end of
the linear ssDNA that is ligated to obtain a circular ssDNA must
have a 5'-phosphate group or the 5'-end must be phosphorylated
using a polynucleotide kinase, such as, but not limited to T4
polynucleotide kinase, during the processes of the method of the
invention.
[0277] A ligase that catalyzes non-homologous intramolecular
ligation, such as, but not limited to ThermoPhage.TM. RNA Ligase II
(Prokaria, Ltd., Reykjavik, Iceland), is a suitable ligase for
ligating linear ssDNA to form a circular ssDNA using the reaction
conditions of the manufacturer.
[0278] NAD-dependent DNA ligases that are not active on blunt ends,
such as, but not limited to Ampligase.RTM. Thermostable DNA Ligase
(Epicentre Technologies, Madison, Wis., USA), Tth DNA ligase, Tfl
DNA ligase, and Tsc DNA Ligase (Prokaria Ltd., Reykjavik, Iceland)
can be used to ligate the 5'-phosphate and 3'-hydroxyl termini of
DNA ends that are adjacent to one another when annealed to a
complementary DNA molecule, and are suitable ligases in embodiments
of the invention that use a ligation splint oligo comprising DNA.
However, the invention is not limited to the use of a particular
ligase and any suitable ligase can be used. For example, T4 DNA
ligase can be used in embodiments of the invention that use a
ligation splint. Still further, Faruqui discloses in U.S. Pat. No.
6,368,801 that T4 RNA ligase can efficiently ligate DNA ends of
nucleic acids that are adjacent to each other when hybridized to an
RNA strand. Thus, T4 RNA ligase is a suitable ligase of the
invention in embodiments in which DNA ends are ligated on a
ligation splint oligo comprising RNA or modified RNA, such as, but
not limited to modified RNA that contains 2'-F-dCTP and 2'-F-dUTP
made using the DuraScribe.TM. T7 Transcription Kit (Epicentre
Technologies, Madison, Wis., USA).
[0279] The invention is also not limited to the use of a ligase for
covalently joining the 5'-end to the 3'-end of the same or
different nucleic acid molecules in the various embodiments of the
invention. By way of example, other ligation methods such as, but
not limited to, topoisomerase-mediated ligation (e.g., U.S. Pat.
No. 5,766,891, incorporated herein by reference) can be used.
[0280] E. Modes of Performance of the Methods of the Invention
[0281] Depending on the application and its requirements and
constraints, the methods of the invention can be performed in a
stepwise fashion, with one set of reactions being performed,
followed by purification of a reaction product or removal of
reagents or inactivation of enzymes or addition of reagents before
proceeding to the next set of reactions, or, in other embodiments
for other applications, the methods can be performed as a
continuous set of multiple reactions in a single reaction mixture.
By way of example, but not of limitation, in some embodiments, each
of the separate reactions for transcription using
promoter-containing first-strand cDNA as a template for
transcription can be performed separately. Still by way of example,
in some embodiments in which the methods of the invention are used
as part of a diagnostic assay, all of the reactions can be carried
out in a single reaction mixture and the products of the
transcription reaction may be detected, without ever being
isolated.
[0282] The invention also comprises parts or subsets of the methods
and compositions of the invention. Thus, the invention comprises
all of the individual steps of the methods of the invention that
are enabled thereby, in addition to the overall methods.
[0283] F. Examples of the Scope of Applications of the
Invention
[0284] Those with knowledge in the art will understand that the
present invention is novel and very broad in scope and provides
improvements in methods, processes, compositions and kits related
to making transcription products corresponding to a target nucleic
acid sequence comprising RNA, including mRNA, or DNA in a
biological sample for many applications. These methods are useful
for applications such as, but not limited to, making full-length
cDNA and cDNA libraries, improving gene expression analysis, and
detecting a target sequence. By way of further example, but not of
limitation, the present invention comprises improved methods,
processes, compositions and kits that improve upon methods and
applications to: [0285] 1. amplify nucleic acid molecules in vitro
[0286] 2. amplify a DNA sequence in vitro [0287] 3. amplify a
genomic DNA sequence in vitro [0288] 4. amplify an RNA sequence in
vitro [0289] 5. amplify mRNA [0290] 6. amplify rRNA [0291] 7.
synthesize RNA [0292] 8. synthesize modified RNA, such as, but not
limited to RNA containing 2'-fluoro-nucleotides [0293] 9.
synthesize DNA [0294] 10. detect the presence of a nucleic acid
sequence in a sample [0295] 11. detect the presence of a target
nucleic acid sequence in a sample that is indicative of the
presence of a target organism [0296] 12. detect the presence of a
target nucleic acid analyte in a sample [0297] 13. detect the
presence of a DNA sequence in a sample [0298] 14. detect the
presence of an RNA sequence in a sample [0299] 15. detect the
presence (or absence at a detectable level) of a gene in a sample
[0300] 16. detect the presence of a target organism in a sample
[0301] 17. detect the presence of a virus in a sample [0302] 18.
detect the presence of a bacterium in a sample [0303] 19. detect
the presence of a pathogenic organism in a sample [0304] 20. detect
the presence of a beneficial organism in a sample [0305] 21.
identify and quantify nucleic acids associated with RNA and DNA
binding proteins [0306] 22. detect an oncogene [0307] 23. detect an
anti-oncogene [0308] 24. quantify the level of a virus, a
microorganism, a gene, an mRNA, an rRNA, a nucleic acid analyte, or
any other nucleic acid of whatever type for whatever purpose.
[0309] 25. use as a probe [0310] 26. use as a probe for an array or
microarray [0311] 27. make dsRNA or modified dsRNA that can be
introduced into human, animal or other eukaryotic cells in serum
and without use of a transfection agent. [0312] 28. synthesize
dsRNA or modified dsRNA in vitro for use as RNAi [0313] 29.
synthesize dsRNA or modified dsRNA in vitro for use siRNA [0314]
30. make RNAi or modified RNAi that silences a gene encoded by a
virus or other infectious agent. [0315] 31. make siRNAi or modified
siRNA that silences a gene encoded by a virus or other infectious
agent. [0316] 32. make RNAi or modified RNAi that silences a gene
encoded by a plant, animal, human, fungal or other eukaryotic host
gene that is involved with and/or interacts with a biological
molecule encoded by a virus or other infectious agent. [0317] 33.
make siRNAi or modified siRNA that silences a gene encoded by a
plant, animal, human, fungal or other eukaryotic host gene that is
involved with and/or interacts with a biological molecule encoded
by a virus or other infectious agent. [0318] 34. make RNAi or
modified RNAi that silences a non-essential disease-susceptibility
gene encoded by a plant, animal, human, fungal or other eukaryotic
host gene. [0319] 35. make siRNAi or modified siRNA that silences a
non-essential disease-susceptibility gene encoded by a plant,
animal, human, fungal or other eukaryotic host gene. [0320] 36.
make RNAi or modified RNAi that silences a non-essential gene
encoded by a plant, animal, human, fungal or other eukaryotic host
gene, wherein the gene silencing results in a beneficial effect.
[0321] 37. make siRNAi or modified siRNA that silences a
non-essential gene encoded by a plant, animal, human, fungal or
other eukaryotic host gene, wherein the gene silencing results in a
beneficial effect. [0322] 38. make RNAi or modified RNAi that
silences a non-essential gene encoded by a plant, animal, fungal or
other eukaryotic host gene, wherein the gene silencing results in
improved yield, production of a biological molecule, flavor, or
other commercially beneficial effect, such as, but not limited to,
cold-hardiness, salt-tolerance, shortened growing season, or
increased efficiency of utilization of a nutrient. [0323] 39. make
siRNAi or modified siRNA that silences a non-essential gene encoded
by a plant, animal, fungal or other eukaryotic host gene, wherein
the gene silencing results in improved yield, production of a
biological molecule, flavor, or other commercially beneficial
effect, such as, but not limited to, cold-hardiness,
salt-tolerance, shortened growing season, or increased efficiency
of utilization of a nutrient. [0324] 40. diagnose the presence
and/or level of an infectious organism [0325] 41. diagnose a
disease [0326] 42. detect the presence of a nucleic acid in an
environmental sample [0327] 43. differentiate, both qualitatively
and quantitatively, between which mRNA molecules are present in
different types of cells or in the same type of cells under
different conditions or in the same or different types of cells
under the same or different conditions or in response to specific
stimuli or treatments [0328] 44. analyze, both qualitatively and
quantitatively, gene expression profiles in cells under different
defined conditions [0329] 45. analyze, both qualitatively and
quantitatively, gene expression profiles in different types of
cells under the same defined environmental conditions [0330] 46.
analyze, both qualitatively and quantitatively, gene expression
profiles in cells over time [0331] 47. analyze, both qualitatively
and quantitatively, gene expression in response to specific stimuli
[0332] 48. make a library or libraries of mRNA molecules and/or
cDNA molecules that are present in one type of cell that are not
present in another type of cell, or that are present in one type of
cell under certain conditions but not under other conditions (i.e.,
a subtraction library). [0333] 49. map and clone sequences
corresponding to the 5'-ends of mRNA's, including, but not limited
to, those generated from a specific gene by alternative splicing
and promoter usage [0334] 50. generate improved templates for more
accurate rapid amplification of cDNA ends ("RACE") techniques
(e.g., see Flouriot et al., Nucleic Acids Res. 27:e8 (1-iv), 1999).
[0335] 51. make and/or amplify mRNA for in vitro or in vivo
translation, including, but not limited to coupled or step-wise
transcription and translation. [0336] 52. amplify RNA and/or DNA or
modified RNA and/or DNA present in living cells, such as, but not
limited to, tumor or cancer cells from a patient for introduction
into dendritic cells (e.g., see U.S. Pat. Nos. 5,994,126 and
6,475,483 of Steinman et al., incorporated herein by reference) or
other cells from the patient in order to boost or increase an in
vivo response, such as, but not limited to, an immune response in
the patient, with the goal of decreasing the size or the number of
cells in the tumor or cancer in the patient. [0337] 53. make and/or
amplify RNA and/or DNA or modified RNA and/or DNA from a patient
and or present in a virus or other infectious agent, wherein the
RNA and/or DNA is for use as an RNA vaccine and/or a DNA vaccine,
respectively. [0338] 54. make RNA or modified RNA that can be
introduced into human, animal or other eukaryotic cells in serum
and without use of a transfection agent. [0339] 55. make modified
RNA containing 2'-fluoro-2' deoxynucleotides, such as, but not
limited to, 2'-F-dCMP and 2'-F-dUTP, in place of the corresponding
canonical nucleotides. [0340] 56. produce arrays or microarrays of
amplified nucleic acids by attaching the amplification products
onto a solid substrate. [0341] 57. detect a mutation or a mutated
form of a target nucleic acid sequence in a sample [0342] 58.
quantify the amount of a target nucleic acid or target nucleic
acids in a sample.
[0343] Modified RNA molecules that contain 2'-F-dCMP and 2'-F-dUTP
are resistant to RNase A-type ribonucleases (Sousa et al., U.S.
Pat. No. 5,849,546), included herein by reference. Capodici et al.,
(J. Immunology 169:5196-5201, 2002), included herein by reference,
showed that 2'-fluoro-containing dsRNA molecules made using the
DuraScribe.TM. T7 Transcription Kit (Epicentre Technologies,
Madison, Wis., USA) did not require transfection reagents for
delivery into cells, even in the presence of serum. Kakiuchi et al.
(J. Biol. Chem. 257:1924-1928, 1982), included herein by reference,
showed that use of [(2'-F-dI).sub.n: (2'-F-dC).sub.n. duplexes were
40-100 times less antigenic than [(rI).sub.sub.n.: (rC.sub.n.]
duplexes, and did not induce an interferon response like
[(rI).sub.n: (rC).sub.n.] duplexes.
I. Kits and Compositions for Embodiments of the Invention for
Making a Transcription Product Using a Transcription Substrate
Comprising a Target Nucleic Acid Sequence in a Sample
[0344] Important compositions of the invention are Sense Promoter
Primers. A Sense Promoter Primer can be provided for primer
extension of one specific target sequence or a Sense Promoter
Primer can be provided for amplifying a multiplicity of target
sequences, such as, but not limited to target sequences comprising
all mRNA targets in a sample. In the latter case, a Sense Promoter
Primer can be provided that comprises an oligo(dT), sequence, or an
anchored oligo(dT).sub.nX sequence, or a randomized sequence, such
as a random hexamer or random octamer sequence. Still further,
multiple specific Sense Promoter Primers can be provided in order
to permit amplification of multiple different target sequences in
the same sample. Also, multiple different Sense Promoter Primers
can be provided which encode different sense promoter sequences
that are recognized by different RNA polymerase, such as, but not
limited to Sense Promoter Primers that encode sense promoters for
T7, T3 and SP6 RNAPs.
[0345] Another composition of the invention can be an Anti-Sense
Promoter Oligo that is annealed to a complementary sense promoter
in order to obtain a circular or linear transcription substrate
having a functional double-stranded promoter.
[0346] Still another composition of an anti-sense promoter oligo of
the invention can be an oligonucleotide comprising an anti-sense
promoter that is immobilized or attached to a solid support.
Preferably, the anti-sense promoter oligo comprising the anti-sense
promoter is immobilized on the solid support at or near its 5'-end
and the anti-sense promoter sequence is at a sufficient distance
from the surface of the solid support so that the sense promoter in
a circular sense promoter-containing first-strand cDNA or a linear
sense promoter-containing first-strand cDNA can anneal to the
anti-sense sequence so as to make a functional immobilized circular
or linear transcription substrate, respectively, when the support
is incubated with an RNA polymerase that uses the double-stranded
promoter to make a transcription product in a reaction medium under
suitable transcription conditions. Preferably, the solid support
has a chemical composition and structure so that it does not
non-specifically bind nucleic acid from a sample or that comprises
a composition of the invention, such as, but not limited to a sense
promoter primer. Preferably, the solid support has a chemical
composition and structure so that it does not non-specifically bind
enzymes, co-factors or other substances in reactions comprising
methods of the invention. Without limiting the invention, solid
supports can comprise dipsticks, membranes, such as nitrocellulose
or nylon membranes, beads, chips or slides used for making arrays
or microarrays, and the like. Some solid supports and methods for
immobilizing or attaching an anti-sense promoter oligo on a surface
or solid support, which can be used for the present invention, are
disclosed by Marble et al. in U.S. Pat. No. 5,700,667 and in
references therein, all of which methods are incorporated herein by
reference. Other solid supports which can be used for the present
invention are also known in the art and can be used. Numerous other
methods for attaching a molecule comprising an oligonucleotide to a
surface or other substance are known in the art, and any known
method for attaching or immobilizing a molecule comprising an
anti-sense promoter oligo can be used to make a composition
comprising an immobilized anti-sense promoter oligo is included in
the present invention. The composition comprising an
oligonucleotide comprising an anti-sense promoter that is
immobilized on a solid support can also be used to make functional
transcription substrates for making anti-sense transcription
products by annealing linear sense promoter-containing
second-strand cDNA obtained by rolling circle transcription of a
circular anti-sense promoter-containing first-strand cDNA.
[0347] A kit of the invention can comprise one or more Sense
Promoter Primers, an Anti-Sense Promoter Oligo and instructions for
their use in a method of the invention. The Anti-Sense Promoter
Oligo can be in solution, or it can comprise an oligonucleotide
that is immobilized on a solid support, as discussed above.
[0348] Another kit of the invention can comprise one or more
Anti-Sense Promoter Primers and an Anti-Sense Promoter Oligo, along
with instructions for their use in a method of the invention, such
as a method for making transcription substrates to obtain
anti-sense transcription products. Similarly, the Anti-Sense
Promoter Oligo in the kit can be in solution, or it can comprise an
oligonucleotide that is immobilized on a solid support, as
discussed above.
[0349] Still another kit of the invention can comprise one or more
Sense Promoter Primers or one or more Anti-Sense Promoter Primers,
an Anti-Sense Promoter Oligo, whether in solution or immobilized on
a solid support, and an optimized composition of a ligase enzyme
for circularizing linear promoter-containing first-strand cDNA, as
well as instructions for use.
[0350] Yet another kit of the invention can comprise one or more
Sense Promoter Primers, an Anti-Sense Promoter Oligo, whether in
solution or immobilized on a solid support, an optimized
composition of a ligase enzyme for circularizing linear
promoter-containing first-strand cDNA, and an optimized
composition, such as but not limited to a uracil-N-glycosylase
enzyme, for linearizing a circular sense promoter-containing
first-strand cDNA having a dUMP residue that was introduced using a
Sense Promoter Primer having the dUMP residue, as well as
appropriate instructions for use.
[0351] Still another kit of the invention can comprise one or more
Sense Promoter Primers or one or more Anti-Sense Promoter Primers,
an Anti-Sense Promoter Oligo, whether in solution or immobilized on
a solid support, an optimized composition of a ligase enzyme for
circularizing linear promoter-containing first-strand cDNA,
optionally, an optimized composition for linearizing a circular
sense promoter-containing first-strand cDNA, and an optimized
composition of the RNA polymerase that uses the double-stranded
promoter in the transcription substrates obtained to make a
transcription products, as well as instructions for use.
[0352] Another kit can comprise all compositions needed, as
individual compositions or as a single optimized combined
composition or as a small number of compositions to perform a
continuous transcription amplification reaction of the invention,
such as but not limited to a reaction as shown in the schematic in
FIG. 3.
[0353] Other kits can comprise the compositions above, individually
or in combination and/or other compositions, such as, but not
limited to:
[0354] (a) a suitable DNA polymerase or reverse transcriptase for
making a promoter-containing first-strand cDNA using a Sense or
Anti-Sense Promoter Primer;
[0355] (b) a suitable strand-displacing DNA polymerase, such as,
but not limited to IsoTherm.TM. DNA Polymerase (EPICENTRE
Technologies, Madison, Wis.), for rolling circle replication of a
circular anti-sense promoter-containing first-strand cDNA;
[0356] Enzymes can be provided in a kit separately or combined into
a single ready-to-use solution containing the optimal ratio of each
enzyme. A kit comprising enzymes that are used in a method that
uses a Promoter Primer can be provided with the Promoter Primer or
without a Promoter Primer for customers who wish to prepare their
own Promoter Primers for a specific target sequence.
[0357] A kit can be for a specific method, such as, but not limited
to, a kit for making a transcription product corresponding to mRNA
from a particular type of cell or different cells, whether under
the same conditions or different environmental conditions, for gene
expression profiling, including generation of sense or anti-sense
probes for microarrays, a kit for amplification of an RNA or DNA
sequence, including a kit for performing multiple rounds of
transcription of a target nucleic acid sequence in a single
reaction mixture, a kit for making a transcription product
corresponding to a particular target nucleic acid sequence, if
present in a sample, that is diagnostic or indicative of a
pathogen, a disease gene, a mutated allele, or the like, a kit for
an analyte-specific assay, wherein the analyte is a nucleic acid, a
kit for making RNAi, including siRNA, or modified RNAi or siRNA,
including, but not limited to, 2'-F-dCMP- and 2'-F-dUMP-containing
RNAi or siRNA using a DuraScribe.TM. T7 Transcription Kit
(EPICENTRE Technologies, Madison, Wis.), or any of a broad range of
kits that will be understood by those with knowledge in the art by
a reading of the description of the invention herein.
[0358] In general, a kit of the invention will also comprise a
description of the components of the kit and instructions for their
use in a particular process or method or methods of the invention.
In general, a kit of the present invention will also comprise other
components, such as, but not limited to, buffers, ribonucleotides
and/or deoxynucleotides, including modified nucleotides in some
embodiments, DNA polymerization or reverse transcriptase enhancers,
such as, but not limited to betaine (trimethylglycine), and salts
of monvalent or divalent cations, such as but not limited to
potassium acetate or chloride and/or magnesium chloride, enzyme
substrates and/or cofactors, such as, but not limited to, ATP or
NAD, and the like which are needed for optimal conditions of one or
more reactions or processes of a method or a combination of methods
for a particular application. A kit of the invention can comprise a
a set of individual reagents for a particular process or a series
of sets of individual reagents for multiple processes of a method
that are performed in a stepwise or serial manner, or a kit can
comprise a multiple reagents combined into a single reaction
mixture or a small number of mixtures of multiple reagents, each of
which perform multiple reactions and/or processes in a single tube.
In general, the various components of a kit for performing a
particular process of a method of the invention or a complete
method of the invention will be optimized so that they have
appropriate amounts of reagents and conditions to work together in
the process and/or method.
[0359] A kit of the invention can also comprise additional
components, such as reaction buffers, control substrates, size
markers, detection compositions or detection reagents, and the
like, all of which can be provided in quantities to match the need
for each component for the proscribed number of intended reactions,
but a kit need not contain these components. Still further, a kit
can optionally contain detailed instructions and troubleshooting
guides.
[0360] Components of a kit may be provided as solutions or as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent, in which case, the solvent may also be provided
in another container.
[0361] As used herein in the specification, "a" or "an" may mean
one or more. As used herein in the claim(s), when used in
conjunction with the word "comprising," the words "a" or "an" may
mean one or more than one. As used herein "another" may mean at
least a second or more.
IX. Signaling System of the Invention
[0362] With respect to this aspect of the invention, a "signaling
system" means and comprises the multitude of substances,
compositions and environmental conditions comprising a method for
detecting the presence or quantity of an analyte in a sample by
detecting a transcript that results from transcription of a
oligonucleotide that is operably complexed with an analyte-binding
substance.
[0363] Other methods for detecting an analyte using transcription
are known in the art, including the methods described by Zhang et
al. (Proc. Natl. Acad. Sci. USA 98: 5497-5502, 2001), by Eberwine
in PCT Patent Application No. WO 02/14476, and by Hudson et al. in
U.S. Pat. No. 6,100,024.
[0364] However, the present applicants believe that the methods,
compositions and kits presented herein are easier to use and make,
and can be more easily used for detecting a wide variety of
analytes than those which have been described previously by
others.
A. Signal Probes of the Invention
[0365] FIG. 7 and FIG. 8 show two embodiments of methods of the
invention that use Signal Probes as part of a signaling system of
the invention. As defined herein, a Signal Probe comprises a sense
promoter for an RNA polymerase that recognizes a double-stranded
promoter for transcription under transcription conditions, and a
single-stranded template that is joined 5'- of the sense promoter.
If the Signal Probe is complexed or annealed with an anti-sense
promoter sequence that is complementary to the sense promoter
sequence, a functional promoter is obtained for a cognate RNA
polymerase that permits transcription of the template under
transcription conditions. The RNA polymerase can be any RNA
polymerase that requires a double-stranded promoter, so long as
promoter sequences that comprise a double-stranded promoter are
used. That is, a single-stranded pseudopromoter or synthetic
promoter that is recognized by the RNA polymerase is not suitable
for aspects of the invention that used Signal Probe. A suitable RNA
polymerase is a T7-type RNA polymerase, preferably, an RNA
polymerase chosen from among T7 RNAP, T3 RNAP and SP6 RNAP, and the
promoter is a cognate double-stranded promoter that is recognized
by the respective RNAP. A "cognate promoter" for a particular RNA
polymerase is a promoter that is recognized by that particular RNA
polymerase with specifity. Similarly, a "cognate RNA polymerase"
for a particular promoter is one that recognizes the particular
promoter with substantially greater specifity than another RNA
polymerase that recognizes one or more other promoter sequences,
thus permitting transcription of the template that is joined with
the promoter with specificity even in the presence of other
sequences that are not recognized as a promoter by the particular
RNA polymerase. The template thus encodes a "signal sequence" and
can, but need not, encode one or more other sequences for a
particular purpose. By way of example but not of limitation, the
template can also encode one or more transcription termination
sequences.
[0366] The invention comprises two kinds of Signal Probes. An RCT
Signal Probe, which is used in FIG. 7, is defined herein as a
circular Signal Probe wherein the template strand is joined to both
the 3'-end and the 5'-end of a sequence that comprises the sense
promoter sequence. An RCT Signal Probe can be transcribed by
rolling circle transcription (RCT), although, as mentioned above,
the template of an RCT Signal Probe can also encode transcription
termination sequences. The second kind of Signal Probe of the
invention, a LINT Signal Probe, is used in FIG. 8 and is so named
to refer to "LINear Transcription." A LINT Signal Probe is a linear
Signal Probe wherein the sense promoter sequence is 3'- of the
template. Although the template of a LINT Signal Probe can also
encode one or more other sequences in addition to the signal
sequence, unless it is desired to terminate transcription from one
sequence to another, a transcription terminator sequence is not
needed because "run-off" transcription occurs on the linear
template. In general, both RCT and LINT Signal Probes comprise
deoxyribonucleotides although other nucleotides, including modified
nucleotides that do not affect transcription of the signal sequence
can be used for a particular purpose. Similarly, modified linkages,
such as, but not limited to alpha-thiophosphate sugar linkages that
are resistant to certain nucleases can be used for a particular
purpose.
B. Analyte-Binding Substances (ABS) Joined to an Oligonucleotide
Comprising an Anti-Sense Promoter
[0367] Another important composition of this aspect of the
invention is an analyte-binding substance that is joined to an
oligonucleotide comprising a sequence for an anti-sense promoter,
wherein the anti-sense promoter comprises one strand of a
functional double-stranded promoter. Thus, the anti-sense promoter
sequence comprising the oligonucleotide that is joined to the
analyte-binding substance can complex or anneal with the sense
promoter sequence of the Signal Probe to obtain a functional
double-stranded promoter for a cognate RNA polymerase. In addition,
the anti-sense promoter sequence provides a binding site for the
sense promoter sequence in the Signal Probe, thereby enabling
complexing or annealing of the Signal Probe to the analyte-binding
substance, which in turn can be immobilized or bound to a surface
or solid support.
[0368] The template sequence in the Signal Probe encodes a sequence
that, once transcribed by the RNA polymerase under transcription
conditions, is detectable using any of a variety of methods known
in the art. By way of example, but not of limitation, the
transcription product could be detected using one or a multiplicity
of molecular beacons that anneal complementary sequence of the
transcription products. Alternatively, the template sequence can
encode a substrate for Q-beta replicase, which can replicate the
substrate and thereby, further amplify the signal. From these
examples, it should be clear that the invention is not limited with
respect to the transcription products that can be encoded by the
template sequence that is joined 5'- of the sense transcription
promoter in the Signal Probe. As discussed herein below an
analyte-binding substance can be any of a wide variety of
compositions for detecting a wide variety of analytes, and all of
these are included in the present invention. In general, the
oligonucleotide (or "oligo") that is joined to the ABS comprises
deoxyribonucleotides, although other nucleotides, including
modified nucleotides that do not affect binding of the sense
promoter sequence or transcription of the signal sequence can be
used for a particular purpose. Similarly, modified linkages, such
as, but not limited to alpha-thiophosphate sugar linkages that are
resistant to certain nucleases can be used for a particular
purpose. The oligo that is joined to the ABS can be of any length,
so long as it has sufficient length to comprise an anti-sense
promoter sequence that, when annealed to a Signal Probe of the
invention, makes a functional double-stranded promoter that can be
used by an RNA polymerase under transcription conditions to make a
transcription product. The oligo comprising the anti-sense promoter
can comprise additional nucleotides that are 3'- of or 5'- of the
anti-sense promoter sequence so long as the additional nucleotides
do not bind the Signal Probe or another component of a method or
assay of the invention in a manner that is independent of
complexing of the anti-sense promoter sequence with the sense
promoter sequence of the Signal Probe or another component of a
method or assay of the invention, or otherwise negatively affect
the results of the method or assay. Any suitable method, whether
covalent or non-covalent can be used to join the 5'-end or
5'-portion of the oligonucleotide to the ABS or to another chemical
that serves as a linker to bind the oligonucleotide to the ABS. By
way of example, but without limiting the invention, the
oligonucleotide can have a biotin moiety that is joined to an ABS
that has an attached streptavidin or avidin moiety. It is important
that the method and site of joining the oligonucleotide does not
substantially affect the ability of the ABS to bind to the analyte,
or to another molecule such as a first antibody that is required
for the functioning of a particular assay.
[0369] The invention comprises methods, compositions and kits for
using the RNA polymerases of the invention as a signaling system
for an analyte of any type, including analytes such as, but not
limited to, antigens, antibodies or other substances, in addition
to an analyte that is a target nucleic acid.
C. Methods for Using Signal Probes and ABS-Oligos to Detect an
Analyte
[0370] Thus, the invention comprises a method for detecting an
analyte in or from a sample, the method comprising:
[0371] 1. obtaining an analyte-binding substance-oligonucleotide
("ABS-oligo"),
[0372] wherein the ABS-oligo comprises an ABS that is joined to a
oligonucleotide comprising a sequence for an anti-sense promoter
portion of a double-stranded promoter for an RNA polymerase that
recognizes the promoter;
[0373] 2. obtaining a Signal Probe, wherein the Signal Probe
comprises a sense promoter that is joined to the 3'-end of a
template, wherein the sense promoter is sufficiently complementary
to the anti-sense promoter of the ABS-oligo to form a complex that
can be used for transcription of the template using an RNA
polymerase that binds to the complex;
[0374] 3. contacting an ABS-oligo with a surface to which an
analyte is bound if present in a sample under analyte-binding
conditions that permit the ABS-oligo to bind the analyte if present
on said surface;
[0375] 4. washing the surface under conditions that permit removal
of unbound ABS-oligo;
[0376] 5. contacting the surface with a Signal Probe under
complexing conditions that permit complexing of the Signal Probe
with the ABS-oligo if present on the surface;
[0377] 6. optionally, washing the surface under conditions that
permit removal of unbound Signal Probe;
[0378] 7. contacting the surface with an RNA polymerase under
conditions that permit transcription of a product encoded by the
template using the complex between the ABS-oligo and the Signal
Probe;
[0379] 8. detecting a transcription product encoded by the
template, if present.
[0380] The Signal Probe used in the above described method can be
an RCT Signal Probe or a LINT Signal Probe. The step for washing
the surface under conditions that permit removal of unbound Signal
Probe is optional based on the particular Signal Probe used and
whether any "background" transcription products (i.e., "signal")
are detected using the method with a sample that does not contain
the analyte. If a background signal is obtained without the washing
step, then the washing step to remove unbound Signal Probe should
be performed. Since a double-stranded promoter sequence is
generally required for transcription by an RNA polymerase of the
present invention, the presence of unbound Signal Probes may not
cause background signal, in which case, the washing step is not
required. A washing step may not be required for methods that use
LINT Signal Probes. However, it is known that some RNA polymerases
can synthesize RNA by rolling circle transcription using a template
comprising a circular ssDNA molecule that is less than about 150
nucleotides, even if the template does not comprise a promoter
sequence (U.S. Pat. Nos. 5,714,320; 6,077,668; 6,096,880; and
6,368,802). By way of example, but without limitation, the
transcription activity by a T7 RNAP on circular ssDNA templates
that lack a promoter sequence is higher on smaller circles, such as
circular templates that comprise about 25 to about 50 nucleotides.
Therefore, a washing step to remove unbound RCT Signal Probes may
be required, particularly with RCT Signal Probes up to about 150
nucleotides, and, particularly, with RCT Signal Probes up to about
50 nucleotides. However, whether or not this washing step is
required for a particular assay will depend on many factors,
including the level of detection, the amount of analyte in the
sample, the template used in the Signal Probe, and the level of
acceptable background signal for the particular assay. Therefore,
the need for the washing step is determined for each particular
method (or "assay") of the invention.
[0381] The invention also comprises additional methods,
compositions and kits for amplifying the amount of transcription
product obtained from transcription of the complex between a Signal
Probe and an ABS-oligo. Thus, one embodiment of the invention
comprises a method for amplifying the amount of
template-complementary transcription product, the method
comprising:
[0382] a. obtaining a transcription product by transcription of the
template of a Signal Probe that is complexed with an ABS-oligo;
[0383] b. obtaining a sense promoter primer comprising a 3'-end
portion that is complementary to the 3'-end of the transcription
product and optionally, a phosphate group or a topoisomerase moiety
on its 5-end;
[0384] c. annealing the promoter primer to the transcription
product;
[0385] d. primer-extending the promoter primer annealed to the
transcription product with an RNA-dependent DNA polymerase under
DNA synthesis conditions so as to obtain first-strand cDNA;
[0386] e. optionally, removing the RNA that is annealed to the
first-strand cDNA;
[0387] f. ligating the first-strand cDNA, wherein the 5'-end is
covalently joined to the 3'-end of the first-strand cDNA so as to
obtain circular sense promoter-containing first-strand cDNA;
[0388] g. annealing an anti-sense promoter oligo to the circular
sense promoter-containing first-strand cDNA so as to obtain a
circular substrate for transcription;
[0389] h. contacting the circular substrate for transcription with
an RNA polymerase under transcription conditions so as to obtain
additional transcription product; and
[0390] i. obtaining the additional transcription product.
[0391] In another embodiment, the circular sense-promoter
first-strand cDNA is linearized to obtain linear
sense-promoter-containing first-strand cDNA, and then an anti-sense
promoter primer is annealed to the sense promoter sequence therein
to obtain a linear substrate for transcription. Then, the linear
substrate for transcription is contacted with an RNA polymerase
under transcription conditions so as to obtain additional
transcription product, and additional transcription product is
obtained.
[0392] Although a single-stranded promoter cannot be used to obtain
a substrate for transcription that comprises a Signal
Probe-ABS-oligo complex, the method described above for amplifying
the amount of transcription product obtained from transcription of
the complex between a Signal Probe and an ABS-oligo using a sense
promoter primer to copy the template can use a sense promoter
primer comprising a single-stranded sense promoter that is
recognized by an RNA polymerase that can make transcription
products using the single-stranded promoter. Thus, a sense promoter
primer comprising a single-stranded pseudopromoter or synthetic
promoter obtained by a method such as that described by Ohmichi et
al. (Proc. Natl. Acad. Sci. USA 99:54-59, 2002) or a
single-stranded phage N4 promoter, such as, but not limited to the
P2 promoter, can be used in embodiments of the method above for
amplifying the amount of transcription product. If a
single-stranded promoter is used, the cognate RNA polymerase for
the promoter is used for transcription of the circular sense
promoter-containing first-strand cDNA or linear sense
promoter-containing first-strand cDNA, which is a substrate for
transcription. Thus, in these embodiments which use a
single-stranded promoter, an anti-sense promoter oligo is not
needed to the obtain a circular or linear substrate for
transcription for amplifying the transcription products obtained
from transcription of the complex between a Signal Probe and an
ABS-oligo.
[0393] Still another method of the invention is to amplify the
signal of a transcription product from transcription of the complex
between a Signal Probe and an ABS-oligo is to use a template
sequence in the Signal Probe that encodes a substrate for a
replicase such as, but not limited to Q-beta replicase, and to
contact the transcription product from transcription of the complex
between a Signal Probe and an ABS-oligo with the replicase under
replication conditions, and to obtain replicated transcription
product, which is detected.
D. Detection of Signal Sequences Encoded by the Template of a
Signal Probe
[0394] A transcription product from transcription of the complex
between a Signal Probe and an ABS-oligo can be detected by any
method known in the art. By way of example, but not of limitation,
can comprise a substrate for Q-beta replicase, which is detectable,
following replication by the replicase under replication
conditions, using an intercalating dye such as, but not limited to
ethidium bromide. A transcription product can also comprise a
sequence that encodes a protein, such as green fluorescent protein,
that is detectable following translation of the signal sequence.
Without limitation, it can also comprise a sequence that is
detectable by a probe, such as, but not limited to a molecular
beacon, as described by Tyagi et al. (U.S. Pat. Nos. 5,925,517 and
6,103,476 of Tyagi et al. and 6,461,817 of Alland et al., all of
which are incorporated herein by reference).
E. Use of the Signaling System with Different Analytes
[0395] A signaling system of the invention can be used for a broad
range of analytes and analyte-binding substances. By way of
example, but not of limitation, the analyte can be an antigen and
the analyte-binding substance can be antibody, or the analyte can
be a nucleic acid and the analyte-binding substance can be another
complementary nucleic acid. As discussed below, a large number of
other substances exist for which a specific-binding pair can be
found, all of which are within the scope of the invention.
[0396] In order to detect an analyte in a sample, an assay of this
aspect of the invention uses an analyte-binding substance that
"binds" to the analyte under "binding conditions." The
analyte-binding substance, which can also be referred to as an
"affinity molecule," an "affinity substance," a "specific binding
substance," or a "binding molecule" for the analyte, is in turn
detected by making a transcription product using a transcription
signaling system that is joined to the analyte-binding
substance.
[0397] An "analyte" can be any substance whose presence,
concentration or amount in a sample is determined in an assay. By
way of example, but not of limitation, an analyte can be a
biochemical molecule or a biopolymer or a segment of a biopolymer,
such as a protein or peptide, including a glycoprotein or
lipoprotein, an enzyme, hormone, receptor, antigen or antibody,
nucleic acid (DNA or RNA), polysaccharide, or lipid.
[0398] An analyte-binding substance that is a nucleic acid,
polynucleotide, oligonucleotide or a segment of a nucleic acid or
polynucleotide, including nucleic acids composed of either DNA or
RNA, or both DNA and RNA mononucleosides, including modified DNA or
RNA mononucleosides, can also be used according to the invention to
detect an analyte that does not comprise nucleic acid. For example,
a method termed "SELEX," as described by Gold and Tuerk in U.S.
Pat. No. 5,270,163, can be used to select a nucleic acid for use as
an analyte-binding substance according to the invention. SELEX
permits selection of a nucleic acid molecule that has high affinity
for a specific analyte from a large population of nucleic acid
molecules, at least a portion of which have a randomized sequence.
For example, a population of all possible randomized 25-mer
oligonucleotides (i.e., having each of four possible nucleic acid
bases at every position) will contain 4.sup.25 (or 10.sup.15)
different nucleic acid molecules, each of which has a different
three-dimensional structure and different analyte binding
properties. SELEX can be used, according to the methods described
in U.S. Pat. Nos. 5,270,163; 5,567,588; 5,580,737; 5,587,468;
5,683,867; 5,696,249; 5,723,594; 5,773,598; 5,817,785; 5,861,254;
5,958,691; 5,998,142; 6,001,577; 6,013,443; and 6,030,776,
incorporated herein by reference, in order to select an
analyte-binding nucleic acid with high affinity for a specific
analyte that is not a nucleic acid or polynucleotide. Once selected
using SELEX, nucleic acid affinity molecules can be made by any of
numerous known in vivo or in vitro techniques, including; by way of
example, but not of limitation, automated nucleic acid synthesis
techniques, PCR, or in vitro transcription.
[0399] Naturally occurring nucleic acid or polynucleotide sequences
that have affinity for other naturally occurring molecules such as,
but not limited to, protein molecules, are also known in the art.
Examples include, but are not limited to certain nucleic acid
sequences such as operators, promoters, origins of replication,
sequences recognized by steroid hormone-receptor complexes,
restriction endonuclease recognition sequences, ribosomal nucleic
acids, and so on, which are known to bind tightly to certain
proteins. For example, in two well-known systems, the lac repressor
and the bacteriophage lambda repressor each bind to their
respective specific nucleic acid sequences called "operators" to
block initiation of transcription of their corresponding mRNA
molecules. Nucleic acids containing such specific sequences can be
used in the invention as analyte-binding substances for the
respective proteins or other molecules for which the nucleic acid
has affinity. In these cases, the nucleic acid with the specific
sequence can be used according to this aspect of the invention as
the analyte-binding substance for the respective specific protein,
glycoprotein, lipoprotein, small molecule or other analyte that it
binds. One of several techniques which are generally called
"footprinting" (e.g., see Galas, D. and Schmitz, A, Nucleic Acids
Res. 5:3161, 1978) can be used to identify sequences of nucleic
acids which bind to a protein. Other methods are also known to
those with skill in the art and can be used to identify nucleic
acid sequences for use as specific analyte-binding substances for
use in the invention.
[0400] A peptide nucleic acid (PNA) or a molecule comprising both a
nucleic acid and a PNA can also be used according to the invention
as an analyte-binding substance for an analyte that is a nucleic
acid or polynucleotide. PNA as an analyte-binding substance of the
invention provides tighter binding (and greater binding stability)
in assays for a nucleic acid analyte (e.g., see U.S. Pat. No.
5,985,563). Also, since PNA is not naturally occurring, PNA
molecules are highly resistant to protease and nuclease activity.
Antibodies to PNA/DNA or PNA/RNA complexes can be used in the
invention for capture, recognition, detection, identification, or
quantitation of nucleic acids in biological samples, via their
ability to bind specifically to the respective complexes without
binding the individual molecules (U.S. Pat. No. 5,612,458).
[0401] The invention also contemplates that a combinatorial library
of randomized peptide nucleic acids prepared by a method such as,
but not limited to, the methods described in U.S. Pat. Nos.
5,539,083; 5,831,014; and 5,864,010, can be used to prepare
analyte-binding substances for use in assays for analytes of all
types, including analytes that are nucleic acids, proteins, or
other analytes, without limit. As is the case for the SELEX method
with nucleic acids, randomized peptide or peptide nucleic acid
libraries are made to contain molecules with a very large number of
different binding affinities for an analyte. After selection of an
appropriate affinity molecule for an analyte from a library, the
selected affinity molecule can be used in the invention as an
analyte-binding substance.
[0402] An analyte-binding substance can also be an oligonucleotide
or polynucleotide with a modified backbone that is not an amino
acid, such as, but not limited to modified oligonucleotides
described in U.S. Pat. Nos. 5,602,240; 6,610,289; 5,696,253; or
6,013,785.
[0403] The invention also contemplates that an analyte-binding
substance can be prepared from a combinatorial library of
randomized peptides (i.e., comprising at least four
naturally-occurring amino acids). One way to prepare the randomized
peptide library is to place a randomized DNA sequence, prepared as
for SELEX, downstream of a phage T7 RNA polymerase promoter, or a
similar promoter, and then use a method such as, but not limited
to, coupled transcription-translation, as described in U.S. Pat.
Nos. 5,324,637; 5,492,817; or 5,665,563, or stepwise transcription,
followed by translation. Alternatively, a randomized DNA sequence,
prepared as for SELEX, can be cloned into a site in a DNA vector
that, once inserted, encodes a recombinant MDV-1 RNA containing the
randomized sequence that is replicatable by Q-beta replicase (e.g.,
between nucleotides 63 and 64 in MDV-1 (+) RNA; see U.S. Pat. No.
5,620,870). The recombinant MDV-1 DNA containing the randomized DNA
sequence is downstream from a T7 RNA polymerase promoter or a
similar promoter in the DNA vector. Then, following transcription,
the recombinant MDV-1 RNA, containing the randomized sequence can
be used to make a randomized peptide library comprising at least
four naturally-occurring amino acids by coupled
replication-translation as described in U.S. Pat. No. 5,556,769. An
analyte-binding substance can be selected from the library by
binding peptides in the library to an analyte, separating the
unbound peptides, and identifying one or more peptides that is
bound to analyte by means known in the art. Alternatively, high
throughput screening methods can be used to screen all individual
peptides in the library to identify those which can be used as
analyte-binding substances. Although the identification of an
analyte-binding peptide by these methods is difficult and tedious,
the methods in the art are improving for doing so, and the
expenditure of time and effort required may be warranted for
identifying analyte-binding substances for use in assays of the
invention that will be used routinely in large numbers.
[0404] A variety of other analyte-binding substances can also be
used in methods for this aspect of the invention.
[0405] For an antigen analyte (which itself may be an antibody),
antibodies, including monoclonal antibodies, are available as
analyte-binding substances. For certain antibody analytes in
samples which include only one antibody, an antibody binding
protein such as Staphylococcus aureus Protein A can be employed as
an analyte-binding substance.
[0406] For an analyte, such as a glycoprotein or class of
glycoproteins, or a polysaccharide or class of polysaccharides,
which is distinguished from other substances in a sample by having
a carbohydrate moiety which is bound specifically by a lectin, a
suitable analyte-binding substance is the lectin.
[0407] For an analyte which is a hormone, a receptor for the
hormone can be employed as an analyte-binding substance.
Conversely, for an analyte which is a receptor for a hormone, the
hormone can be employed as the analyte-binding substance.
[0408] For an analyte which is an enzyme, an inhibitor of the
enzyme can be employed as an analyte-binding substance. For an
analyte which is an inhibitor of an enzyme, the enzyme can be
employed as the analyte-binding substance.
[0409] Usually, an analyte molecule and an affinity molecule for
the analyte molecule are related as a specific "binding pair",
i.e., their interaction is only through non-covalent bonds such as
hydrogen-bonding, hydrophobic interactions (including stacking of
aromatic molecules), van der Waals forces, and salt bridges.
Without being bound by theory, it is believed in the art that these
kinds of non-covalent bonds result in binding, in part due to
complementary shapes or structures of the molecules involved in the
binding pair.
[0410] The term "binding" according to this aspect of the invention
refers to the interaction between an analyte-binding substance or
affinity molecule and an analyte as a result of non-covalent bonds,
such as, but not limited to, hydrogen bonds, hydrophobic
interactions, van der Waals bonds, and ionic bonds.
[0411] Based on the definition for "binding," and the wide variety
of affinity molecules and analytes which can be used in the
invention, it is clear that "binding conditions" vary for different
specific binding pairs. Those skilled in the art can easily
determine conditions whereby, in a sample, binding occurs between
affinity molecule and analyte that may be present. In particular,
those skilled in the art can easily determine conditions whereby
binding between affinity molecule and analyte, which would be
considered in the art to be "specific binding," can be made to
occur. As understood in the art, such specificity is usually due to
the higher affinity of affinity molecule for analyte than for other
substances and components (e.g., vessel walls, solid supports) in a
sample. In certain cases, the specificity might also involve, or
might be due to, a significantly more rapid association of affinity
molecule with analyte than with other substances and components in
a sample.
[0412] "Hybridization" is the term used to refer to the process of
incubating an affinity molecule comprising a nucleic acid, or a
peptide nucleic acid (PNA) molecule, or a covalently linked, joined
or attached nucleic acid-PNA molecule with an analyte comprising a
nucleic acid under "binding conditions," which are also called
"hybridization conditions." The ability of two polymers of nucleic
acid containing complementary sequences to find each other and
anneal through base pairing interaction is a well-recognized
phenomenon. The initial observations of the "hybridization" process
by Marmur and Lane (Proc. Nat. Acad. Sci. USA 46:453, 1960) and
Doty, et al. (Proc. Nat. Acad. Sci. USA 46:461, 1960) have been
followed by the refinement of this process into an essential tool
of modern biology. "Hybridization" also refers to the "binding" or
"pairing" of complementary nucleic acid bases in a single-stranded
nucleic acid, PNA, or linked nucleic acid-PNA affinity molecule
with a single-stranded nucleic acid analyte, which occurs according
to base pairing rules (e.g., adenine pairs with thymine or uracil
and guanine pairs with cytosine). Those with skill in the art will
be able to develop and make conditions which comprise binding
conditions or hybridization conditions for particular nucleic acid
analytes of an assay. In developing and making binding conditions
for particular nucleic acid analytes analyte-binding substances, as
well as in developing and making hybridization conditions for
particular analytes and capture probes, certain additives can be
added in the hybridization solution. By way of example, but not of
limitation, dextran sulfate or polyethylene glycol can be added to
accelerate the rate of hybridization (e.g., Chapter 9, Sambrook, et
al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory Press, 1989), or betaine can be added to
the hybridization solution to eliminate the dependence of T.sub.m
on basepair composition (Rees, W. A., et al., Biochemistry
32:137-144, 1993).
[0413] The terms "degree of homology" or "degree of
complementarity" are used to refer to the extent or frequency at
which the nucleic acid bases on one strand (e.g., of the affinity
molecule) are "complementary with" or "able to pair" with the
nucleic acid bases on the other strand (e.g., the analyte).
Complementarity may be "partial," meaning only some of the nucleic
acid bases are matched according to base pairing rules, or
complementarity may be "complete" or "total." The length (i.e., the
number of nucleic acid bases comprising the nucleic acid and/or PNA
affinity molecule and the nucleic acid analyte), and the degree of
"homology" or "complementarity" between the affinity molecule and
the analyte have significant effects on the efficiency and strength
of binding or hybridization when the nucleic acid bases on the
affinity molecule are maximally "bound" or "hybridized" to the
nucleic acid bases on the analyte. The terms "melting temperature"
or "T.sub.m" are used as an indication of the degree of
complementarity. The T.sub.m is the temperature at which a
population of double-stranded nucleic acid molecules becomes half
dissociated into single strands under defined conditions. Based on
the assumption that a nucleic acid molecule that is used in
hybridization will be approximately completely homologous or
complementary to a target polynucleotide, equations have been
developed for estimating the T.sub.m for a given single-stranded
sequence that is hybridized or "annealed" to a complementary
sequence. For example, a common equation used in the art for
oligodeoxynucleotides is: T.sub.m=81.5.degree. C.+0.41 (% G+C) when
the nucleic acid is in an aqueous solution containing 1 M NaCl (see
e.g., Anderson and Young, Quantitative Filter Hybridization, in
Nucleic Acid Hybridization, 1985). Other more sophisticated
equations available for nucleic acids take nearest neighbor and
other structural effects into account for calculation of the
T.sub.m. Binding is generally stronger for PNA affinity molecules
than for nucleic acid affinity molecules. For example the T.sub.m
of 10-mer homothymidine PNA binding to its complementary 10-mer
homoadenosine DNA is 73.degree. C., whereas the T.sub.m for the
corresponding 10-mer homothymidine DNA to the same complementary
10-mer homoadenosine DNA is only 23.degree. C. Equations for
calculating the T.sub.m for a nucleic acid are not appropriate for
PNA. Preferably, a T.sub.m that is calculated using an equation in
the art, is checked empirically and the hybridization or binding
conditions are adjusted by empirically raising or lowering the
stringency of hybridization as appropriate for a particular assay.
As used herein the term "stringency" is used in reference to the
conditions of temperature, ionic strength, and the presence of
other compounds, under which nucleic acid hybridizations are
conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"weak" or "low" stringency are often required when it is desired
that nucleic acids that are not completely complementary to one
another be hybridized or annealed together.
[0414] With regard to complementarity, it is important for some
assays of the invention to determine whether the hybridization
represents complete or partial complementarity. For example, where
it is desired to detect simply the presence or absence of pathogen
DNA (such as from a virus, bacterium, fungi, mycoplasma,
protozoan), it is only important that the hybridization method
ensures hybridization when the relevant sequence is present. In
those embodiments of the invention, conditions can be selected
where both partially complementary probes and completely
complementary probes will hybridize.
F. Kits and Compositions for Signaling Systems of the Invention
[0415] Important compositions of this aspect of the invention are
Signal Probes. Compositions comprising Signal Probes can comprise
an RCT Signal Probe or a LINT Signal Probe as described above.
Still further, multiple specific Signal Probes, each comprising
either a sense promoter for a different RNA polymerase, such as,
but not limited to sense a promoter for T7, T3 or SP6 RNAP and/or a
different template can be provided in order to detect and/or
quantify multiple different analytes in the same sample, or to
detect multiple different parts of the same analyte in the sample.
By way of example, but without limiting the invention, different
Signal Probes can be provided that detect different analyte-binding
substances comprising antibodies that recognize antigens comprising
different proteins or antigens comprising different antigenic
determinants on the same protein. If Signal Probes comprising
multiple different templates are used, the templates can encode
transcription products that are detectable by multiple different
detection molecules, such as, but not limited to multiple different
molecular beacons that anneal to different transcription products
in order to make a detectable fluorescent signal. Each different
molecular beacon can, but need not, have a different fluorescent
moiety that is quenched by the quenching moiety, wherein the signal
from one fluorescent moiety is distinguishable from the signal from
another fluorescent moiety.
[0416] Another important composition of the invention is an
analyte-binding substance that is joined to an oligonucleotide
comprising a sense promoter sequence (i.e., an "ABS-oligo) as
described above. Also as discussed elsewhere herein, the
analyte-binding substance (ABS) can be any substance that binds the
analyte tightly and with specificity so as to obtain specific
binding to the analyte in the presence of other substances to which
the ABS does not bind.
[0417] A kit can comprise a Signal Probe and an RNA polymerase and
other compositions that are used in a method that uses a Signal
Probe, but without an ABS-oligo for customers who wish to prepare
their own ABS-oligo for a particular analyte.
[0418] A kit of the invention can comprise one or more Signal
Probes and an ABS-oligo for detecting an analyte and/or for
quantifying an analyte in a sample and instructions for their use
in a method of the invention.
[0419] Another kit of the invention can comprise one or more Signal
Probes, one or more ABS-oligos for detecting one or more analytes
and/or for quantifying one or more analytes in a sample, and an
optimized composition of the RNA polymerase that uses the
double-stranded promoter in the transcription substrates obtained
by complexing a Signal Probe with an ABS-oligo to make a
transcription products, as well as instructions for use.
[0420] Still another kit of the invention comprises a kit for
amplifying the amount of template-complementary transcription
product obtained from transcription of the complex between a Signal
Probe and an ABS-oligo that is bound to an analyte, the kit
comprising:
[0421] 1. a sense promoter primer comprising a 3'-end portion that
is complementary to the 3'-end of the transcription product encoded
by the template of the Signal Probe, and optionally comprising a
phosphate group or a topoisomerase moiety on its 5-end;
[0422] 2. optionally, an RNA-dependent DNA polymerase for primer
extension of the sense promoter primer using a transcription
product encoded by the template of the Signal Probe as a
template;
[0423] 3. optionally, a ligase for ligating a first-strand cDNA
obtained by primer extension of the sense promoter primer;
[0424] 4. an anti-sense promoter oligo that can complex with the
sense promoter of the first-strand cDNA primer extension product so
as to obtain a functional double-stranded transcription promoter
for an RNA polymerase that binds the promoter and initiates
transcription therefrom under transcription conditions;
[0425] 5. optionally, the RNA polymerase that uses the
double-stranded promoter;
[0426] 6. optionally, other optimized reaction buffers and
compositions for the method, either as individual compositions or
as a single optimized combined composition or as a small number of
compositions; and
[0427] 7. instructions for their use in a method of the
invention.
[0428] Enzymes can be provided in the kit separately or combined
into a single ready-to-use solution containing the optimal ratio of
each enzyme.
[0429] A kit comprising enzymes and/or other compositions that are
used in a method that uses a Signal Probe can be provided with the
Signal Probe or without a Signal Probe for customers who wish to
prepare their own Signal Probes comprising a different template
sequence.
[0430] A kit can be for detecting and/or quantifying a specific
analyte, such as, but not limited to, a kit for an analyte-specific
assay comprising a pathogen, a disease gene, a mutated allele, or
the like.
[0431] In general, a kit of the invention will also comprise a
description of the components of the kit and instructions for their
use in a particular process or method or methods of the invention.
In general, a kit of the present invention will also comprise other
components, such as, but not limited to, buffers, ribonucleotides
and/or deoxynucleotides, including modified nucleotides in some
embodiments, DNA polymerization or reverse transcriptase enhancers,
such as, but not limited to betaine (trimethylglycine), and salts
of monvalent or divalent cations, such as but not limited to
potassium acetate or chloride and/or magnesium chloride, enzyme
substrates and/or cofactors, such as, but not limited to, ATP or
NAD, and the like which are needed for optimal conditions of one or
more reactions or processes of a method or a combination of methods
for a particular application. A kit of the invention can comprise a
a set of individual reagents for a particular process or a series
of sets of individual reagents for multiple processes of a method
that are performed in a stepwise or serial manner, or a kit can
comprise a multiple reagents combined into a single reaction
mixture or a small number of mixtures of multiple reagents, each of
which perform multiple reactions and/or processes in a single tube.
In general, the various components of a kit for performing a
particular process of a method of the invention or a complete
method of the invention will be optimized so that they have
appropriate amounts of reagents and conditions to work together in
the process and/or method.
[0432] A kit of the invention can also comprise additional
components, such as reaction buffers, control substrates, size
markers, detection compositions or detection reagents, and the
like, all of which can be provided in quantities to match the need
for each component for the proscribed number of intended reactions,
but a kit need not contain these components. Still further, a kit
can optionally contain detailed instructions and troubleshooting
guides.
[0433] Components of a kit may be provided as solutions or as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent, in which case, the solvent may also be provided
in another container.
[0434] A broad range of other kits included within the invention
will be understood by those with knowledge in the art by a reading
of the description of the invention herein.
[0435] As used herein in the specification, "a" or "an" may mean
one or more. As used herein in the claim(s), when used in
conjunction with the word "comprising," the words "a" or "an" may
mean one or more than one. As used herein "another" may mean at
least a second or more.
Sequence CWU 1
1
5118DNAArtificialSynthetic Oligonucleotides 1taatacgact cactatag
18218DNAArtificialSynthetic Oligonucleotides 2ctatagtgag tcgtatta
18320DNAArtificialSynthetic Oligonucleotides 3caacgaagcg ttgaatacct
20422DNAArtificialSynthetic Oligonucleotides 4ttcttcgagg cgaagaaaac
ct 22520DNAArtificialSynthetic Oligonucleotides 5cgacgaggcg
tcgaaaacca 20
* * * * *