U.S. patent application number 13/352237 was filed with the patent office on 2012-08-02 for workflow for detection of ligands using nucleic acids.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Shiaw-Min Chen, Stephen Hendricks, Julia Lu, David Ruff, Mark Shannon, Elana Swartzman.
Application Number | 20120196294 13/352237 |
Document ID | / |
Family ID | 45561104 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120196294 |
Kind Code |
A1 |
Chen; Shiaw-Min ; et
al. |
August 2, 2012 |
WORKFLOW FOR DETECTION OF LIGANDS USING NUCLEIC ACIDS
Abstract
This application relates to methods for ligating
oligonucleotides having complementarity to a target nucleic acid,
and amplifying the ligated oligonucleotides, where ligation and
amplification occur in the same reaction mixture.
Inventors: |
Chen; Shiaw-Min; (Fremont,
CA) ; Swartzman; Elana; (Alameda, CA) ; Ruff;
David; (San Francisco, CA) ; Shannon; Mark;
(San Francisco, CA) ; Lu; Julia; (US) ;
Hendricks; Stephen; (Los Gatos, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
45561104 |
Appl. No.: |
13/352237 |
Filed: |
January 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61433475 |
Jan 17, 2011 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6855 20130101;
C12Q 2561/125 20130101; C12Q 1/6804 20130101; C12Q 1/6804 20130101;
C12Q 2561/101 20130101; C12Q 2537/101 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for ligating at least two oligonucleotides to produce a
ligated oligonucleotide and amplifying the ligated oligonucleotide,
wherein ligation and amplification occur in a single reaction
mixture.
2. The method of claim 1 whereby a ligand is detected in a test
sample, the method comprising the steps of, in combination: a)
contacting a target protein or analyte with at least a first and
second probe, each probe having binding specificity for the protein
or analyte, and being adjoined to at least one type of
oligonucleotide; b) ligating the oligonucleotides on the first and
second probes to one another using a ligase to produce a target
nucleic acid and amplifying the target nucleic acid; and, c)
detecting the amplified target nucleic acid.
3. The method of claim 1 or 2, wherein a portion of at least one of
said probes is an antibody.
4. The method of claim 3, wherein a portion of each of said first
and second probes are antibodies.
5. The method of any one of claims 1-4, wherein at least one of
said oligonucleotides comprises at least three nucleotides.
6. The method of any one of claims 1-5, wherein at least one of
said oligonucleotides consists of three nucleotides.
7. The method of any one of claims 1-6, wherein said
oligonucleotides are ligated using a small footprint ligase
(SFL).
8. The method of claim 7, wherein said small footprint ligase is
contacted with adenosine triphosphate prior to use.
9. The method of any one of claims 1-8, wherein said ligated
oligonucleotide is amplified using the polymerase chain reaction
(PCR).
10. The method claim 9, wherein said amplified ligated
oligonucleotide is detected using quantitative PCR (qPCR).
11. The method of any one of claims 1-10, wherein a ligase is used
for ligation, and said ligase is inactivated prior to
amplification.
12. The method of claim 11, wherein said ligase is inactivated by
heat.
13. The method of claim 11 or 12, wherein said ligase is selected
from the group consisting of a nucleic acid ligase, oligonucleotide
ligase, DNA ligase, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7
DNA ligase, vaccinia virus DNA ligase, E. coli DNA ligase,
mammalian DNA ligase I, mammalian DNA ligase II, mammalian DNA
ligase III, Tth DNA ligase, KOD DNA ligase, a thermostable DNA
ligase, RNA ligase, T4 RNA ligase, bacteriophage RB69 RNA ligase,
Autographa californica nuclearpolyhedrosis virus RNA ligase, a
thermophilic RNA ligase, bacteriophage RM378 RNA ligase,
bacteriophage TS2126 RNA ligase, a small-footprint DNA ligase
(SFL), the ligase of SEQ ID NO.: 77, the ligase of SEQ ID NO.: 78,
the ligase of SEQ ID NO.: 79, the ligase of SEQ ID NO.: 80, the
ligase of SEQ ID NO.: 81, the ligase of SEQ ID NO.: 82, a
derivative thereof, a fragment thereof, and combinations
thereof.
14. A method for detecting a target in a sample wherein: a) binding
a first and a second probe, each of which binds specifically to the
target, wherein each of the probes comprises an oligonucleotide
portion or tail; b) ligating the first and second oligonucleotide
tails thereby producing a ligated oligonucleotide template; and, c)
performing a polymerase chain reaction (PCR) of the oligonucleotide
template across the first and second oligonucleotide to quantify
the said template.
15. The method of claim 14, wherein steps b and c are performed in
the same reaction mixture.
16. The method of claim 14 or 15, wherein a splint oligonucleotide
is used to ligate said first and second oligonucleotide tails.
17. The method of claim 16, wherein the 3' and 5' ends (which
overlap with said first and second oligonucleotide tails,
respectively) of said splint oligonucleotide are symmetrical or
asymmetrical to one another.
18. The method of claim 16 or 17, wherein said splint
oligonucleotide is blocked at it's 3'-end.
19. The method of any of claims 16-18, wherein said splint
oligonucleotide is at least 6 nucleotides in length.
20. The method of any of claims 16-19, wherein said 3'-end of said
splint oligonucleotide overlaps with at least 3 nucleotides of said
first oligonucleotide tail and/or said 5'-end of said splint
oligonucleotide overlaps with at least 3 nucleotides of said second
oligonucleotide tail.
21. The method of any of claims 14-20, wherein the length of said
first and second oligonucleotide tails is at least 3 nucleotides in
length.
22. The method of any of claims 14-21, wherein a small footprint
ligase (SFL) is used to ligate said first and second
oligonucleotide tails.
23. The method of any of claims 14-22, wherein said first and/or
second probes further comprises an antibody portion specific to
said target.
24. A method for detecting a target in sample comprising: a)
binding a first and a second probe, wherein each probe binds
specifically to the target, each of said probes comprise an
oligonucleotide portion or tail; b) ligating the oligonucleotides
to produce a ligated oligonucleotide template and amplifying the
template by PCR in a single step to produce an amplified template;
and, c) quantitating the amplified template
24. The method of claim 24, wherein the probe further comprises an
antibody.
25. The method of claim 24 or 25, wherein said ligase is selected
from the group consisting of a nucleic acid ligase, oligonucleotide
ligase, DNA ligase, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7
DNA ligase, vaccinia virus DNA ligase, E. coli DNA ligase,
mammalian DNA ligase I, mammalian DNA ligase II, mammalian DNA
ligase III, Tth DNA ligase, KOD DNA ligase, a thermostable DNA
ligase, RNA ligase, T4 RNA ligase, bacteriophage RB69 RNA ligase,
Autographa californica nuclearpolyhedrosis virus RNA ligase, a
thermophilic RNA ligase, bacteriophage RM378 RNA ligase,
bacteriophage TS2126 RNA ligase, a small-footprint DNA ligase
(SFL), the ligase of SEQ ID NO.: 77, the ligase of SEQ ID NO.: 78,
the ligase of SEQ ID NO.: 79, the ligase of SEQ ID NO.: 80, the
ligase of SEQ ID NO.: 81, the ligase of SEQ ID NO.: 82, a
derivative thereof, a fragment thereof, and combinations
thereof.
26. The method of claim 26, wherein said ligase is a T4 DNA
ligase.
27. The method of claim 26, wherein the ligase is a small-footprint
DNA ligase (SFL).
28. The method of any one of claims 24-27, wherein said
oligonucleotides are ligated using a splint oligonucleotide.
29. The method of claim 28, wherein said splint oligonucleotide
comprises a 3' end of four to nine bases in length and a 5' end of
four to nine bases in length.
30. The method of claim 28 or 29, wherein said 3' and 5' ends
(which overlap with said first and second tails, respectively) of
said splint oligonucleotide are symmetrical or asymmetrical to one
another.
31. The method of claim 28-30, wherein said splint oligonucleotide
is blocked at it's 3'-end.
32. The method of any one of claims 23-31, wherein the ligase is
pre-enriched using ATP.
33. The method of any one of claims 23-32, wherein said ligase is
inactivated after ligation using a protease or heat.
34. The method of any one of claims 23-33, wherein said template is
quantified using real-time PCR.
35. The method of claim 34, wherein said real-time PCR assay is a
TaqMan assay.
36. The method of claim 1, 14, or 24, wherein said method provides
at least about a one to three-fold dCt improvement over a typical
PLA method or process.
37. The method of claim 1, 14, or 24, wherein said method provides
about a two- to ten-fold improvement/increase in sensitivity over
the typical process.
38. The method of claim 36 or 37, wherein said typical method or
process includes the use of a protease and/or dilution of the
reaction mixture prior to PCR and said improved method of claim 1,
14, or 24 does not.
39. The method of claim 2, wherein said oligonucleotides on said
first and second probes, are at least partially complementary to
one another
Description
FIELD OF THE DISCLOSURE
[0001] This application relates to methods for ligating
oligonucleotides having complementarity to a target nucleic acid,
and amplifying the ligated oligonucleotides, where ligation and
amplification occur in the same reaction mixture.
BACKGROUND OF THE DISCLOSURE
[0002] The correlation of gene and protein expression changes in
biological systems has been hampered by the need for separate
sample handling and analysis platforms for nucleic acids and
proteins. In contrast to the simple, rapid, and flexible workflow
of quantitative PCR (qPCR) methods, which enable characterization
of several classes of nucleic acid biomarkers (e.g., DNA, mRNA, and
microRNAs), protein analysis methods such as Western blotting are
cumbersome, laborious, and much less quantitative. Proximity
Ligation Assays (PLAs) have been shown to eliminate some of these
problems. However, improvements to PLAs are desired by those of
skill in the art.
[0003] Typical or conventional PLAs usually involve at least three
or four steps. The first step is typically the binding of first and
second probes (e.g., antibody probes) to a ligand (e.g., a protein
of interest) such that the probes are in close proximity to
another. Each of the probes typically contain an oligonucleotide.
The oligonucleotides are brought into proximity to one another with
the binding of the probes and, in the second step, are then ligated
to one another (e.g., the ligation event). The ligated
oligonucleotides may then be amplified and detected to determine
the presence of the ligand with a test sample (e.g., a biological
sample). This step is typically accomplished by adding ligation
components, such as ligase, adenosine triphosphate (ATP) and
buffer-salt mixture, to the binding reaction. In the third step,
the ligase is typically then deactivated (e.g., by protease
digestion) to prevent any further ligation of unbound
oligonucleotides. In the fourth step, the reaction mixture is
transferred to a real-time polymerase chain reaction (PCR) mixture
and the quantity of the amplified product determined by
quantitative PCR (qPCR). As described below, it has been
surprisingly found that the third step (ligase digestion) may be
eliminated, thereby allowing ligation and amplification to occur in
the same reaction mixture without inactivation of the ligase. These
and other features and advantages of the methods described herein
will be apparent to the skilled artisan from this disclosure.
SUMMARY OF THE DISCLOSURE
[0004] Provided herein are methods for ligating and amplifying
oligonucleotides. In some embodiments, the oligonucleotides are
attached to ligand-specific probes, and amplification of the
oligonucleotides indicates that the probes have bound a ligand in
the sample. In one embodiment, a method for ligating at least two
oligonucleotides to produce a ligated oligonucleotide and
amplifying the ligated oligonucleotide, wherein ligation and
amplification occur in a single reaction mixture (e.g., that may be
considered undiluted) is provided. In some embodiments, a third
oligonucleotide may be used to bridge the at least two
oligonucleotides that are bound to the probes. In certain
embodiments, the method may comprise detecting a ligand in a test
sample (e.g., a biological sample) comprising contacting the
protein with at least a first and second probe, each probe having
binding specificity for the protein and being adjoined to at least
one type of oligonucleotide, the oligonucleotides on the first and
second probes, respectively, being at least partially complementary
to one another; ligating the oligonucleotides on the first and
second probes to one another using a ligase to produce a target
nucleic acid and amplifying the target nucleic acid; and, detecting
the amplified target nucleic acid. For instance, the method may
comprise detecting a protein in a test sample, the method
comprising contacting the protein with at least two probes having
binding specificity therewith, each of the two agents comprising at
least one oligonucleotide; ligating the oligonucleotides to produce
a ligated oligonucleotide and amplifying the ligated
oligonucleotide in a single reaction mixture; and, detecting
amplification of the ligated oligonucleotide. In some embodiments,
one or more of the probes is an antibody. In certain embodiments,
at least one of the oligonucleotides comprises at least three
nucleotides. Some embodiments provide for the oligonucleotide being
ligated using a small footprint ligase, which may be contacted with
adenosine triphosphate prior to use. Any type of amplification
procedure may be used such as, without limitation, polymerase chain
reaction (PCR) (e.g., quantitative PCR (qPCR)). In some
embodiments, it may be beneficial to inactivate the ligase prior to
amplification (e.g., using a protease). Other embodiments of the
methods described herein will be apparent to the skilled artisan
from the disclosure provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The skilled artisan will understand that the drawings
described below are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0006] FIG. 1. A schematic diagram of an exemplary typical or
conventional PLA process.
[0007] FIG. 2. A schematic diagram of an exemplary improved PLA
process (as disclosed herein).
[0008] FIG. 3. (A) A schematic diagram of an exemplary typical or
conventional PLA workflow. (B) A schematic diagram of an exemplary
improved PLA workflow (as disclosed herein).
[0009] FIG. 4. A schematic diagram of exemplary (A) asymmetrical
(e.g., 3+6) and (B) symmetrical (e.g., 4+4) oligonucleotide splints
("connectors").
[0010] FIG. 5. Comparison of a typical or conventional PLA process
(PLA1) and the improved process (PLA2) (dCT data shown).
[0011] FIG. 6. Use of the improved PLA process with various target
nucleic acids.
[0012] FIG. 7. Comparison of two different splint lengths at
varying concentrations.
[0013] FIG. 8. Comparison of five different splint lengths.
[0014] FIG. 9. Comparison of T4 ligase to two different SF ligases
(e.g., SF and DLxD).
DETAILED DESCRIPTION
[0015] Disclosed herein are methods for performing proximity
ligation assays. In typical or conventional proximity ligation
assay (PLA) processes (FIG. 1), a probe mix and sample are combined
into a binding reaction. Following the binding reaction, the
ligation reaction mixture is added in order to carry out the
ligation reaction. To prepare the ligation reaction mixture, a
ligase and ligation buffer are diluted. Following the ligation
reaction, the ligated product is stabilized by protease digestion;
the protease is then inactivated (e.g, using heat). A portion of
the ligated product is transferred to a real-time PCR reaction
mixture, then placed on a PCR reaction vessel (e.g., plate) in a
qPCR instrument. Detection and quantification of the ligated
product then proceeds using standard techniques.
[0016] In one embodiment of the improved PLA process disclosed
herein, a cell lysate may be prepared and a ligation buffer added
thereto. To that mixture may then be added a proximity probe
mixture, a ligase, and a PCR mixture (which may include, for
example, a thermostable polymerase). This combined reaction mixture
can then incubated for a suitable amount of time (e.g., one hour,
37.degree. C.), the ligase optionally inactivated (e.g., using
heat) and PCR performed directly on the mixture. A schematic of an
exemplary embodiment of the improved PLA process is illustrated in
FIG. 2. As shown therein, the binding reaction is the same as that
shown in FIG. 1. However, in some embodiments of the improved PLA
processes, the ligase is added to the real-time PCR mixture which
is then added directly to the binding reaction. In some
embodiments, this reaction mixture is then deposited onto a
reaction plate and then analyzed by a qPCR instrument. Detection
and quantification of the ligated product then proceeds using
standard techniques.
[0017] In some embodiments, the lesser dilution factor provided
thereby may result in a higher PLA probe concentration in the
ligation reaction. The increased probe concentration may cause
increased background signal, which may be minimized by using a
short splint oligonucleotide at reduced concentration. For
instance, a suitable splint oligonucleotide may be 14 nucleotides
in length (e.g., at least five nucleotides in the 3' end and at
least nine nucleotides in the 5' end; 5+9). To ensure the ligation
efficiency, a small footprint ligase (SFL) may be used. In some
embodiments, to further simplify the ligation-PCR step, the typical
addition of ATP to the ligation reaction may be omitted. Instead,
one may optionally use an ATP-enriched SFL (e.g., an SFL that is
exposed to or contacted with an abundance or additional supply of
ATP for some period of time). This enrichment step may be
especially useful when co-substrates for other ligases are used.
Thus, the binding reaction may be assembled by combining proximity
probes and samples containing target molecule(s), and incubating
the mixture such that binding between the probes and the target
molecule(s) occurs. In some embodiments, after the binding
reaction, a ligation-PCR mix (e.g., comprising a short splint oligo
(e.g., an oligonucleotide that is at least 6 nucleotides, e.g., 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
nucleotides) in length, an SFL, and standard real-time PCR
components) may be added. In some other embodiments, the ligation
reaction may then take place at room-temperature, and the product
amplified and quantitated by real-time PCR. In some embodiments,
the ligase may be deactivated (e.g., using heat). The ligated
product may then be subjected to real-time PCR immediately or
following storage. Thus, the various embodiments of the novel work
flows for improved PLA processes as disclosed herein provide
reduced dilution factors which enable one to accomplish ligation
and PCR in a single reaction mixture (e.g., or in a single step
without intermediate/intervening steps). In some preferred
embodiments, a short splint oligonucleotide and an SFL may also be
used to control any increased background reactions. In additional
embodiments, the SFL may be pre-enriched using ATP.
[0018] Exemplary typical and improved PLA processes are further
compared in FIGS. 3A and 3B. As shown therein, the typical
processes include sample preparation, a binding reaction, ligation,
ligase inactivation using a protease, protease inactivation (e.g.,
using heat), followed by real-time PCR. To carry out the PCR step
in typical PLA processes, a portion of the reaction mixture
containing the inactivated ligase and protease is usually
transferred to a PCR plate, and the "PCR mix" (e.g., containing
primers, dNTPs, polymerase, and the like) added thereto. As shown
in FIG. 3B, the disclosed improved processes can eliminate the use
of a protease and dilution of the reaction mixture prior to PCR. As
shown therein, the ligase may be inactivated using, for example,
heat, and the resultant reaction mixture placed directly into a
qPCR assay/instrument. Thus, simplified, improved PLA work flows
can use entire binding reaction products in a real-time PCR assay
(e.g., in a multi-plate well). This provides an improved work-flow
and reduced dilution of the reaction mixture. As a result, in some
embodiments of the improved PLA processes, the PCR reaction mixture
contains a higher concentration of the ligated product (e.g., the
target nucleic acid). In some embodiments, the improvement provided
by the improved PLA processes can be measured as the dCT of the
reaction (e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more signal
(dCT); see, for example, FIG. 4). Sensitivity of the improved PLA
processes as compared to typical PLA processes can also be observed
(e.g., as fold-change; see, for example, FIG. 5).
[0019] The processes described herein provide for, in some
embodiments, detecting a protein in a test sample, the methods
comprising contacting the protein with at least two probes having
binding specificity therewith, each of the two probes comprising at
least one oligonucleotide; ligating the oligonucleotides to produce
a ligated oligonucleotide; amplifying the ligated oligonucleotide
in a single reaction mixture; and, detecting amplification of the
ligated oligonucleotide. In some embodiments, a third
oligonucleotide may also be used to bridge each of the
oligonucleotides attached to each of the probes. In some
embodiments, one or more of the probes is an antibody. In certain
embodiments, at least one of the oligonucleotides comprises at
least three nucleotides. Some embodiments provide for use of a SFL,
which may optionally be contacted with adenosine triphosphate (ATP)
prior to use, for ligation of the oligonucleotides. Any type of
amplification procedure may be used such as, without limitation,
polymerase chain reaction (PCR) (e.g., quantitative PCR). In some
embodiments, it may be beneficial to inactivate the ligase prior to
amplification (e.g., using a protease, heat, or any other methods
known in the art). Other embodiments of the inventions described
herein will be apparent to the skilled artisan from the disclosure
provided herein.
[0020] In some embodiments, a method for detecting a target in a
sample is provided where the method includes the steps of binding a
first and a second probe, each of which binds specifically to the
target, wherein each of the probes comprises an oligonucleotide
portion (or tail); ligating the first and second oligonucleotide
tails thereby producing a ligated oligonucleotide template; and,
performing a polymerase chain reaction (PCR) of the oligonucleotide
template across the first and second oligonucleotide tails to
quantify the said template. In some embodiments, the ligation and
PCR steps may be performed in the same reaction mixture. In other
embodiments, the method may include binding a first and a second
probe, wherein each probe binds specifically to the target, each of
the probes comprise a oligonucleotide tail; ligating the
oligonucleotide tails to produce a ligated oligonucleotide template
and amplifying the template by PCR in a single step to produce an
amplified template; and, quantitating the amplified template. The
probes may comprise antibodies which specifically bind to the
target. The oligonucleotides may be ligated using a splint
oligonucleotide (e.g., splint oligos of at least 6 nucleotides in
length having 3' and 5' overhangs of, for example, 9+9, 9+8, 9+7,
9+6, 9+5, 8+8, 5+3, 4+7, 3+3 nucleotides, or any other possible
variations in length or symmetry as contemplated and described in
further detail below). In some embodiments, the ligase may be
pre-enriched using ATP and/or inactivated using, for example, one
or more proteases and/or heat. The amplified template may be
quantified by any suitable method including, for example, real-time
PCR (e.g., a TaqMan assay or a molecular beacon assay).
[0021] The improved processes disclosed and/or exemplified herein
provide reduced work times from process start time to collection of
results (e.g., faster), reduced hands-on time (e.g., simpler and
cheaper), reduced lab plasticware usage (e.g., cheaper and more
environmentally sound ("greener")), and increased signals and
sensitivities (e.g., more sensitive). In some embodiments, these
improved processes provide simplified work flows by combining
ligation and PCR steps, reduced dilution factors from the binding
step to the ligation step, reduced binding probe concentrations to
enable reduced dilution factors, use of shorter connector
oligonucleotides (e.g., as few as 6 nucleotides in length) to
control background signals, use of lower connector oligonucleotide
concentrations to control background signals, use of SF ligases to
enable use of shorter connector oligonucleotide lengths,
ATP-enriched SF ligase purification schemes to omit ATP in
ligation-PCR step, and/or enabling use of the entire reaction
volume to improve PLA signal and sensitivity.
[0022] The methods described herein are particularly useful in that
the same may be used with various systems for detecting proteins.
Exemplary of such systems include, for example, TaqMan.RTM. Protein
Assays. TaqMan.RTM. Protein Assays are an adapted form of PLA.TM.,
a proximity ligation assay technology that combines
antibody-protein binding with detection of the reporter nucleic
acid by real-time PCR. Applied Biosystems has optimized this
technique for use with crude cell and tissue lysates and combined
it with TaqMan.RTM. chemistry to create a highly sensitive and
specific process for measuring protein expression in small samples.
Assays have been developed for the detection of OCT3/4, NANOG,
SOX2, and LIN28 in human embryonic stem cells, as well as ICAM1 and
CSTB to measure relative quantification in human cells. The basic
steps of such assays include binding of a protein target by paired
assay probes, ligation of the oligonucleotides by a DNA ligase, and
amplification of the ligation product by TaqMan real-time PCR
assay. The probes used in the first step are typically
target-specific antibodies conjugated to oligonucleotides through a
biotin-streptavidin (SA) linkage. Each oligonucleotide in the pair
presents a 5' or 3' end that are brought into proximity when the
assay probes bind to two different epitopes on the target protein.
The substrate for the ligase is typically a bridge structure formed
by hybridization of a third oligonucleotide to the oligonucleotide
ends of the assay probe pair. This structure forms preferentially
when the assay probes are in proximity to each other. The ligation
product typically serves the template in the TaqMan.RTM. real-time
PCR assay. The systems may be used to, for example, to perform
protein analysis on small samples (e.g., stem cells, germ cell
tumors), correlate and/or validate results from RNA and protein
quantitation, analyze post-translational modifications, validate
siRNA-induced gene silencing, and/or validate gene
transfection/transduction experiments. Data generated using these
systems may be analyzed using software such as, for instance,
ProteinAssist.TM. software package (Applied Biosystems.TM.).
[0023] To more clearly and concisely describe and point out the
subject matter of the present disclosure, the following definitions
are provided for specific terms, which are used in the following
description and the appended claims. Throughout the specification,
exemplification of specific terms should be considered as
non-limiting examples.
[0024] As used herein the terms "nucleotide" or "nucleotide base"
refer to a nucleoside phosphate. It includes, but is not limited
to, a natural nucleotide, a synthetic nucleotide, a modified
nucleotide, or a surrogate replacement moiety or universal
nucleotide (e.g., inosine). The nucleoside phosphate may be a
nucleoside monophosphate, a nucleoside diphosphate or a nucleoside
triphosphate. A "nucleotide" refers to a nucleotide, nucleoside or
analog thereof. Optionally, the nucleotide is an N- or C-glycoside
of a purine or pyrimidine base. (e.g., deoxyribonucleoside
containing 2-deoxy-D-ribose or ribonucleoside containing D-ribose).
Examples of other analogs include, without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides. Nucleotide
bases usually have a substituted or unsubstituted parent aromatic
ring or rings. In certain embodiments, the aromatic ring or rings
contain at least one nitrogen atom. In certain embodiments, the
nucleotide base is capable of forming Watson-Crick and/or Hoogsteen
hydrogen bonds with an appropriately complementary nucleotide base.
Exemplary nucleotide bases and analogs thereof include, but are not
limited to, purines such as 2-aminopurine, 2,6-diaminopurine,
adenine (A), ethenoadenine, N6-.DELTA.2-isopentenyladenine (61A),
N6-.DELTA.2-isopentenyl-2-methylthioadenine (2ms6iA),
N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine
(dmG), 7-methylguanine (7 mG), 2-thiopyrimidine, 6-thioguanine
(6sG) hypoxanthine and O6-methylguanine; 7-deaza-purines such as
7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G);
pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine,
thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine,
O4-methylthymine, uracil (U), 4-thiouracil (4sU) and
5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole
and 4-methylindole; pyrroles such as nitropyrrole; nebularine; base
(Y); etc. In certain embodiments, nucleotide bases are universal
nucleotide bases. Additional exemplary nucleotide bases can be
found, e.g., in Fasman, 1989, Practical Handbook of Biochemistry
and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla.,
and the references cited therein. A "universal base", as used
herein, is a base that is complementary to more than one other
bases. Fully universal bases can pair with any of the bases
typically found in naturally occurring nucleic acids. The base need
not be equally capable of pairing with each of the naturally
occurring bases. Alternatively, the universal base may pair only or
selectively with two or more bases but not all bases. Optionally
the universal base pairs only or selectively with purines, or
alternatively with pyrimidines. If so desired, two or more
universal bases can be included at a particular position in a
probe. A number of universal bases are known in the art including,
but not limited to, hypoxanthine, 3-nitropyrrole, 4-nitroindole,
5-nitroindole, 4-nitrobenzimidazole, 5-nitroindazole,
8-aza-7-deazaadenine,
6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P. Kong Thoo
Lin. and D. M. Brown, Nucleic Acids Res., 1989, 17, 10373-10383),
2-amino-6-methoxyaminopurine (D. M. Brown and P. Kong Thoo Lin,
Carbohydrate Research, 1991, 216, 129-139), etc. Hypoxanthine is
one preferred fully universal base. Nucleosides comprising
hypoxanthine include, but are not limited to, inosine, isoinosine,
2'-deoxyinosine, and 7-deaza-2'-deoxyinosine, 2-aza-2'deoxyinosine.
Naturally occurring and synthetic analogs may also be used,
including for example hypoxanthine, 2-aminoadenine, 2-thiouracil,
2-thiothymine, 5-N.sup.4 ethencytosine,
4-aminopyrrazolo[3,4-d]pyrimidine and
6-amino-4-hydroxy[3,4-d]pyrimidine, among others. The nucleotide
units of the oligonucleotides may also have a cross-linking
function (e.g. an alkylating agent).
[0025] A nucleoside is usually a compound having a nucleotide base
covalently linked to the C-1' carbon of a pentose sugar. In certain
embodiments, the linkage is via a heteroaromatic ring nitrogen.
Typical pentose sugars include, but are not limited to, those
pentoses in which one or more of the carbon atoms are each
independently substituted with one or more of the same or different
--R, --OR, --NRR or halogen groups, where each R is independently
hydrogen, (C.sub.1-C.sub.6) alkyl or (C.sub.5-C.sub.14) aryl. The
pentose sugar may be saturated or unsaturated. Exemplary pentose
sugars and analogs thereof include, but are not limited to, ribose,
2'-deoxyribose, 2'-(C.sub.1-C.sub.6)alkoxyribose,
2'-(C.sub.5-C.sub.14)aryloxyribose, 2',3'-dideoxyribose,
2',3'-didehydroribose, 2'-deoxy-3'-haloribose,
2'-deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose,
2'-deoxy-3'-aminoribose, 2'-deoxy-3'-(C.sub.1-C.sub.6)alkylribose,
2'-deoxy-3'-(C.sub.1-C.sub.6)alkoxyribose and
2'-deoxy-3'-(C.sub.5-C.sub.14)aryloxyribose. One or more of the
pentose carbons of a nucleoside may be substituted with a phosphate
ester, as disclosed in, for example, U.S. Pat. No. 7,255,994. In
certain embodiments, the nucleosides are those in which the
nucleotide base is a purine, a 7-deazapurine, a pyrimidine, a
universal nucleotide base, a specific nucleotide base, or an analog
thereof. Nucleotide analogs include derivatives in which the
pentose sugar and/or the nucleotide base and/or one or more of the
phosphate esters of a nucleoside may be replaced with its
respective analog. Exemplary pentose sugar analogs and nucleotide
base analog are described above. Exemplary phosphate ester analogs
include, but are not limited to, alkylphosphonates,
methylphosphonates, phosphoramidates, phosphotriesters,
phosphorothioates, phosphorodithioates, phosphoroselenoates,
phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates,
phosphoroamidates, boronophosphates, etc., and may include
associated counterions. Other nucleotide analogs are nucleotide
analog monomers which can be polymerized into polynucleotide
analogs in which the DNA/RNA phosphate ester and/or sugar phosphate
ester backbone is replaced with a different type of linkage.
Exemplary polynucleotide analogs include, but are not limited to,
peptide nucleic acids, in which the sugar phosphate backbone of the
polynucleotide is replaced by a peptide backbone. The
internucleoside linkages can be a phosphodiester linkage, although
other linkages (e.g., scissile linkages which can be substantially
cleaved under conditions in which phosphodiester linkages are not
substantially cleaved) can be used. For example, a linkage that
contains an AP endonuclease sensitive site, for example an abasic
residue, a residue containing a damaged base that is a substrate
for removal by a DNA glycosylase, or another residue or linkage
that is a substrate for cleavage by an AP endonuclease, or a
disaccharide nucleoside.
[0026] As used herein, the term "oligonucleotide" ("oligo") or
"polynucleotide" may refer to an oligomer of nucleotides or
derivatives thereof. Polynucleotides include double- and
single-stranded DNA, as well as double- and single-stranded RNA,
DNA:RNA hybrids, peptide-nucleic acids (PNAs) and hybrids between
PNAs and DNA or RNA, and also include known types of modifications,
for example, labels which are known in the art, methylation,
"caps," substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoramidates, carbonates,
etc.), with negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters),
those containing pendant moieties, such as, for example, proteins
(including nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide or oligonucleotide. The oligomers may also include
modified bases, and/or backbones (e.g., modified phosphate linkage
or modified sugar moiety). Non-limiting examples of synthetic
backbones that confer stability and/or other advantages to the
oligomers may include phosphorothioate linkages, peptide nucleic
acid, locked nucleic acid (Singh, et al. Chem Commum 4:455-456
(1998)), xylose nucleic acid, and/or analogues thereof. In other
cases, the polynucleotide can contain non-nucleotidic backbones,
for example, polyamide (e.g., peptide nucleic acids (PNAs)) and
polymorpholino (commercially available from the Anti-Virals, Inc.,
Corvallis, Oreg., as Neugene.TM. polymers), and other synthetic
sequence-specific nucleic acid polymers providing that the polymers
contain nucleobases in a configuration which allows for base
pairing and base stacking, such as is found in DNA and RNA.
[0027] Oligonucleotides and/or polynucleotides may be any length
"n." For example, n may be any of 1, 2, 4, 6, 8, 12, 16, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40 etc. number of nucleotides. The
polynucleotide structure (N).sub.n represents an oligonucleotide
consisting of n number of nucleotides N (e.g., (I).sub.8 is
representative of an oligonucleotide having the sequence IIIIIIII;
or (A).sub.12 is representative of an oligonucleotide having the
sequence AAAAAAAAAAAA). Other types of oligonucleotides or
polynucleotides may also be suitable for use as would be understood
to one of skill in the art from this disclosure.
[0028] Oligonucleotides and/or polynucleotides may optionally be
attached to one or more non-nucleotide moieties such as labels and
other small molecules, large molecules such proteins, lipids,
sugars, and solid or semi-solid supports, for example through
either the 5' or 3' end. Labels include any moiety that is
detectable using a detection method of choice, and thus renders the
attached nucleotide or polynucleotide similarly detectable using a
detection method of choice (e.g., using a SGC and/or detectable
label). Optionally, the label emits electromagnetic radiation that
is optically detectable or visible. In some cases, the nucleotide
or polynucleotide is not attached to a label, and the presence of
the nucleotide or polynucleotide is directly detected.
[0029] As used herein, the term "nucleic acid" refers to polymers
of nucleotides or derivatives thereof. As used herein, the term
"target nucleic acid" refers to a nucleic acid that is desired to
be amplified in a nucleic acid amplification reaction. For example,
the target nucleic acid comprises a nucleic acid template. In some
embodiments, the target nucleic acid may be the product of the
ligation of at least two oligonucleotides to one another.
[0030] As used herein, the term "sequence" refers to a nucleotide
sequence of an oligonucleotide or a nucleic acid. Throughout the
specification, whenever an oligonucleotide/nucleic acid is
represented by a sequence of letters, the nucleotides are in 5' to
3' order from left to right. For example, if the polynucleotide
contains bases Adenine, Guanine, Cytosine, Thymine, or Uracil, the
polynucleotide sequence can be represented by a corresponding
succession of letters A, G, C, T, or U), e.g., a DNA or RNA
molecule. And, an oligonucleotide represented by a sequence
(I).sub.n(A).sub.n wherein n=1, 2, 3, 4 and so on, represents an
oligonucleotide where the 5' terminal nucleotide(s) is inosine and
the 3' terminal nucleotide(s) is adenosine.
[0031] Oligonucleotides and/or polynucleotides can optionally be
regarded as having "complementary" sequences if the same may
hybridize to one another. The term "hybridization" typically refers
to the process by which oligonucleotides and/or polynucleotides
become hybridized to each other. The adjectival term "hybridized"
refers to two polynucleotides which are bonded to each other by two
or more sequentially adjacent base pairings. Typically, these terms
refer to "specific hybridization". Two oligonucleotides and/or
polynucleotides may selectively (or specifically) hybridize to each
other if they bind significantly or detectably to each other under
stringent hybridization conditions when present in a complex
polynucleotide mixture such as total cellular or library DNA. In
some embodiments, for selective or specific hybridization, a
positive signal is at least two times background, preferably 10
times background hybridization. Optionally, stringent conditions
are selected to be about 5-10.degree. C. lower than the thermal
melting point for the specific sequence at a defined ionic strength
pH. Stringent conditions are optionally in which the salt
concentration is less than about 1.0 M sodium ion, typically about
0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0
to 8.3 and the temperature is at least about 30.degree. C. for
short probes (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C. Nucleic acids that do
not hybridize to each other under stringent conditions are still
substantially identical if the polypeptides which they encode are
substantially identical. "Nonspecific hybridization" is used to
refer to any unintended or insignificant hybridization, for example
hybridization to an unintended polynucleotide sequence other than
the intended target polynucleotide sequence. The unintended
polynucleotide sequence can be on the same or different
polynucleotide from the intended target. In some cases, the only
intended hybridization can be from Watson-Crick base pairing
between two polynucleotides. Other kinds of intended base pairings
can include base pairing between corresponding analogs of such
nucleotides or between iso-cytidine and iso-guanine. In some cases
where hybridization is only intended between complementary bases,
any bonding between non-complementary bases is considered to be
non-specific hybridization.
[0032] In some embodiments, complementary sequences may be those
that, when hybridized together, may be efficiently ligated to a
third polynucleotide that has hybridized adjacently to it.
Similarly, nucleotide residues can be regarded as complementary if
when both are base-paired with each other within two hybridized
polynucleotides, either nucleotide can be ligated in a
template-driven ligation reaction when situated as the terminal
nucleotide in its polynucleotide. Nucleotides that are efficiently
incorporated by DNA polymerases opposite each other during DNA
replication under physiological conditions are also considered
complementary. In an embodiment, complementary nucleotides can form
base pairs with each other, such as the A-T/U and G-C base pairs
formed through specific Watson-Crick type hydrogen bonding between
the nucleobases of nucleotides and/or polynucleotides positions
antiparallel to each other. The complementarity of other artificial
base pairs can be based on other types of hydrogen bonding and/or
hydrophobicity of bases and/or shape complementarity between bases.
In appropriate instances, polynucleotides can be regarded as
complementary when the same may undergo cumulative base pairing at
two or more individual corresponding positions in antiparallel
orientation, as in a hybridized duplex. Optionally there can be
"complete" or "total" complementarity between a first and second
polynucleotide sequence where each nucleotide in the first
polynucleotide sequence can undergo a stabilizing base pairing
interaction with a nucleotide in the corresponding antiparallel
position on the second polynucleotide. "Partial" complementarity
describes polynucleotide sequences in which at least 20%, but less
than 100%, of the residues of one polynucleotide are complementary
to residues in the other polynucleotide. A "mismatch" is present at
any position in the two opposed nucleotides that are not
complementary. In some ligation assays, a polynucleotide can
undergo substantial template-dependent ligation even when it has
one or more mismatches to its hybridized template. Optionally, the
polynucleotide has no more than 4, 3, or 2 mismatches, e.g., 0 or 1
mismatch, with its template. In some assays, the polynucleotide
will not undergo substantial template-dependent ligation unless it
is at least 60% complementary, e.g., at least about 70%, 80%, 85%,
90%, 95% or 100% complementary to its template.
[0033] "Degenerate", with respect to a position in a polynucleotide
that is one of a population of polynucleotides, means that the
identity of the base of the nucleoside occupying that position
varies among different members of the population. A population of
polynucleotides in this context is optionally a mixture of
polynucleotides within a single continuous phase (e.g., a fluid).
The "position" can be designated by a numerical value assigned to
one or more nucleotides in a polynucleotide, generally with respect
to the 5' or 3' end. For example, the terminal nucleotide at the 3'
end of an extension probe may be assigned position 1. Thus in a
pool of extension probes of structure 3'-XXXNXXXX-5', the N is at
position 4. A position is said to be k-fold degenerate if it can be
occupied by nucleosides having any of k different identities. For
example, a position that can be occupied by nucleosides comprising
either of 2 different bases is 2-fold degenerate.
[0034] A "solid support", as used herein, typically refers to a
structure or matrix on or in which ligation and/or amplification
reagents (e.g., nucleic acid molecules, microparticles, and/or the
like) may be immobilized so that they are significantly or entirely
prevented from diffusing freely or moving with respect to one
another. The reagents can for example be placed in contact with the
support, and optionally covalently or noncovalently attached or
partially/completely embedded. The terms "microparticle," "beads"
"microbeads", etc., refer to particles (optionally but not
necessarily spherical in shape) having a smallest cross-sectional
length (e.g., diameter) of 50 microns or less, preferably 10
microns or less, 3 microns or less, approximately 1 micron or less,
approximately 0.5 microns or less, e.g., approximately 0.1, 0.2,
0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer, about
1-10 nanometer, about 10-100 nanometers, or about 100-500
nanometers). Microparticles (e.g., Dynabeads from Dynal, Oslo,
Norway) may be made of a variety of inorganic or organic materials
including, but not limited to, glass (e.g., controlled pore glass),
silica, zirconia, cross-linked polystyrene, polyacrylate,
polymethylmethacrylate, titanium dioxide, latex, polystyrene, etc.
Magnetization can facilitate collection and concentration of the
microparticle-attached reagents (e.g., polynucleotides or ligases)
after amplification, and facilitates additional steps (e.g.,
washes, reagent removal, etc.). In certain embodiments of the
invention a population of microparticles having different shapes
sizes and/or colors can be used. The microparticles can optionally
be encoded, e.g., with quantum dots such that each microparticle
can be individually or uniquely identified.
[0035] As used herein the term "reaction mixture" refers to the
combination of reagents or reagent solutions, which are used to
carry out a chemical analysis or a biological assay. In some
embodiments, the reaction mixture comprises all necessary
components to carry out a nucleic acid (DNA)
synthesis/amplification reaction. As described above, such reaction
mixtures may include at least one amplification primer pair
suitable for amplifying a nucleic acid sequence of interest (e.g.,
target nucleic acid). As described above, a suitable reaction
mixture may also include a "master mix" containing the components
(e.g., typically not including the primer pair) needed to perform
an amplification reaction (e.g., detergent, magnesium, buffer
components, etc.). Other embodiments of reaction mixtures are also
contemplated herein as would be understood by one of skill in the
art.
[0036] As used herein, the terms "reagent solution" or "solution
suitable for performing a DNA synthesis reaction" refer to any or
all solutions, which are typically used to perform an amplification
reaction or DNA synthesis. They include, but are not limited to,
solutions used in DNA amplification methods, solutions used in PCR
amplification reactions, or the like. The solution suitable for DNA
synthesis reaction may comprise buffer, salts, and/or nucleotides.
It may further comprise primers and/or DNA templates to be
amplified. One or more reagent solutions are typically included in
the reactions mixtures or master mixes described herein.
[0037] As used herein, the term "primer" or "primer sequence"
refers to a short linear oligonucleotide that hybridizes to a
target nucleic acid sequence (e.g., a DNA template to be amplified)
to prime a nucleic acid synthesis reaction. The primer may be a RNA
oligonucleotide, a DNA oligonucleotide, or a chimeric sequence
(e.g., comprising RNA and DNA). The primer may contain natural,
synthetic, or modified nucleotides. Both the upper and lower limits
of the length of the primer are empirically determined. The lower
limit on primer length is the minimum length that is required to
form a stable duplex upon hybridization with the target nucleic
acid under nucleic acid amplification reaction conditions. Very
short primers (usually less than 3 nucleotides long) do not form
thermodynamically stable duplexes with target nucleic acid under
such hybridization conditions. The upper limit is often determined
by the possibility of having a duplex formation in a region other
than the pre-determined nucleic acid sequence in the target nucleic
acid. Generally, suitable primer lengths are in the range of about
any of, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, (and so on) nucleotides in
length.
[0038] In some embodiments, the terms "probe(s)",
"oligonucleotide(s)" and/or "primer(s)" may be interchangeable
terms herein, so that any one of these may be taken as a reference
to another. The terms "polynucleotide," "oligonucleotide", "probe",
"primer", "template", "nucleic acid" and the like may be taken to
refer to a populations or pools of individual molecules that are
substantially identical across their entire length or across a
relevant portion of interest. For example, the term "template" may
indicate a plurality of template molecules that are substantially
identical, etc. In the case of polynucleotides that are degenerate
at one or more positions, it will be appreciated that the
degenerate polynucleotide may comprise a plurality of
polynucleotide molecules, which have sequences that are
substantially identical only at the nondegenerate position(s) and
differ in sequence at the degenerate positions. Thus, reference to
"a" polynucleotide (e.g., "a" primer, probe, oligonucleotide,
template, etc.) may be taken to mean a population of substantially
identical polynucleotide molecules, such that the plural nature of
a population of substantially identical nucleic acid molecules need
not be explicitly indicated, but may if so desired. These terms are
also intended to provide adequate support for a claim that
explicitly specifies a single polynucleotide molecule itself.
[0039] "Ligation" involves the formation of a covalent bond or
linkage between the termini of two or more nucleic acids, e.g.
oligonucleotides and/or polynucleotides, optionally in a
template-driven reaction. Exemplary ligations may be carried out
enzymatically to form a phosphodiester linkage between a 5' carbon
of a terminal nucleotide of one oligonucleotide with 3' carbon of
another oligonucleotide (e.g., using a ligase). The nature of the
bond or linkage may vary widely and the ligation is preferably
achieved enzymatically. The efficiency of ligation refers to the
rate of ligation. Where the relative efficiency of ligation is
specified in comparative or relative terms by comparison to a
reference ligation assay, it is implicit that all other reagents
and conditions (e.g., temperature, concentration of all reagents,
pH, concentration of requisite ions such as Mg++ and Mn++,
concentration of requisite cofactors such as NAD and/or ATP, salts,
buffers, molar concentrations of all reagents, including enzyme,
template, probe primer, oligonucleotides, etc) are otherwise kept
identical. For example, a proviso that a ligase (e.g., an SFL (see
below)) can ligate a short (e.g., less than 6 nucleotides) probe at
least X % (e.g., where X is 100 or less; 100, 99, 95, 90, 85, 80,
75, 70, 50, 25, 10 1, 0.5, 0.1, etc., or any increment in between)
as efficiently as the ligase can ligate a corresponding
octanucleotide, may be understood to mean that the rate of ligation
of the shorter probe occurs at a rate that is at least X % of the
rate of ligation of the octanucleotide, where all reagents except
for the probes (e.g., primer, template, enzymes and any other
reagents) and all reaction conditions (e.g., temperature, reagent
concentrations, concentrations of any other reagents, etc) are kept
invariant for practical purposes. It is understood that ligation
efficiency in absolute or relative terms may increase or decrease
depending on the exact reaction conditions used.
[0040] Optionally, ligation is performed under in-vitro conditions
that have been experimentally determined to be suitable or optimal
for ligase activity. Preferably, reaction conditions are kept
substantially similar to in-vivo or physiological conditions in
which a naturally-occurring form of the ligase being used is
naturally active. Most preferably, the reaction conditions for a
particular ligase are matched as closely as possible to exemplary
in vitro ligation conditions described herein for that ligase. In
other embodiments, the conditions are such that the reference
ligation assay produces significant or detectable ligation within
30 minutes, within 10 minutes, within 1 minute, or within ten
seconds. Another non-limiting example of a significant or
detectable rate of ligation generates in the range of 100 pM of
ligation product, optionally about 1000 pM or 10,000 pM, in an
appropriate amount of time (e.g., 10 minutes.)
[0041] Along similar lines, it should be understood that a
statement that a result has occurred (e.g., ligation, binding) is
intended to indicate that the result has occurred at a significant
or substantial level or an enhanced level compared to when it has
not occurred. For example, ligation is said to have not occurred if
it is not significant, insubstantial or greatly reduced (e.g.,
reduced by at least 80%, 90%, 95% or 99% compared to when ligation
does occur (e.g., under the conditions described in the last
paragraph). In reference to ligation of two polynucleotides, the
"proximal" terminus of either polynucleotide is other terminus that
is intended to be ligated to the other polynucleotide. It is
generally the terminus that is closer to the other polynucleotide,
or the terminus that is contacted by the active site of the ligase,
or other terminus that is eventually ligated to the other
polynucleotide, while the opposite terminus is the "distal"
terminus. The terminal nucleotide residue at the proximal terminus
can be termed the proximal nucleotide, and the proximal nucleotide
position optionally designated as position 1, the penultimate
nucleotide position as position 2, etc. In some non-limiting
instances of template-dependent ligation, the proximal termini of
both polynucleotides are hybridized adjacently to each other.
[0042] An exemplary type of enzymatic ligation (double-stranded
ligation) includes the formation of a covalent bond between
nucleotides of a polynucleotide (e.g., resulting in
circularization) or between two or more polynucleotides (e.g., a
first double-stranded terminus of a first polynucleotide and a
second different double-stranded terminus of a second
polynucleotide). The polynucleotides may be different, or may be
the same. Polynucleotides may also be ligated using a "splint"
oligonucleotide which may be used to link nucleotides that the user
desires to ligate (e.g., on the same or different polynucleotides).
Optionally, the ends of both double-stranded termini may be joined
irrespective of their sequences (e.g., blunt-end ligation, or
non-homologous end joining).
[0043] In another variation, two double-stranded polynucleotides
with protruding single-stranded ends that are complementary to each
other can be ligated (e.g., cohesive-end ligation). In other
instances, the ligation can ligate two single-stranded
polynucleotides, either or both of which has optionally hybridized
(annealed) to another nucleotide sequence. In template-dependent
ligation, ligation between a first polynucleotide and a second
polynucleotide occurs upon hybridization of at least a portion of
either or both polynucleotides to a target sequence. The target
sequence can be a portion of either polynucleotide (e.g.,
self-hybridization or hybridization to each other) or to a sequence
on a third different polynucleotide (e.g., a "splint"
oligonucleotide). The hybridized portion of the polynucleotide may
be, for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 or 20
nucleotides long. The hybridized portion is optionally a terminal
portion of the nucleotide (e.g., includes the 5' or 3' nucleotide).
For example, the hybridized portion can consist of the 5' or 3'
terminal nucleotide, or the terminal 2, 3, 4, 5, 6, 7, 8, 10, 15 or
20 nucleotides of the 5' or 3' end. Optionally, ligation occurs
when no mismatch is present within the hybridized portions.
[0044] In other cases, ligation occurs when one, two or three
mismatches can be present within the hybridized portion. In some
cases ligation does not occur when the terminal nucleotide and/or
second-most terminal nucleotide and/or third-most terminal
nucleotide is mismatched. As mentioned, the terminal nucleotides
can be the 5'- or 3'-terminal nucleotides of the polynucleotide. An
exemplary type of assay makes use of template-dependent ligation
between a first single-stranded polynucleotide and a second
single-stranded polynucleotide, where ligation can be effected when
either or both polynucleotides is/are hybridized to a third
different single-stranded polynucleotide. In some instances, both
probes must hybridize to the template for significant ligation to
occur. For ease of reference, the first polynucleotide is called
the "initializing probe," the second polynucleotide called the
"extension probe" and the third polynucleotide called the
"template."
[0045] In some variations, (e.g., "nick ligation"), both probes
must hybridize adjacently to each other on the template for
ligation to occur. In some assays, the probes are adjacently
hybridized and can be ligated only when a terminal nucleotide of
the initializing probe is hybridized to a first nucleotide of the
template and a terminal nucleotide of the extension probe is
hybridized to a second nucleotide of the template, where the first
and second nucleotides on the template are not separated by an
intervening nucleotide of the template. In other embodiments, a few
intervening nucleotides may be present between the first and second
nucleotides on the template (e.g., 1, 2, 3 or more nucleotides). In
such embodiments, a "gap-filling" step can be performed to extend
the 3' terminus of one probe before it can be ligated to the 5'
terminus of the other probe.
[0046] In the methods described herein, the terminal nucleotide of
the initializing probe can be the 5' terminal nucleotide and the
terminal nucleotide of the extension probe can be the 3' terminal
nucleotide. Alternatively, the terminal nucleotide of the
initializing probe can be the 3' terminal nucleotide and the
terminal nucleotide of the extension probe can be the 5' terminal
nucleotide. The ligation product of any one reaction can optionally
be subjected to further ligation and/or non-ligation reactions in
turn. For example, the ligation product can be used as the
initializing probe or extension probe or template in a subsequent
ligation. Also for example, it can be used as a template or primer
for polymerase extension, such as in polymerase chain reaction
(PCR). It can be cleaved enzymatically or chemically (for example
when it has scissile linkages), treated with exo- or endonucleases,
kinases, phosphatases, etc. The ends of a double-stranded product
can be blunt-ended or filled in, capped, or adenylated, etc.
[0047] As used herein, "splint oligonucleotide," "splint oligo," or
"connector" refers to an oligonucleotide that is used to provide an
annealing site or a "ligation template" for joining two ends of a
nucleic acid molecule or molecules 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
the 3'-end of a first probe oligo (oligo A) to the 5'-end of a
second probe oligo, the ligation splint oligo has a sequence
complementary to the 3'-end of oligo A (e.g., oligo tail sequence,
and a second neighboring sequence (e.g., an adjacent sequence) that
is complementary to the 5'-end of oligo B (FIG. 4).
[0048] In some embodiments of the improved PLA processes splint
oligos can be either symmetrical or asymmetrical depending on the
number of nucleotides that hybridize to each of the two oligo
probes it is connecting or ligating. FIG. 4 diagrams asymmetrical
and symmetrical splint types for use in the improved PLA processes
as described herein. In some embodiments, asymmetrical splints (or
"connectors") span across the two separate oligo probes (e.g.,
probe oligo A and B) with one of the ends of the splint (e.g.,
either the 3'-end or the 5'-end) having more nucleotides that
hybridize to one of the probe oligos than the other end of the
splint has nucleotides that hybridize to the alternative probe
oligo (FIG. 4A). In other embodiments, symmetrical splints span
across the two separate oligo probes (e.g., probe oligo A and B)
with both ends of the splint (e.g., the 3' end and the 5' end)
having equal number of nucleotides that hybridize to each of the
two probe oligos (FIG. 4BA).
[0049] Both asymmetrical and symmetrical splints can have any
number of intervening nucleotides between each of it's 3' and 5'
ends that hybridize to the separate probe oligos. Alternatively,
there may be no intervening nucleotides between each of the 3' and
5' ends that hybridize to the probe oligos. In preferred
embodiments, splint oligonucleotides (oligos) are at least 6
nucleotides long (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more). In certain other embodiments, each of the 3'-
or 5'-ends of the splint oligo will comprise at least 3 (e.g., 3,
4, 5, 6, 7, 8, 9, 10 or more) nucleotides that separately hybridize
to (or "overlap") an oligo probe, herein referred to as the
"overhang" region.
[0050] In some embodiments of the improved PLA processes the splint
oligo is blocked at it's 3'-end. The blocking agent can be any
covalently connected moiety that prevents polymerase activity. This
3' blocked splint oligo is then prevented from interfering with the
PCR reaction part of the improved PLA process. In some embodiments,
for example, the 3' blocking agent can include, but is not limited
to, 3'-fluoro-, 3'-bromo-, 3'-iodo-, 3'-deoxy-, 3'-methyl-,
3'-methoxy, 3'-phosphate, 3'-aminolink, 3'-abasic amidite, or any
other 3' modification groups. Those of ordinary skill in the art
would be able to further contemplate other blocking agents for use
as disclosed herein.
[0051] Any one or more of the ligation methods provided herein can
be used in a ligation assay. Non-limiting example of ligation
assays include a oligonucleotide ligation assay (OLA), a ligase
chain reaction (LCR), a ligase detection reaction (LDR) and
combination assays such as the OLA coupled with the polymerase
chain reaction (PCR), e.g., OLA-PCR and PCR-OLA, the Combined Chain
Reaction (CCR; a combination of PCR and LCR) and PCR-LDR (see,
e.g., Landegren et al., Science 241:1077-80, 1988; Barany, Proc.
Natl. Acad. Sci. 88:189-93, 1991; Grossman et al., Nucl. Acids Res.
22(21):4527-34, 1994; Bi and Stambrook, Nucl. Acids Res.
25(14):2949-51, 1997; Zirvi et al., Nucl. Acids Res., 27(24):e40,
1999; U.S. Pat. No. 4,988,617; and PCT Publication Nos. WO 97/31256
and WO 01/92579. Such assays have been used for single nucleotide
polymorphism (SNP) analysis, SNP genotyping, mutation detection,
identification of single copy genes, detecting microsatellite
repeat sequences, and DNA adduct mapping, among other things. See
also Whitely et al, U.S. Pat. No. 4,883,750; Letsinger et al, U.S.
Pat. No. 5,476,930; Fung et al, U.S. Pat. No. 5,593,826; Kool, U.S.
Pat. No. 5,426,180; Landegren et al, U.S. Pat. No. 5,871,921; Xu
and Kool, Nucleic Acids Research, 27: 875-881 (1999); Higgins et
al, Methods in Enzymology, 68: 50-71 (1979); Engler et al, The
Enzymes, 15-3-29 (1982); and Namsaraev, U.S. Pat. Pub.
2004/0110213. The fidelity of several known ligases, based on for
example the evaluation of mismatch ligation or ligation rates, has
been reported. For example, the NAD+-dependent ligase from the
hyperthermophilic bacteria Aquifex aeolicus reportedly generates
detectable 3' misligation products with C:A, T:G, and G:T
mismatches (Tong et al., Nucl. Acids Res. 28(6):1447-54, 2000); a
partially purified preparation of bovine DNA ligase III reportedly
generated detectable 3' misligation products with C:T, G:T, and T:G
mismatches, while human ligase I generated detectable 3'
misligation products with C:T and G:T mismatches, but not T:G
mismatches (Husain et al., J. Biol. Chem. 270(16):9683-90, 1995);
and the DNA ligase from the thermophilic bacteria Thermus
thermophilus (Tth) reportedly generates detectable levels of 3'
misligation products with T:G and G:T mismatches (Luo et al., Nucl.
Acids Res. 24(14):3071-78, 1996). Bacteriophage T4 DNA ligase
reportedly generates detectable misligation products with a wide
range of mismatched substrates and appears to have lower fidelity
than Thermus species ligases by at least one to two orders of
magnitude (Landegren et al., Science 241:1077-80, 1988; Tong et
al., Nucl. Acids Res. 27(3):788-94, 1999).
[0052] A particularly useful assay is the oligonucleotide ligation
assay (OLA). The OLA is a convenient, highly-stringent method that
permits distinction among known DNA sequence variants (Landegren,
1988). For instance, multiplex analysis of highly polymorphic loci
is useful for identification of individuals, e.g., for paternity
testing and in forensic science, organ transplant donor-receiver
matching, genetic disease diagnosis, prognosis, and pre-natal
counseling, and other genetic-based testing which depend on the
discrimination of single-base differences at a multiplicity of loci
(Delahunty, 1996). Products of a multiplex OLA may be resolved
electrophoretically from one another and from unligated probes
under denaturing conditions with fluorescence detection (Grossman,
1994). For example, two PNA-DNA chimeras, a wild-type (WT) sequence
chimera and a mutant sequence chimera, may bear different
fluorescent dyes. Only when the mutant sequence is present in the
target sample, will the mutant sequence chimera ligate to the
adjacently annealed second probe (oligo) if the mutant base pair is
at the ligation site. The ligation products may be discriminated by
separation based on: (i) size using electrophoresis or
chromatography and/or (ii) detectable labels (Grossman, 1994). With
a plurality of fluorescent dyes labeled to chimeras with sequences
targeting unique target sequences, multiplexed OLA can be conducted
on a single sample in a single vessel. Requirements for efficient
multiplex OLA include probes that anneal and ligate in a highly
specific and rapid manner. The chimeras and second probe sequences
may be selected such that the mutant base, or single base
polymorphism, may be at the 5'-phosphate of the second probe or the
3'-terminus of the chimera. It is contemplated that OLA experiments
of the present invention may be conducted on solid supports where
the template nucleic acid, PNA-DNA chimeric probe, or the second
probe may be immobilized on a solid particle or bead, or a solid
porous or non-porous surface. When immobilized, the template,
chimera or second probe is preferably covalently attached to the
solid substrate, e.g. via a terminal monomer unit. The solid
substrate may be polystyrene, controlled-pore-glass, silica gel,
silica, polyacrylamide, magnetic beads, polyacrylate,
hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy,
and copolymers and grafts of any of the above solid substrates. The
configuration or format of the solid substrate may be small
particles or beads of approximately 1 to 50 .mu.m in diameter,
membranes, frits, slides, plates, micromachined chips,
alkanethiol-gold layers, non-porous surfaces, and
polynucleotide-immobilizing media.
[0053] As described above, enzymatic ligation is typically
accomplished using a ligase, which may be a polypeptide. Suitable
ligases include, for example, nucleic acid ligase, oligonucleotide
ligase, DNA ligase, RNA ligase, and the like. Suitable RNA ligases
include those described or used in, for example, any of U.S. Pat.
Nos. 4,582,802; 5,665,545; 6,194,637; 6,444,429; 6,455,274;
6,576,453; 6,635,425; 6,855,523; 7,811,753; and the like, and/or
any of U.S. Pat. Pubs. 2004/0171047A1, 2004/0191871A1,
US20050266487A1, 2006/0223098A1, 2007/0037190A1, 20080160526A1;
2009/0061481A1, 2010/0099683A1, and/or 2010/0184618A1; and the
like, all of which are incorporated by reference in their entirety
into this application. Exemplary DNA ligases may include, for
example, T3 DNA ligase, T4 DNA ligase, T5 DNA ligase, T7 DNA
ligase, vaccinia virus DNA ligase, E. coli DNA ligase, mammalian
DNA ligase I, mammalian DNA ligase II, mammalian DNA ligase III,
Tth DNA ligase, KOD DNA ligase, a thermostable DNA ligase, and/or
derivatives, fragments, and/or combinations thereof. Suitable RNA
ligases include those described or used in, for example, any of
U.S. Pat. Nos. 4,661,450; 5,516,664; 5,602,000; 5,807,674;
6,368,801; 6,492,161; 6,635,453; and the like, or any of U.S. Pat.
Pub. Nos. 2003/0082536A1; 2004/0058330A1; 2005/0266439A1;
2005/0074774A1; 2008/0045418A1; 2010/00159526A1; and the like, all
of which are incorporated by reference in their entirety into this
application. Exemplary RNA ligases may include, for example, T4 RNA
ligase, bacteriophage RB69 RNA ligase, Autographa californica
nuclearpolyhedrosis virus RNA ligase, a thermophilic RNA ligase,
bacteriophage RM378 RNA ligase, bacteriophage TS2126 RNA ligase,
and/or derivatives, fragments, and/or combinations thereof.
[0054] In some embodiments, the ligase is a "small footprint
ligase" (SFL). A SFL has the ability to ligate short
polynucleotides (e.g., at least about 3 nucleotides). As described
herein, a SFL may ligate oligonucleotides having a connector oligo
length of as short as 3 base of hybridized DNA adjacent to
5'-phosphate hybridized DNA. In some embodiments, the SFL may be
used to ligate oligonucleotides comprising short overlap sequences
(e.g., short connector oligo length). For instance, the SFL may be
used to ligate oligonucleotides of various nucleotides in length,
whereby each oligo has at least a 3 nucleotide overlap with the
splint oligo. Typical ligases would be better suited for ligating
longer oligonucleotides (e.g., comprising 9 or more nucleotides;
comprising 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, etc nucleotides).
In this way, oligonucleotide concentration may also be reduced to
minimize chance of solution hybridization promoted
non-antigen-binding ligation. For combining the ligation and PCR
reaction into one step, ATP (cofactor for the ligase) can be
omitted from the reaction mixture. In order to maintain the ligase
function, the SFL may be pre-enriched with ATP prior to its
purification and use.
[0055] A SFL may be a naturally occurring or non-naturally
occurring (e.g., artificial, synthetic) ligase. A SFL can comprise
a polypeptide sequence that is homologous to or a variant of a
known ligase sequence or any portion thereof. Exemplary SFLs can
have amino acid sequence identity of at least 70%, optionally at
least 85%, optionally at least 90 or 95%, with a known ligase, and
possesses one or more functional activities of a ligase. A SFL can
thus comprise a polypeptide having any one or more of the following
activities: (1) nucleophilic attack on ATP or NAD resulting in
release of PPi or NMN and formation of a covalent ligase-adenylate
intermediate; (2) transferring the adenylate to the 5'-end of the
5'-phosphate-terminated DNA strand to form DNA-adenylate (e.g., the
5'-phosphate oxygen of the DNA strand attacks the phosphorus of
ligase-adenylate); and, (3) formation of a covalent bond joining
the polynucleotide termini and liberation of AMP (e.g., by the
attack by the 3'-OH on DNA-adenylate). Optionally, the SFL can
mediate any one or more of the following bond transformations: from
phosphoanhydride (ATP) to phosphoramidate (ligase-adenylate); from
phosphoramidate (ligase-adenylate) to phosphoanhydride
(DNA-adenylate); and/or from phosphoanhydride (DNA-adenylate) to
phosphodiester (sealed DNA). The SFL in one aspect is an enzyme
that can mediate the formation of a covalent bond between two
polynucleotide termini, e.g., a 3'-OH terminus and a 5'-PO.sub.4
terminus are joined together to form a phosphodiester bond. In some
instances, DNA ligation entails any one or more of three sequential
nucleotidyl transfer steps, discussed below. All three chemical
steps depend on a divalent cation cofactor. In one aspect, the SFL
is an ATP-dependent ligase or a NAD.sup.+-dependent ligase.
[0056] For example, the SFL can comprise any one or more domains
characteristic of a ligase (e.g., an N-terminal
nucleotidyltransferase (NTase) domain and/or a C-terminal OB
domain). The OB domain optionally comprises a five-stranded
antiparallel beta-barrel plus an alpha-helix. Within the NTase
domain is an adenylate-binding pocket composed of the six peptide
motifs that define the covalent NTase enzyme family of
polynucleotide ligases. Optionally, the NTase domain can comprise
any one or more of the ligase amino acid motifs I, III, IIIa, IV,
and/or V, and preferably all six motifs. Motif I (e.g., KxDGxR or a
"KXDG" motif) optionally contains a lysine. Exemplary sequences for
each motif in CV ligase are ATPKIDGIR (motif I) (SEQ ID NO.: 1),
SRT (motif Ia), EGSDGEIS (motif III) (SEQ ID NO.: 2), YWFDY (motif
Ma) (SEQ ID NO.: 3), EGVMIR (motif IV) (SEQ ID NO.: 4), LLKMK
(motif V) (SEQ ID NO.:5). Motif 1 pfy contains a lysine residue.
Other examples of motif I include CELKLDGLA (SEQ ID NO.: 6),
VEHKVDGLS (SEQ ID NO.: 7), CEPKLDGLA (SEQ ID NO.: 8), CELKLDGVA
(SEQ ID NO.: 9), AEIKYDGVR (SEQ ID NO.: 10), CEYKYDGQR (SEQ ID NO.:
11), VDYKYDGER (SEQ ID NO.: 12), FEIKYDGAR (SEQ ID NO.: 13),
FEGKWDGYR (SEQ ID NO.: 14), AREKIHGTN (SEQ ID NO.: 15), ACEKVHGTN
(SEQ ID NO.: 16), ILTKEDGSL (SEQ ID NO.: 17), and VEEKVDGYN (SEQ ID
NO.: 18). Examples of motif Ia include TRG, SRT, SRR, SRN, SRS,
KRT, KRS, SKG and TRG. Examples of motif III include LEVRGEVF (SEQ
ID NO.: 19), VEVRGECY (SEQ ID NO.: 20), LEVRGEVY (SEQ ID NO.: 21),
LEARGEAF (SEQ ID NO.: 22), FMLDGELM (SEQ ID NO.: 23), EGSDGEIS (SEQ
ID NO.: 24), FILDTEAV (SEQ ID NO.: 25), FIIEGEIV (SEQ ID NO.: 26),
AIVEGELV (SEQ ID NO.: 27), VVLDGEAV (SEQ ID NO.: 28), YQVFGEFA (SEQ
ID NO.: 29), LVLNGELF (SEQ ID NO.: 30), FTANFEFV (SEQ ID NO.: 31)
and LILVGEMA (SEQ ID NO.: 32). Examples of motif Ma include FCYGV
(SEQ ID NO.: 33), FLYTV (SEQ ID NO.: 34), TFYAL (SEQ ID NO.: 35),
ICHGL (SEQ ID NO.: 36), NAYGI (SEQ ID NO.: 37), FVYGL (SEQ ID NO.:
38), KLYAI (SEQ ID NO.: 39), YWFDY (SEQ ID NO.: 40), YAFDI (SEQ ID
NO.: 41), FLFDL (SEQ ID NO.: 42), NLFDV (SEQ ID NO.: 43), WAFDL
(SEQ ID NO.: 44), YVFDI (SEQ ID NO.: 45), FAFDI (SEQ ID NO.: 46),
ILLNA (SEQ ID NO.: 47), and FLFDV (SEQ ID NO.: 48). Examples of
motif IV include DGVVIK (SEQ ID NO.: 49), DGIVIK (SEQ ID NO.: 50),
DGVVVK (SEQ ID NO.: 51), DGTVLK (SEQ ID NO.: 52), EGLIVK (SEQ ID
NO.: 53), EGVMIR (SEQ ID NO.: 54), EGLMVK (SEQ ID NO.: 55), EGVMVK
(SEQ ID NO.: 56), EGLMAK (SEQ ID NO.: 57), EGVIAK (SEQ ID NO.: 58),
EGYVLK (SEQ ID NO.: 59), EGVVIR (SEQ ID NO.: 60), EGYVAV (SEQ ID
NO.: 61), and EGIIMK (SEQ ID NO.: 62). Examples of motif V include
AVAFK (SEQ ID NO.: 63), AIAYK (SEQ ID NO.: 64), ALAYK (SEQ ID NO.:
65), AIAYK (SEQ ID NO.: 66), WWKMK (SEQ ID NO.: 67), LLKMK (SEQ ID
NO.: 68), WLKLK (SEQ ID NO.: 69), WIKLK (SEQ ID NO.: 70), WLKIK
(SEQ ID NO.: 71), WVKDK (SEQ ID NO.: 72), AIKCK (SEQ ID NO.: 73),
IIKLR (SEQ ID NO.: 74), HFKIK (SEQ ID NO.: 75) and IVKYV (SEQ ID
NO.: 76). The SFL optionally comprises all six motifs. Optionally
all six motifs are found together in a naturally-occurring ligase,
such as a SFL identified herein. In some embodiments, the SFL is
not an RNA-capping enzyme. The ligase optionally comprises any
functional portion of a SFL. The ligase can be homologous to a SFL
or any functional portion thereof, for example more than 75%, 85%,
90%, 95% or 99% homologous at the amino acid level. An exemplary
SFL is a Chlorella virus DNA ligase (ChVLig) (Ho, et al., J Virol,
71(3):1931-19374 (1997)) or functional fragment or variant thereof.
Representative examples of SFLs include CV ligase, DLX, DLXd, DLXd2
and MnM ligase. A preferred SFL is Chlorella Virus ligase. Some
exemplary ligases are identified and their GI or accession numbers
are provided in Table 1 below:
TABLE-US-00001 TABLE 1 PRK08224 B. Acidobacteria Bacteria;
Fibrobacteres/Acidobacteria group; Acidobacteria; unclassifed
Acidobacteria; Candidatus Koribacter; Candidatus Koribacter
versatilis Candidatus Solibacter usitatus Ellin6076Candidatus
ATP-Dependent YP_826317 Solibacter (1 proteins) DNA Ligase C.
Actinobacteria Bacteria; Actinobacteria; Actinobacteria (class);
Actinobacteridae; Actinomycetales; Corynebacterineae;
Mycobacteriaceae; Mycobacterium; Mycobacterium marinum
Mycobacterium gilvum PYR-GCKMycobacterium (26 ATP-Dependent
YP_001132524 proteins) DNA Ligase Mycobacterium vanbaalenii
PYR-1Mycobacterium (26 ATP-Dependent YP_956315 proteins) DNA Ligase
Mycobacterium sp. MCSMycobacterium (26 proteins) ATP-Dependent
YP_642076 DNA Ligase F. Chlamydiae/Verrucomicrobia Bacteria;
Chlamydiae/Verrucomicrobia group; Verrucomicrobia; Opitutae;
Opitutales; Opitutaceae; Opitutus; Opitutus terrae Opitutus terrae
PB90-1Opitutus (1 proteins) ATP-Dependent YP_001821013 DNA Ligase
Organism Protein name Accession PRK09125 O. Betaproteobacteria
Neisseria meningitidis Z2491Neisseria (7 proteins) DNA ligase
YP_002341892 Thiobacillus denitrificans ATCC 25259Thiobacillus (1
DNA ligase YP_314570 proteins) Variovorax paradoxus S110Variovorax
(1 proteins) DNA ligase YP_002944627 Verminephrobacter eiseniae
EF01-2Verminephrobacter DNA ligase YP_998235 (1 proteins) -- -- P.
Deltaproteobacteria Desulfobacterium autotrophicum LigA2
YP_002604477 HRM2Desulfobacterium (1 proteins) Myxococcus xanthus
DK 1622Myxococcus (1 DNA ligase YP_628883 proteins) -- -- Q.
Epsilonproteobacteria Campylobacter jejuni subsp. jejuni NCTC
ATP-dependent YP_002345037 11168Campylobacter (10 proteins) DNA
ligase Sulfurimonas denitrificans DSM 1251Sulfurimonas (1 DNA
ligase YP_393098 proteins) -- -- R. Gammaproteobacteria
Aggregatibacter aphrophilus NJ8700Aggregatibacter ATP-dependent
YP_003007537 (2 proteins) DNA ligase Haemophilus influenzae
PittEEHaemophilus (3 ATP-dependent YP_001290961 proteins) DNA
ligase Shewanella baltica OS195Shewanella (18 proteins)
ATP-dependent YP_001554317 DNA ligase Shewanella loihica
PV-4Shewanella (18 proteins) ATP-dependent YP_001093713 DNA ligase
Vibrio cholerae M66-2Vibrio (9 proteins) DNA ligase YP_002810248 --
-- PHA0454 b. Viruses Viruses; dsDNA viruses, no RNA stage;
Caudovirales; Podoviridae; Autographivirinae; phiKMV-like viruses
Pseudomonas phage LKD16phiKMV-like viruses (7 ATP-dependent
YP_001522807 proteins) DNA ligase CLSZ2445448 a. Eukaryota
Eukaryota; Alveolata; Ciliophora; Intramacronucleata;
Oligohymenophorea; Peniculida; Parameciidae; Paramecium; Paramecium
tetraurelia Paramecium tetraurelia strain d4-2Paramecium (5 DNA
ligase XP_001347270 proteins) PRK07636 J. Firmicutes Bacteria;
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus
clausii Bacillus subtilis subsp. subtilis str. 168Bacillus
ATP-dependent NP_389932 DNA ligase Bacteria; Firmicutes; Bacilli;
Bacillales; Bacillaceae; Geobacillus Geobacillus sp.
Y412MC10Geobacillus ATP dependent YP_003240778 DNA ligase
CLSK2551528 J. Firmicutes Bacteria; Firmicutes; Bacilli;
Bacillales; Bacillaceae; Geobacillus Geobacillus sp.
Y412MC10Geobacillus (1 proteins) ATP dependent YP_003245332 DNA
ligase -- -- CLSK2470953 C. Actinobacteria Bacteria;
Actinobacteria; Actinobacteria (class); Actinobacteridae;
Actinomycetales; Micrococcineae; Micrococcaceae; Arthrobacter;
Arthrobacter chlorophenolicus Arthrobacter chlorophenolicus A6 ATP
dependent YP_002478427 (plasmid)Arthrobacter (2 proteins) DNA
ligase CLSK2469924 J. Firmicutes Bacteria; Firmicutes; Bacilli;
Bacillales; Alicyclobacillaceae; Alicyclobacillus; Alicyclobacillus
acidocaldarius; Alicyclobacillus acidocaldarius subsp.
acidocaldarius Alicyclobacillus acidocaldarius subsp.
acidocaldarius ATP dependent YP_003185050 DSM 446Alicyclobacillus
DNA ligase CLSK2340991 N. Alphaproteobacteria Bacteria;
Proteobacteria; Alphaproteobacteria; Caulobacterales;
Caulobacteraceae; Phenylobacterium; Phenylobacterium zucineum
Phenylobacterium zucineum HLK1 ATP-dependent YP_002128631
(plasmid)Phenylobacterium (2 proteins) DNA ligase -- -- CLSK2333706
J. Firmicutes Bacteria; Firmicutes; Clostridia; Clostridiales;
Peptococcaceae; Candidatus Desulforudis; Candidatus Desulforudis
audaxviator Candidatus Desulforudis audaxviator ATP dependent
YP_001716762 MP104CCandidatus Desulforudis (1 proteins) DNA ligase
-- -- CLSK962101 C. Actinobacteria Bacteria; Actinobacteria;
Actinobacteria (class); -- -- Actinobacteridae; Actinomycetales;
Micromonosporineae; Micromonosporaceae; Salinispora; Salinispora
arenicola Salinispora arenicola CNS-205Salinispora (2 proteins) DNA
polymerase YP_001539124 LigD ligase region Salinispora tropica
CNB-440Salinispora (2 proteins) ATP dependent YP_001160776 DNA
ligase -- -- CLSK915249 C. Actinobacteria See CLSK2303611 above
Bacteria; Actinobacteria; Actinobacteria (class); Actinobacteridae;
Actinomycetales; Streptomycineae; Streptomycetaceae; Streptomyces;
Streptomyces coelicolor Streptomyces avermitilis MA-4680 putative
ATP- NP_828839 (plasmid)Streptomyces (2 proteins) dependint DNA
ligase Streptomyces sp. HK1 (plasmid)Streptomyces (2 putative ATP-
YP_001661618 proteins) dependent DNA ligase CLSK862724 A. Archaea
Archaea; Euryarchaeota; Archaeoglobi; Archaeoglobales;
Archaeoglobaceae; Archaeoglobus; Archaeoglobus fulgidus
Archaeoglobus fulgidus DSM 4304Archaeoglobus (1 DNA ligase,
NP_070553 proteins) putative J. Firmicutes Pelotomaculum
thermopropionicum SIPelotomaculum ATP-dependent YP_001211793 (1
proteins) DNA ligase Thermoanaerobacter pseudethanolicus ATCC ATP
dependent YP_001664477 33223Thermoanaerobacter (2 proteins) DNA
ligase CLSK820690 A. Archaea Archaea; Euryarchaeota; environmental
samples uncultured methanogenic archaeon RC-Ienvironmental
ATP-dependent YP_686457 samples (1 proteins) DNA ligase N.
Alphaproteobacteria Bacteria; Proteobacteria; Alphaproteobacteria;
Rhizobiales; Bradyrhizobiaceae; Bradyrhizobium; Bradyrhizobium
japonicum Bradyrhizobium japonicum USDA 110Bradyrhizobium DNA
ligase NP_774671 (2 proteins) Bradyrhizobium sp.
BTAi1Bradyrhizobium (2 putative ATP- YP_001243518 proteins)
dependent DNA ligase CLSK808255 N. Alphaproteobacteria Bacteria;
Proteobacteria; Alphaproteobacteria; Rhizobiales; Rhizobiaceae;
Sinorhizobium/Ensifer group; Sinorhizobium; Sinorhizobium medicae
Sinorhizobium medicae WSM419Sinorhizobium (2 DNA polymerase
YP_001326990 proteins) LigD ligase region Sinorhizobium meliloti
1021 (plasmid)Sinorhizobium putative ATP- NP_437750 (2 proteins)
dependent DNA ligase protein CLSK806855 N. Alphaproteobacteria
Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales;
Rhizobiaceae; Rhizobium/Agrobacterium group; Agrobacterium;
Agrobacterium tumefaciens Agrobacterium tumefaciens str. C58
ATP-dependent NP_396032 (plasmid)Agrobacterium (3 proteins) DNA
ligase Rhizobium leguminosarum bv. trifolii WSM1325 DNA polymerase
YP_002973496 (plasmid)Rhizobium (10 proteins) LigD, ligase domain
protein Rhizobium leguminosarum bv. trifolii WSM2304 DNA polymerase
YP_002278005 (plasmid)Rhizobium (10 proteins) LigD, ligase domain
protein CLSK390680 N. Alphaproteobacteria Bacteria; Proteobacteria;
Alphaproteobacteria; Rhizobiales; Phyllobacteriaceae;
Mesorhizobium; Mesorhizobium loti Mesorhizobium loti
MAFF303099Mesorhizobium (3 hypothetical NP_108282 proteins)
protein
[0057] Additional exemplary SFLs may include, for example:
TABLE-US-00002 (SEQ ID NO.: 77)
MAITKPLLAATLENIEDVQFPCLATPKIDGIRSVKQTQMLSRTFKPIRN
SVMNRLLTELLPEGSDGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWF
DYVTDDPLKKYIDRVEDMKNYITVHPHILEHAQVKIIPLIPVEINNITE
LLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMKQFKDAEAT
IISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSIEVDYDGVVF
SIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRH EEDR (SF DNA
Ligase, GenBank ID AAC96909.1, from Paramecium bursaria Chlorella
virus 1); (SEQ ID NO.: 78)
MSGVPYGFKPNLAATLTKPELIKFPVWASPKIDGIRCVFFGGVAYSRSL
KPIPNPVVQEFAKAYANLLEGLDGELTVGSPTDANCMQNSMAVMSKAAA
PDFTFHVFDWFHPAQAHIEFWQRSDVVEDRIVQFYDRYPEVDIRAAPQV
LCTSLAHLDTNEARWLADGYEGMMIRDHCGRYKFGRSTEREGGLVKVKR
FTDAEAIVIGFEEEMHNANEAKRDATGRTERSTSKAGLHGKGTLGALVV
KNERGIVFNIGTGFTAAQRADYWANHPSLFGKMVKFKHFDHGTVDAPRH PVFIGFRHPEDM (MnM
DNA Ligase, GenBank ID YP_333052.1, from Burkholderia pseudomallei
1710b (equivalent sequence to ABA50091)); (SEQ ID NO.: 79)
MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYW
NGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEISTITKS
FKGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIP
VKDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTARGEE
CTVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSV
ITYKYRGITNSGKPRFATYWREKK (Hin DNA Ligase, GenBank ID P44121, from
Haemophilus influenza); (SEQ ID NO.: 80)
MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYW
NGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEISSITKSF
KGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPV
KDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTARGEEC
TVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVI
TYKYRGITNSGKPRFATYWREKK (DLX DNA Ligase, artificial ligase derived
from Hin DNA ligase from Haemophilus influenza); (SEQ ID NO.: 81)
MKFYRTLLLFFASSFAFANSDLMLLHTYNNQPIEGWVMSEKLDGVRGYW
NGKQLLTRQGQRLSPPAYFIKDFPPFAIDGELFSERNHFEEISSITKSF
KGDGWEKLKLYVFDVPDAEGNLFERLAKLKAHLLEHPTTYIEIIEQIPV
KDKTHLYQFLAQVENLQGEGVVVRNPNAPYERKRSSQILKLKTARDEEC
TVIAHHKGKGQFENVMGALTCKNHRGEFKIGSGFNLNERENPPPIGSVI
TYKYRGITNSGKPRFATYWREKK (DLXd DNA Ligase, artificial ligase derived
from Hin DNA ligase from Haemophilus influenza); and, (SEQ ID NO.:
82) MLLHTYNNQPIEGWVMSEKLDGVRGYWNGKQLLTRQGQRLSPPAYFIK
DFPPFAIDGELFSERNHFEEISSITKSFKGDGWEKLKLYVFDVPDAEGNL
FERLAKLKAHLLEHPTTYIEIIEQIPVKDKTHLYQFLAQVENLQGEGVV
VRNPNAPYERKRSSQILKLKTARDEECTVIAHHKGKGQFENVMGALTCKN
HRGEFKIGSGFNLNERENPPPIGSVITYKYRGITNSGKPRFATYWREKK (DLXd2 DNA Ligase
(Gammaproteobacteria, Haemophilus influenza) (modified)).
[0058] As used herein, the terms "amplification", "nucleic acid
amplification", or "amplifying" refer to the production of multiple
copies of a nucleic acid template, or the production of multiple
nucleic acid sequence copies that are complementary to the nucleic
acid template. The amplification reaction may be a
polymerase-mediated extension reaction such as, for example, a
polymerase chain reaction (PCR). However, any of the known
amplification reactions may be suitable for use as described
herein. The term "amplifying" that typically refers to an
"exponential" increase in target nucleic acid may be used herein to
describe both linear and exponential increases in the numbers of a
select target sequence of nucleic acid. The term "amplification
reaction mixture" and/or "master mix" may refer to an aqueous
solution comprising the various (some or all) reagents used to
amplify a target nucleic acid. Such reactions may also be performed
using solid supports (e.g., an array). The reactions may also be
performed in single or multiplex format as desired by the user.
These reactions typically include enzymes, aqueous buffers, salts,
amplification primers, target nucleic acid, and nucleoside
triphosphates. Depending upon the context, the mixture can be
either a complete or incomplete amplification reaction mixture. The
method used to amplify the target nucleic acid may be any available
to one of skill in the art. Any in vitro means for multiplying the
copies of a target sequence of nucleic acid may be utilized. These
include linear, logarithmic, and/or any other amplification method.
While this disclosure may generally discuss PCR as the nucleic acid
amplification reaction, it is expected that other types of nucleic
acid amplification reactions, including both polymerase-mediated
amplification reactions (such as HDA, RPA, and RCA), as well as
ligase-mediated amplification reactions (such as LDR, LCR, and
gap-versions of each), and combinations of nucleic acid
amplification reactions such as LDR and PCR (see for example U.S.
Pat. No. 6,797,470) may also be suitable. For example, in addition
to those described elsewhere herein, various ligation-mediated
reactions, where for example ligation probes are employed as
opposed to PCR primers. Additional exemplary methods include
polymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos.
4,683,202; 4,683,195; 4,965,188; and/or 5,035,996), isothermal
procedures (using one or more RNA polymerases (see, e.g., WO
2006/081222), strand displacement (see, e.g., U.S. Pat. No.
RE39,007E), partial destruction of primer molecules (see, e.g.,
WO2006087574)), ligase chain reaction (LCR) (see, e.g., Wu, et al.,
Genomics 4: 560-569 (1990)), and/or Barany, et al. PNAS USA
88:189-193 (1991)), Q.beta. RNA replicase systems (see, e.g.,
WO/1994/016108), RNA transcription-based systems (e.g., TAS, 3SR),
rolling circle amplification (RCA) (see, e.g., U.S. Pat. No.
5,854,033; U.S. Pub. No. 2004/265897; Lizardi et al. Nat. Genet.
19: 225-232 (1998); and/or Bailer et al. Nucleic Acid Res., 26:
5073-5078 (1998)), and strand displacement amplification (SDA)
(Little, et al. Clin Chem 45:777-784 (1999)), among others. These
systems, along with the many other systems available to the skilled
artisan, may be suitable for use in amplifying target nucleic acids
for use as described herein.
[0059] "Amplification efficiency" may refer to any product that may
be quantified to determine copy number (e.g., the term may refer to
a PCR amplicon, an LCR ligation product, and/or similar product).
Reactions may be compared by carrying out at least two separate
amplification reactions, each reaction being carried out in the
absence and presence, respectively, of a reagent and/or step and
quantifying amplification that occurs in each reaction.
[0060] Also provided are methods for amplifying a nucleic acid
using at least one polymerase, at least one primer, dNTPs, and
ligating and amplifying the target nucleic acid. In some
embodiments of such methods, at least one primer is utilized. In
certain embodiments, a nucleic acid amplification reaction
mixture(s) comprising at least one polymerase, dNTPs, and at least
one primer is provided. In other embodiments, methods for using
such mixture(s) are provided. Target nucleic acids may be amplified
using any of a variety of reactions and systems. Exemplary methods
for amplifying nucleic acids include, for example,
polymerase-mediated extension reactions. For instance, the
polymerase-mediated extension reaction can be the polymerase chain
reaction (PCR). In other embodiments, the nucleic acid
amplification reaction is a multiplex reaction. For instance,
exemplary methods for amplifying and detecting nucleic acids
suitable for use as described herein are commercially available as
TaqMan.RTM. (see, e.g., U.S. Pat. Nos. 4,889,818; 5,079,352;
5,210,015; 5,436,134; 5,487,972; 5,658,751; 5,210,015; 5,487,972;
5,538,848; 5,618,711; 5,677,152; 5,723,591; 5,773,258; 5,789,224;
5,801,155; 5,804,375; 5,876,930; 5,994,056; 6,030,787; 6,084,102;
6,127,155; 6,171,785; 6,214,979; 6,258,569; 6,814,934; 6,821,727;
7,141,377; and/or 7,445,900, all of which are hereby incorporated
herein by reference in their entirety). TaqMan.RTM. assays are
typically carried out by performing nucleic acid amplification on a
target polynucleotide using a nucleic acid polymerase having 5'-3'
nuclease activity, a primer capable of hybridizing to said target
polynucleotide, and an oligonucleotide probe capable of hybridizing
to said target polynucleotide 3' relative to said primer. In some
embodiments, the oligonucleotide probe includes a detectable label
(e.g., a fluorescent reporter molecule) and a quencher molecule
capable of quenching the fluorescence of said reporter molecule. In
certain embodiments, the detectable label and quencher molecule are
part of a single probe. As amplification proceeds, the polymerase
digests the probe to separate the detectable label from the
quencher molecule. The detectable label (e.g., fluorescence) can be
monitored during the reaction, where detection of the label
corresponds to the occurrence of nucleic acid amplification (e.g.,
the higher the signal the greater the amount of amplification).
Variations of TaqMan.RTM. assays (e.g., LNA.TM. spiked TaqMan.RTM.
assay) are known in the art and would be suitable for use in the
methods described herein.
[0061] Another exemplary system suitable for use as described
herein utilizes double-stranded probes in displacement
hybridization methods (see, e.g., Morrison et al. Anal. Biochem.,
18:231-244 (1989); and/or Li, et al. Nucleic Acids Res., 30(2,e5)
(2002)). In such methods, the probe typically includes two
complementary oligonucleotides of different lengths where one
includes a detectable label and the other includes a quencher
molecule. When not bound to a target nucleic acid, the quencher
suppresses the signal from the detectable label. The probe becomes
detectable upon displacement hybridization with a target nucleic
acid. Multiple probes may be used, each containing different
detectable labels, such that multiple target nucleic acids may be
queried in a single reaction.
[0062] Additional exemplary methods for amplifying and detecting
target nucleic acids suitable for use as described herein involve
"molecular beacons", which are single-stranded hairpin shaped
oligonucleotide probes. In the presence of the target sequence, the
probe unfolds, binds and emits a signal (e.g., fluoresces). A
molecular beacon typically includes at least four components: 1)
the "loop", an 18-30 nucleotide region which is complementary to
the target sequence; 2) two 5-7 nucleotide "stems" found on either
end of the loop and being complementary to one another; 3) at the
5' end, a detectable label; and 4) at the 3' end, a quencher dye
that prevents the detectable label from emitting a single when the
probe is in the closed loop shape (e.g., not bound to a target
nucleic acid). Thus, in the presence of a complementary target, the
"stem" portion of the beacon separates out resulting in the probe
hybridizing to the target. Other types of molecular beacons are
also known and may be suitable for use in the methods described
herein. Molecular beacons may be used in a variety of assay
systems. One such system is nucleic acid sequence-based
amplification (NASBA.RTM.), a single step isothermal process for
amplifying RNA to double stranded DNA without temperature cycling.
A NASBA reaction typically requires avian myeloblastosis virus
(AMV), reverse transcriptase (RT), T7 RNA polymerase, RNase H, and
two oligonucleotide primers. After amplification, the amplified
target nucleic acid may be detected using a molecular beacon. Other
uses for molecular beacons are known in the art and would be
suitable for use in the methods described herein.
[0063] The Scorpion system is another exemplary assay format that
may be used in the methods described herein. Scorpion primers are
bi-functional molecules in which a primer is covalently linked to
the probe, along with a detectable label (e.g., a fluorophore) and
a quencher. In the presence of a target nucleic acid, the
detectable label and the quencher separate which leads to an
increase in signal emitted from the detectable label. Typically, a
primer used in the amplification reaction includes a probe element
at the 5' end along with a "PCR blocker" element (e.g., a
hexethylene glycol (HEG) monomer (Whitcombe, et al. Nat. Biotech.
17: 804-807 (1999)) at the start of the hairpin loop. The probe
typically includes a self-complementary stem sequence with a
detectable label at one end and a quencher at the other. In the
initial amplification cycles (e.g., PCR), the primer hybridizes to
the target and extension occurs due to the action of polymerase.
The Scorpion system may be used to examine and identify point
mutations using multiple probes that may be differently tagged to
distinguish between the probes. Using PCR as an example, after one
extension cycle is complete, the newly synthesized target region
will be attached to the same strand as the probe. Following the
second cycle of denaturation and annealing, the probe and the
target hybridize. The hairpin sequence then hybridizes to a part of
the newly produced PCR product. This results in the separation of
the detectable label from the quencher and causes emission of the
signal. Other uses for molecular beacons are known in the art and
would be suitable for use in the methods described herein.
[0064] The nucleic acid polymerases that may be employed in the
disclosed nucleic acid amplification reactions may be any that
function to carry out the desired reaction including, for example,
a prokaryotic, fungal, viral, bacteriophage, plant, and/or
eukaryotic nucleic acid polymerase. As used herein, the term "DNA
polymerase" refers to an enzyme that synthesizes a DNA strand de
novo using a nucleic acid strand as a template. DNA polymerase uses
an existing DNA or RNA as the template for DNA synthesis and
catalyzes the polymerization of deoxyribonucleotides alongside the
template strand, which it reads. The newly synthesized DNA strand
is complementary to the template strand. DNA polymerase can add
free nucleotides only to the 3'-hydroxyl end of the newly forming
strand. It synthesizes oligonucleotides via transfer of a
nucleoside monophosphate from a deoxyribonucleoside triphosphate
(dNTP) to the 3'-hydroxyl group of a growing oligonucleotide chain.
This results in elongation of the new strand in a 5' to 3'
direction. Since DNA polymerase can only add a nucleotide onto a
pre-existing 3'-OH group, to begin a DNA synthesis reaction, the
DNA polymerase needs a primer to which it can add the first
nucleotide. Suitable primers may comprise oligonucleotides of RNA
or DNA, or chimeras thereof (e.g., RNA/DNA chimerical primers). The
DNA polymerases may be a naturally occurring DNA polymerases or a
variant of natural enzyme having the above-mentioned activity. For
example, it may include a DNA polymerase having a strand
displacement activity, a DNA polymerase lacking 5' to 3'
exonuclease activity, a DNA polymerase having a reverse
transcriptase activity, or a DNA polymerase having an endonuclease
activity.
[0065] Suitable nucleic acid polymerases may also comprise
holoenzymes, functional portions of the holoenzymes, chimeric
polymerase, or any modified polymerase that can effectuate the
synthesis of a nucleic acid molecule. Within this disclosure, a DNA
polymerase may also include a polymerase, terminal transferase,
reverse transcriptase, telomerase, and/or polynucleotide
phosphorylase. Non-limiting examples of polymerases may include,
for example, T7 DNA polymerase, eukaryotic mitochondrial DNA
Polymerase .gamma., prokaryotic DNA polymerase I, II, III, IV,
and/or V; eukaryotic polymerase .alpha., .beta., .gamma., .delta.,
.epsilon., .eta., .zeta., .tau., and/or .kappa.; E. coli DNA
polymerase I; E. coli DNA polymerase III alpha and/or epsilon
subunits; E. coli polymerase IV, E. coli polymerase V; T. aquaticus
DNA polymerase I; B. stearothermophilus DNA polymerase I;
Euryarchaeota polymerases; terminal deoxynucleotidyl transferase
(TdT); S. cerevisiae polymerase 4; translesion synthesis
polymerases; reverse transcriptase; and/or telomerase. Non-limiting
examples of suitable thermostable DNA polymerases that may be used
include Taq, Tfl, Pfu, and Vent.TM. DNA polymerases, any
genetically engineered DNA polymerases, any having reduced or
insignificant 3' to 5' exonuclease activity (e.g., SuperScript.TM.
DNA polymerase), and/or genetically engineered DNA polymerases
(e.g., those having the active site mutation F667Y or the
equivalent of F667Y (e.g., in Tth), AmpliTaqFS,
ThermoSequenase.TM.), Therminator I, Therminator II, Therminator
III, Therminator Gamma (all available from NEB), and/or any
derivatives and fragments thereof. Other nucleic acid polymerases
may also be suitable as would be understood by one of skill in the
art.
[0066] In another aspect, the present disclosure provides reaction
mixtures for amplifying a nucleic acid sequence of interest (e.g.,
a target sequence). In some embodiments, the reaction mixture may
further comprise a signal-generating compound (SGC) and/or
detectable label. The methods may also include one or more steps
for detecting the SGC and/or detectable label to quantitate the
amplified nucleic acid.
[0067] A SGC may be a substance that is itself detectable in an
assay of choice, or capable of reacting to form a chemical or
physical entity (e.g., a reaction product) that is detectable in an
assay of choice. Representative examples of reaction products
include precipitates, fluorescent signals, compounds having a
color, and the like. Representative SGC include e.g.,
bioluminescent compounds (e.g., luciferase), fluorophores (e.g.,
below), bioluminescent and chemiluminescent compounds,
radioisotopes (e.g., .sup.131I, .sup.125I, .sup.14C, .sup.3H,
.sup.35S, .sup.32P and the like), enzymes (e.g., below), binding
proteins (e.g., biotin, avidin, streptavidin and the like),
magnetic particles, chemically reactive compounds (e.g., colored
stains), labeled oligonucleotides; molecular probes (e.g., CY3,
Research Organics, Inc.), and the like. Representative fluorophores
include fluorescein isothiocyanate, succinyl fluorescein, rhodamine
B, lissamine, 9,10-diphenylanthracene, perylene, rubrene, pyrene
and fluorescent derivatives thereof such as isocyanate,
isothiocyanate, acid chloride or sulfonyl chloride, umbelliferone,
rare earth chelates of lanthanides such as Europium (Eu) and the
like. Representative SGC's useful in a signal generating conjugate
include the enzymes in: TUB Class 1, especially 1.1.1 and 1.6
(e.g., alcohol dehydrogenase, glycerol dehydrogenase, lactate
dehydrogenase, malate dehydrogenase, glucose-6-phosphate
dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and the
like); TUB Class 1.11.1 (e.g., catalase, peroxidase, amino acid
oxidase, galactose oxidase, glucose oxidase, ascorbate oxidase,
diaphorase, urease and the like); IUB Class 2, especially 2.7 and
2.7.1 (e.g., hexokinase and the like); TUB Class 3, especially
3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase,
.beta.-galacturonidase, amyloglucosidase, .beta.-glucuronidase,
alkaline phosphatase, acid phosphatase and the like); TUB Class 4
(e.g., lyases); TUB Class 5 especially 5.3 and 5.4 (e.g.,
phosphoglucose isomerase, trios phosphatase isomerase,
phosphoglucose mutase and the like.) SGCs may also generate
products detectable by fluorescent and chemiluminescent
wavelengths, e.g., sequencing dyes, luciferase, fluorescence
emitting metals such as .sup.152Eu, or others of the lanthanide
series; compounds such as luminol, isoluminol, acridinium salts,
and the like; bioluminescent compounds such as luciferin;
fluorescent proteins (e.g., GFP or variants thereof); and the like.
Attaching certain SGC to agents can be accomplished through metal
chelating groups such as EDTA. The subject SGC shares the common
property of allowing detection and/or quantification of an attached
molecule. SGCs are optionally detectable using a visual or optical
method; preferably, with a method amenable to automation such as a
spectrophotometric method, a fluorescence method, a
chemiluminescent method, an electrical nanometric method involving
e.g., a change in conductance, impedance, resistance and the like
and a magnetic field method. Some SGCs are optionally detectable
with the naked eye or with a signal detection apparatus. Some SGCs
are not themselves detectable but become detectable when subject to
further treatment. The SGC can be attached in any manner (e.g.,
through covalent or non-covalent bonds) to a binding agent of
interest (e.g., an antibody or a PDZ polypeptide). SGCs suitable
for attachment to agents such as antibodies include colloidal gold,
fluorescent antibodies, Europium, latex particles, and enzymes. The
agents that bind to NS1 and NP can each comprise distinct SGCs. For
example, red latex particles can be conjugated to anti-NS1
antibodies and blue latex particles can be conjugated to anti-NP
antibodies. Other detectable SGCs suitable for use in a lateral
flow format include any moiety that is detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical,
chemical, or other means. For example, suitable SGCs include biotin
for staining with labeled streptavidin conjugate, fluorescent dyes
(e.g., fluorescein, Texas red, rhodamine, green fluorescent
protein, and the like), radiolabels, enzymes (e.g., horseradish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric SGCs such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex beads).
Patents that described the use of such labels include U.S. Pat.
Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241. See also Handbook of Fluorescent Probes
and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene
Oreg.). Radiolabels can be detected using photographic film or
scintillation counters, fluorescent markers can be detected using a
photodetector to detect emitted light.
[0068] Similarly, the term "detectable label" may refer to any of a
variety of signaling molecules indicative of amplification. For
example, SYBR GREEN and other DNA-binding dyes are detectable
labels. Such detectable labels may comprise or may be, for example,
nucleic acid intercalating agents or non-intercalating agents. As
used herein, an intercalating agent is an agent or moiety capable
of non-covalent insertion between stacked base pairs of a
double-stranded nucleic acid molecule. A non-intercalating agent is
one that does not insert into the double-stranded nucleic acid
molecule. The nucleic acid binding agent may produce a detectable
signal directly or indirectly. The signal may be detectable
directly using, for example, fluorescence and/or absorbance, or
indirectly using, for example, any moiety or ligand that is
detectably affected by proximity to double-stranded nucleic acid is
suitable such as a substituted label moiety or binding ligand
attached to the nucleic acid binding agent. It is typically
necessary for the nucleic acid binding agent to produce a
detectable signal when bound to a double-stranded nucleic acid that
is distinguishable from the signal produced when that same agent is
in solution or bound to a single-stranded nucleic acid. For
example, intercalating agents such as ethidium bromide fluoresce
more intensely when intercalated into double-stranded DNA than when
bound to single-stranded DNA, RNA, or in solution (see, e.g., U.S.
Pat. Nos. 5,994,056; 6,171,785; and/or 6,814,934). Similarly,
actinomycin D fluoresces red fluorescence when bound to
single-stranded nucleic acids, and green when bound to
double-stranded nucleic acids. And in another example, the
photoreactive psoralen 4-aminomethyl-4-5'8-trimethylpsoralen (AMT)
has been reported to exhibit decreased absorption at long
wavelengths and fluorescence upon intercalation into
double-stranded DNA (Johnson et al. Photochem. & Photobiol.,
33:785-791 (1981). For example, U.S. Pat. No. 4,257,774 describes
the direct binding of fluorescent intercalators to DNA (e.g.,
ethidium salts, daunomycin, mepacrine and acridine orange,
4'6-diamidino-.alpha.-phenylindole). Non-intercalating agents
(e.g., minor groove binders as described herein such as Hoechst
33258, distamycin, netropsin) may also be suitable for use. For
example, Hoechst 33258 (Searle, et al. Nuc. Acids Res.
18(13):3753-3762 (1990)) exhibits altered fluorescence with an
increasing amount of target. Minor groove binders are described in
more detail elsewhere herein.
[0069] Other DNA binding dyes are available to one of skill in the
art and may be used alone or in combination with other agents
and/or components of an assay system. Exemplary DNA binding dyes
may include, for example, acridines (e.g., acridine orange,
acriflavine), actinomycin D (Jain, et al. J. Mol. Biol. 68:21
(1972)), anthramycin, BOBO.TM.-1, BOBO.TM.-3, BO-PRO.TM.-1,
chromomycin, DAPI (Kapuseinski, et al. Nuc. Acids Res. 6(112): 3519
(1979)), daunomycin, distamycin (e.g., distamycin D), dyes
described in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts
(e.g., ethidium bromide), fluorcoumanin, fluorescent intercalators
as described in U.S. Pat. No. 4,257,774, GelStar.RTM. (Cambrex Bio
Science Rockland Inc., Rockland, Me.), Hoechst 33258 (Searle and
Embrey, 1990, Nuc. Acids Res. 18:3753-3762), Hoechst 33342,
homidium, JO-PRO.TM.-1, LIZ dyes, LO-PRO.TM.-1, mepacrine,
mithramycin, NED dyes, netropsin,
4'6-diamidino-.alpha.-phenylindole, proflavine, POPO.TM.-1,
POPO.TM.-3, PO-PRO.TM.-1, propidium iodide, ruthenium polypyridyls,
S5, SYBR.RTM. Gold, SYBR.RTM. Green I (U.S. Pat. Nos. 5,436,134 and
5,658,751), SYBR.RTM. Green II, SYTOX blue, SYTOX green, SYTO.RTM.
43, SYTO.RTM. 44, SYTO.RTM. 45, SYTOX.RTM. Blue, TO-PRO.RTM.-1,
SYTO.RTM. 11, SYTO.RTM. 13, SYTO.RTM. 15, SYTO.RTM. 16, SYTO.RTM.
20, SYTO.RTM. 23, thiazole orange (Aldrich Chemical Co., Milwaukee,
Wis.), TOTO.TM.-3, YO-PRO.RTM.-1, and YOYO.RTM.-3 (Molecular
Probes, Inc., Eugene, Oreg.), among others. SYBR.RTM. Green I (see,
e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or 6,569,927), for
example, has been used to monitor a PCR reactions. Other DNA
binding dyes may also be suitable as would be understood by one of
skill in the art.
[0070] For use as described herein, one or more detectable labels
and/or quenching agents may be attached to one or more primers
and/or probes (e.g., detectable label). The detectable label may
emit a signal when free or when bound to one of the target nucleic
acids. The detectable label may also emit a signal when in
proximity to another detectable label. Detectable labels may also
be used with quencher molecules such that the signal is only
detectable when not in sufficiently close proximity to the quencher
molecule. For instance, in some embodiments, the assay system may
cause the detectable label to be liberated from the quenching
molecule. Any of several detectable labels may be used to label the
primers and probes used in the methods described herein. As
mentioned above, in some embodiments the detectable label may be
attached to a probe, which may be incorporated into a primer, or
may otherwise bind to the amplified target nucleic acid (e.g., a
detectable nucleic acid binding agent such as an intercalating or
non-intercalating dye). When using more than one detectable label,
each should differ in their spectral properties such that the
labels may be distinguished from each other, or such that together
the detectable labels emit a signal that is not emitted by either
detectable label alone. Exemplary detectable labels include, for
instance, a fluorescent dye or fluorophore (e.g., a chemical group
that can be excited by light to emit fluorescence or
phosphorescence), "acceptor dyes" capable of quenching a
fluorescent signal from a fluorescent donor dye, and the like.
Suitable detectable labels may include, for example, fluorosceins
(e.g., 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein
(5-FAM); S-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT);
6-JOE; 6-carboxyfluorescein (6-FAM); FITC;
6-carboxy-1,4-dichloro-2',7'-dichlorofluorescein (TET);
6-carboxy-1,4-dichloro-2',4',5',7'-tetrachlorofluorescein (HEX);
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE);); Alexa
fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,
594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores
(e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563,
558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676,
FL, FL ATP, FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE,
TR, TR ATP, TR-X SE), coumarins (e.g., 7-amino-4-methylcoumarin,
AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin
phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein,
calcein AM, calcein blue, calcium dyes (e.g., calcium crimson,
calcium green, calcium orange, calcofluor white), Cascade Blue,
Cascade Yellow; Cy.TM. dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7),
cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins
(e.g., green fluorescent protein (e.g., GFP. EGFP), blue
fluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1),
cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow
fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET
donor/acceptor pairs (e.g., fluorescein/tetramethylrhodamine,
IAEDANS/fluorescein, EDANS/dabcyl, fluorescein/fluorescein, BODIPY
FL/BODIPY FL, Fluorescein/QSY7 and QSY9), LysoTracker and
LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White
DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26,
LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green
DND-189, LysoSensor Green DND-153, LysoSensor Yellow/Blue DND-160,
LysoSensor Yellow/Blue 10,000 MW dextran), Oregon Green (e.g., 488,
488-X, 500, 514); rhodamines (e.g., 110, 123, B, B 200, BB, BG, B
extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD,
6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B,
Phallicidine, Phalloidine, Red, Rhod-2, ROX
(6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine),
Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA
(6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT),
Texas Red, Texas Red-X, VIC and other labels described in, e.g., US
Pub. No. 2009/0197254 (incorporated herein by reference in its
entirety), among others as would be known to those of skill in the
art. Other detectable labels may also be used (see, e.g., US Pub.
No. 2009/0197254 (incorporated herein by reference in its
entirety)), as would be known to those of skill in the art. Any of
these systems and detectable labels, as well as many others, may be
used to detect amplified target nucleic acids.
[0071] Some detectable labels can be sequence-based (also referred
to herein as a "locus-specific detectable label"), for example 5'
nuclease probes. Such probes may comprise one or more detectable
labels. Various detectable labels are known in the art, for example
(TaqMan.RTM. probes described herein (See also U.S. Pat. No.
5,538,848 (incorporated herein by reference in its entirety))
various stem-loop molecular beacons (See, e.g., U.S. Pat. Nos.
6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature
Biotechnology 14:303-308), stemless or linear beacons (See, e.g.,
WO 99/21881; U.S. Pat. No. 6,485,901), PNA Molecular Beacons.TM.
(See, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA
beacons (See, e.g., Kubista et al., 2001, SPIE 4264:53-58),
non-FRET probes (See, e.g., U.S. Pat. No. 6,150,097),
Sunrise.RTM./Amplifluor.RTM. probes (U.S. Pat. No. 6,548,250),
stem-loop and duplex Scorpion.TM. probes (Solinas et al., 2001,
Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge
loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S.
Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB
Eclipse.TM. probe (Epoch Biosciences), hairpin probes (U.S. Pat.
No. 6,596,490), peptide nucleic acid (PNA) light-up probes
(Svanvik, et al. Anal Biochem 281:26-35 (2001)), self-assembled
nanoparticle probes, ferrocene-modified probes described, for
example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods
25:463-471; Whitcombe et al., 1999, Nature Biotechnology.
17:804-807; Isacsson et al., 2000, Molecular Cell Probes.
14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs
et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,
Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002,
Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai.
34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612;
Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al.,
2002, Chem Res. Toxicol. 15:118-126; and Yu et al., 2001, J. Am.
Chem. Soc 14:11155-11161; QuantiProbes (www.qiagen.com), HyBeacons
(French, et al. Mol. Cell. Probes 15:363-374 (2001)), displacement
probes (Li, et al. Nucleic Acids Res. 30:e5 (2002)), HybProbes
(Cardullo, et al. PNAS 85:8790-8794 (1988)), MGB Alert
(www.nanogen.com), Q-PNA (Fiandaca, et al. Genome Res. 11:609-611
(2001)), Plexor (www.Promega.com), LUX primers (Nazarenko, et al.
Nucleic Acids Res. 30:e37 (2002)), DzyNA primers (Todd, et al.
Clin. Chem. 46:625-630 (2000)). Detectable labels can also comprise
black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher
(Molecular Probes), and Dabsy1 and Dabce1 sulfonate/carboxylate
Quenchers (Epoch). Detectable labels can also comprise two probes,
wherein for example a fluor is on one probe, and a quencher on the
other, wherein hybridization of the two probes together on a target
quenches the signal, or wherein hybridization on a target alters
the signal signature via a change in fluorescence. Exemplary
systems may also include FRET, salicylate/DTPA ligand systems (see,
e.g., Oser et al. Angew. Chem. Int. Engl. 29(10):1167 (1990)),
displacement hybridization, homologous probes, and/or assays
described in EP 070685 and/or U.S. Pat. No. 6,238,927. Detectable
labels can also comprise sulfonate derivatives of fluorescein dyes
with SO.sub.3 instead of the carboxylate group, phosphoramidite
forms of fluorescein, phosphoramidite forms of CY5 (available for
example from Amersham).
[0072] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues, or any variant or functional fragment thereof. The terms
apply to amino acid polymers in which one or more amino acid
residue is an artificial chemical mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers and non-naturally occurring amino acid
polymers. The term "amino acid" includes naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0073] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. The following eight groups each contain
amino acids that are conservative substitutions for one another: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); [0078] 4) Arginine (R), Lysine
(K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)). Variants of a given nucleotide
sequence or polypeptide sequence are optionally conservatively
modified variants. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences.
[0074] The term "antibody" or "antibodies" may include whole and/or
fragments and/or derivatives of antibodies in unpurified or
partially purified form (e.g., hybridoma supernatant, ascites,
polyclonal antisera) or in purified form. A "purified" antibody may
be one that is separated from at least about 50% of the proteins
with which it is initially found (e.g., as part of a hybridoma
supernatant or ascites preparation). Preferably, a purified
antibody is separated from at least about 60%, 75%, 90%, or 95% of
the proteins with which it is initially found. Suitable derivatives
may include fragments (e.g., Fab, Fab.sub.2 or single chain
antibodies (Fv for example)), as are known in the art. The
antibodies may be of any suitable origin or form including, for
example, murine (e.g., produced by murine hybridoma cells), or
expressed as humanized antibodies, chimeric antibodies, human
antibodies, and the like. Methods of preparing and utilizing
various types of antibodies are well-known to those of skill in the
art and would be suitable for use (see, for example, Harlow, et al.
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988; Harlow, et al. Using Antibodies: A Laboratory Manual,
Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495
(1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al.
Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol.,
2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988);
Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.
Immunol., 147(1):86-95 (1991); Marks et al., Bio/Technology 10,
779-783 (1992); Lonberg et al., Nature 368 856-859 (1994);
Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature
Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology
14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93
(1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and, 5,661,016).
[0075] In certain applications, the antibodies may be contained
within hybridoma supernatant or ascites and utilized either
directly as such or following concentration using standard
techniques. In other applications, the antibodies may be further
purified using, for example, salt fractionation and ion exchange
chromatography, or affinity chromatography using Protein A, Protein
G, Protein A/G, and/or Protein L ligands covalently coupled to a
solid support such as agarose beads, or combinations of these
techniques. The antibodies may be stored in any suitable format,
including as a frozen preparation (e.g., about -20.degree. C. or
-70.degree. C.), in lyophilized form, or under normal refrigeration
conditions (e.g., about 4.degree. C.). When stored in liquid form,
it is preferred that a suitable buffer such as Tris-buffered saline
(TBS) or phosphate buffered saline (PBS) is utilized. Antibodies
and their derivatives may be incorporated into compositions (e.g.,
attached to oligonucleotides) described herein for use in vitro or
in vivo. Antibodies may also be modified for use by, for example,
biotinylation. Other methods for making and using antibodies
available to one of skill in the art may also be suitable for
use.
[0076] The methods described herein may be useful for detecting
and/or quantifying a variety of target nucleic acids from a test
sample (e.g., biological sample). A target nucleic acid is any
nucleic acid for which an assay system is designed to identify or
detect as present (or not), and/or quantify in a test sample. Such
nucleic acids may include, for example, those of infectious agents
(e.g., virus, bacteria, parasite, and the like), a disease process
such as cancer, diabetes, or the like, or to measure an immune
response. Exemplary "test samples" include various types of
samples, such as biological samples. Exemplary biological samples
include, for instance, a bodily fluid (e.g., blood, saliva, spinal
fluid), a tissue sample, a food (e.g., meat) or beverage (e.g.,
milk) product, or the like. Other examples of biological samples
may include, whole blood, serum, plasma, urine, synovial fluid,
saliva, cerebrospinal fluid, tissue infiltrate, cervical or vaginal
exudate, pleural effusion, bronchioalveolar lavage fluid, gastric
lavage fluid, small or large bowel contents, and swab specimens
from various bodily orifices dispersed in a suitable medium.
Expressed nucleic acids may include, for example, genes for which
expression (or lack thereof) is associated with medical conditions
such as infectious disease (e.g., bacterial, viral, fungal,
protozoal infections) or cancer. The methods described herein may
also be used to detect contaminants (e.g., bacteria, virus, fungus,
and/or protozoan) in pharmaceutical, food, or beverage products.
The methods described herein may be also be used to detect rare
alleles in the presence of wild type alleles (e.g., one mutant
allele in the presence of 10.sup.6-10.sup.9 wild type alleles). The
methods are useful to, for example, detect minimal residual disease
(e.g., rare remaining cancer cells during remission, especially
mutations in the p53 gene or other tumor suppressor genes
previously identified within the tumors), and/or measure mutation
load (e.g., the frequency of specific somatic mutations present in
normal tissues, such as blood or urine).
[0077] Kits for performing the methods described herein are also
provided. The kit may comprise one or more probes (e.g., antibody
conjugated to an oligonucleotide) a pair of oligonucleotides for
amplifying at least one target nucleic acid from a sample, a
biocatalyst (e.g., DNA polymerase) and/or corresponding one or more
probes labeled with a detectable label. The kit may also include
samples containing pre-defined target nucleic acids to be used in
control reactions. The kit may also optionally include stock
solutions, buffers, enzymes, detectable labels or reagents required
for detection, tubes, membranes, and the like that may be used to
complete the amplification reaction. In some embodiments, multiple
primer sets are included. Other embodiments of particular systems
and kits are also contemplated which would be understood by one of
skill in the art.
[0078] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to this Description.
[0079] Unless otherwise apparent from the context, any feature can
be claimed in combination with any other, or be claimed as not
present in combination with another feature. A feature can be any
piece of information that can characterize an invention or can
limit the scope of a claim, for example any variation, step,
feature, property, composition, method, step, degree, level,
component, material, substance, element, mode, variable, aspect,
measure, amount, option, embodiment, clause, descriptive term,
claim element or limitation.
[0080] The singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise.
Approximating language, as used herein throughout the specification
and claims, may be applied to modify any quantitative
representation that could permissibly vary without resulting in a
change in the basic function to which it is related. Accordingly, a
value modified by a term such as "about" is not to be limited to
the precise value specified. Where necessary, ranges have been
supplied, and those ranges are inclusive of all sub-ranges there
between.
[0081] In this disclosure, the use of the singular can include the
plural unless specifically stated otherwise or unless, as will be
understood by one of skill in the art in light of the present
disclosure, the singular is the only functional embodiment. Thus,
for example, "a" may mean more than one, and "one embodiment" may
mean that the description applies to multiple embodiments. The
phrase "and/or" denotes a shorthand way of indicating that the
specific combination is contemplated in combination and,
separately, in the alternative.
[0082] It will be appreciated that there is an implied "about"
prior to the temperatures, concentrations, times, etc. discussed in
the present teachings, such that slight and insubstantial
deviations are within the scope of the present teachings herein.
Also, the use of "comprise", "comprises", "comprising", "contain",
"contains", "containing", "include", "includes", and "including"
are not intended to be limiting. It is to be understood that both
the foregoing general description and detailed description are
exemplary and explanatory only and are not restrictive of the
invention.
[0083] Unless specifically noted in the above specification,
embodiments in the above specification that recite "comprising"
various components are also contemplated as "consisting of" or
"consisting essentially of" the recited components; embodiments in
the specification that recite "consisting of" various components
are also contemplated as "comprising" or "consisting essentially
of" the recited components; and embodiments in the specification
that recite "consisting essentially of" various components are also
contemplated as "consisting of" or "comprising" the recited
components (this interchangeability does not apply to the use of
these terms in the claims).
[0084] Generally, features described herein are intended to be
optional unless explicitly indicated to be necessary in the
specification. Non-limiting examples of language indicating that a
feature is regarded as optional in the specification include terms
such as "variation," "where," "while," "when," "optionally,"
"include," "preferred," "especial," "recommended," "advisable,"
"particular," "should," "alternative," "typical," "representative,"
"various," "such as," "the like," "can," "may," "example,"
"embodiment," or "aspect," "in some," "example," "exemplary",
"instance", "if" or any combination and/or variation of such
terms.
[0085] "Isolated" or "purified" generally refers to isolation of a
substance (compound, polynucleotide, protein, polypeptide,
polypeptide composition) such that the substance comprises a
significant percent (e.g., greater than 2%, greater than 5%,
greater than 10%, greater than 20%, greater than 50%, or more,
sometimes more than 90%, 95% or 99%) of the sample in which it
resides. In certain embodiments, a substantially purified component
comprises at least 50%, 80%-85%, or 90-95% of the sample.
Techniques for purifying polynucleotides and polypeptides of
interest are well-known in the art and include, for example,
ion-exchange chromatography, affinity chromatography and
sedimentation according to density. Generally, a substance is
purified when it exists in a sample in a higher proportion than it
is naturally found.
[0086] Sequence identity (also called homology) refer to similarity
in sequence of two or more sequences (e.g., nucleotide or
polypeptide sequences). In the context of two or more homologous
sequences, the percent identity or homology of the sequences or
subsequences thereof indicates the percentage of all monomeric
units (e.g., nucleotides or amino acids) that are the same (e.g.,
about 70% identity, preferably 75%, 80%, 85%, 90%, 95% or 99%
identity). The percent identity can be over a specified region,
when compared and aligned for maximum correspondence over a
comparison window, or designated region as measured using a BLAST
or BLAST 2.0 sequence comparison algorithms with default parameters
described below, or by manual alignment and visual inspection.
Sequences are said to be "substantially identical" when there is at
least 90% identity at the amino acid level or at the nucleotide
level. This definition also refers to the complement of a test
sequence. Preferably, the identity exists over a region that is at
least about 25, 50, or 100 residues in length, or across the entire
length of at least one compared sequence. A preferred algorithm for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al, Nuc. Acids Res. 25:3389-3402 (1977). Other methods include
the algorithms of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), and Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
etc. Another indication that two nucleic acid sequences are
substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions.
[0087] Any indication that a feature is optional is intended
provide adequate support (e.g., under 35 U.S.C. 112 or Art. 83 and
84 of EPC) for claims that include closed or exclusive or negative
language with reference to the optional feature. Exclusive language
specifically excludes the particular recited feature from including
any additional subject matter. For example, if it is indicated that
A can be drug X, such language is intended to provide support for a
claim that explicitly specifies that A consists of X alone, or that
A does not include any other drugs besides X. "Negative" language
explicitly excludes the optional feature itself from the scope of
the claims. For example, if it is indicated that element A can
include X, such language is intended to provide support for a claim
that explicitly specifies that A does not include X. Non-limiting
examples of exclusive or negative terms include "only," "solely,"
"consisting of," "consisting essentially of," "alone," "without",
"in the absence of (e.g., other items of the same type, structure
and/or function)" "excluding," "not including", "not", "cannot," or
any combination and/or variation of such language.
[0088] Similarly, referents such as "a," "an," "said," or "the,"
are intended to support both single and/or plural occurrences
unless the context indicates otherwise. For example "a dog" is
intended to include support for one dog, no more than one dog, at
least one dog, a plurality of dogs, etc. Non-limiting examples of
qualifying terms that indicate singularity include "a single",
"one," "alone", "only one," "not more than one", etc. Non-limiting
examples of qualifying terms that indicate (potential or actual)
plurality include "at least one," "one or more," "more than one,"
"two or more," "a multiplicity," "a plurality," "any combination
of," "any permutation of," "any one or more of," etc. Claims or
descriptions that include "or" between one or more members of a
group are considered satisfied if one, more than one, or all of the
group members are present in, employed in, or otherwise relevant to
a given product or process unless indicated to the contrary or
otherwise evident from the context.
[0089] In the claims, any active verb (or its gerund) are intended
to indicate the corresponding actual or attempted action, even if
no actual action occurs. For example, the verb "hybridize" and
gerund form "hybridizing" and the like refer to actual
hybridization or to attempted hybridization by contacting nucleic
acid sequences under conditions suitable for hybridization, even if
no actual hybridization occurs. Similarly, "detecting" and
"detection" when used in the claims refer to actual detection or to
attempted detection, even if no target is actually detected.
[0090] Furthermore, it is to be understood that the inventions
encompass all variations, combinations, and permutations of any one
or more features described herein. Any one or more features may be
explicitly excluded from the claims even if the specific exclusion
is not set forth explicitly herein. It should also be understood
that disclosure of a reagent for use in a method is intended to be
synonymous with (and provide support for) that method involving the
use of that reagent, according either to the specific methods
disclosed herein, or other methods known in the art unless one of
ordinary skill in the art would understand otherwise. In addition,
where the specification and/or claims disclose a method, any one or
more of the reagents disclosed herein may be used in the method,
unless one of ordinary skill in the art would understand
otherwise.
[0091] All publications and patents cited in this specification are
herein incorporated by reference in their entirety into this
application as if each individual publication or patent were
specifically and individually indicated to be incorporated by
reference. Genbank records referenced by GID or accession number,
particularly any polypeptide sequence, polynucleotide sequences or
annotation thereof, are incorporated by reference herein. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0092] Where ranges are given herein, the endpoints are included.
Furthermore, it is to be understood that unless otherwise indicated
or otherwise evident from the context and understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value or subrange within the stated ranges in
different embodiments of the invention, to the tenth of the unit of
the lower limit of the range, unless the context clearly dictates
otherwise.
[0093] Certain embodiments are further described in the following
examples. These embodiments are provided as examples only and are
not intended to limit the scope of the claims in any way.
EXAMPLES
Example 1
[0094] In an exemplary embodiment of typical proximity ligation
assay (PLA) processes (FIG. 1), the probe mix (e.g., comprising two
probes, A and B, (each probe comprising a streptavidin oligo "SAO"
component and an antibody "Ab" component) in probe dilution buffer
"PDB") and test sample (in sample dilution buffer "SDB") are
combined into a binding reaction. Following the binding reaction
(e.g., at 37 C for 1 hour), the ligation reaction mixture is added
in order to carry out the ligation reaction. To prepare the
ligation reaction mixture, the ligase and ligation buffer are
diluted. Following the ligation reaction (e.g., at 37 C for 10
minutes), the ligated product is stabilized by protease digestion;
the protease is then inactivated (e.g., using heat by incubation at
37 C for 10 minutes followed by 95 C for 5 minutes). Usually, a
portion of the ligated product is transferred to the real-time PCR
reaction mixture (comprising PCR primers and proximity probe mix
"PCR-PP"), then placed on the PCR reaction plate in a qPCR
instrument. Detection and quantification of the ligated product can
then proceed using standard techniques.
[0095] A schematic of an exemplary improved PLA process is
illustrated in FIG. 2. As shown therein, the binding reaction is
the same as shown in FIG. 1. However, in some embodiments of the
improved processes disclosed herein, as shown in FIG. 2, ligase is
added to a real-time PCR mixture (comprising PCR primers, proximity
probe and splint mix "PCR-PPS") which is then added directly to the
binding reaction. In certain embodiments of the improved PLA
processes, a test sample (e.g., cell lysate) is prepared, a binding
reaction is allowed to take place and then a ligation buffer added
directly thereto. To that mixture is then added a proximity probe
mixture, and a PCR mixture. This combined reaction mixture is then
incubated for a suitable amount of time (e.g., room temperature for
20 minutes and then 96.degree. C. for 5 min) and PCR is performed.
The PCR reaction mixture is then deposited onto the reaction plate
in a qPCR instrument and detection and quantification of the
ligated product can then proceed using standard techniques (as in
typical PLA processes).
Example 2
[0096] Some exemplary embodiments of typical and improved PLA
processes are also compared in FIGS. 3A and 3B. As shown in the
embodiments illustrated therein, the typical process includes
sample preparation, a binding reaction, ligation, ligase
inactivation using a protease, protease inactivation (e.g., using
heat), followed by real-time PCR. To carry out the PCR step, a
portion of the reaction mixture containing the inactivated ligase
and protease is transferred to the PCR plate, and the "PCR mix"
(e.g., containing primers, dNTPs, polymerase, and the like) added
thereto.
[0097] As shown in FIG. 3B, the improved process may eliminate the
use of a protease and dilution of the reaction mixture prior to
PCR. As shown therein, the ligase may be inactivated using heat,
and the resultant reaction mixture placed directly into the qPCR
assay. Thus, some embodiments of the improved PLA work flow uses
entire binding reaction products in the real-time PCR well. This
provides a simplified work-flow and reduced dilution of the
reaction mixture. As a result, in some preferred embodiments of the
improved PLA processes, the PCR reaction mixture contains a higher
concentration of the ligated product (e.g., the target nucleic
acid).
[0098] In order to reduce non-binding probe ligation, the probe
concentration may be reduced. The splint (e.g., connector) oligo
length and concentration may also be reduced to minimize chance of
solution hybridization promoted by non-antigen-binding ligation
(e.g., connector oligonucleotides of at least 14 bases in length
(e.g., 9 bases overlapping a first oligo probe and 5 bases
overlapping a second oligo probe (9+5)) vs. connector
oligonucleotides of at least 18 bases in length (e.g., 9 bases
overlapping a first oligo probe and 9 bases overlapping a second
oligo probe (9+9)). In such embodiments, a small footprint ligase
(SFL) may be used. As described herein, a SFL may ligate
oligonucleotides having a connector oligo length of as short as 3
bases of hybridized DNA adjacent to 5'-phosphate hybridized DNA.
For combining the ligation and PCR reaction into one step, in some
embodiments, ATP (cofactor for the ligase) can be optionally
omitted from the reaction mixture. In order to maintain the ligase
function, in other embodiments, the SFL may be pre-enriched with
ATP prior to its purification and use.
[0099] In some embodiments of the improved PLA processes splint
oligos can be used that are either considered to be symmetrical
splints or asymmetrical splints depending on the number of
nucleotides that hybridize to each of the two oligo probes it is
connecting. FIG. 4 diagrams asymmetrical and symmetrical splint
types for use in the improved PLA processes as described herein.
Asymmetrical splints (or "connectors") span across the two separate
oligo probes (e.g., probe oligo A and B) with one of the ends of
the splint (e.g., either the 3' end or the 5' end) having more
nucleotides that hybridize to one of the probe oligos than the
other end of the splint has nucleotides that hybridize to the
alternative probe oligo (FIG. 4A). Symmetrical splints span across
the two separate oligo probes (e.g., probe oligo A and B) with both
ends of the splint (e.g., the 3' end and the 5' end) having equal
number of nucleotides that hybridize to each of the two probe
oligos (FIG. 4BA).
[0100] Both asymmetrical and symmetrical splints can have any
number of intervening nucleotides between each of it's 3' and 5'
ends that hybridize to the separate probe oligos. Alternatively,
there may be no intervening nucleotides between each of the 3' and
5' ends that hybridize to the probe oligos.
Example 3
[0101] FIG. 5 provides a comparison between results obtained using
exemplary embodiments of a typical process ("TaqMan Protein Assay
Open Kit from Life Technologies, Inc.; "PLA1") and an improved
process (using methods disclosed herein; "PLA2"). Both assays were
set up to target CSTB in NTera2 cell lysate. The binding reaction
was identical for both PLA1 and PLA2 using the manufacturer's (Life
Technologies, Inc.) recommend reagents and protocol in 4 .mu.l
volume binding reactions. After the binding reaction, PLA1
proceeded following the manufacturer's protocol and reagents. The
PLA2 reaction was combined with 16 .mu.l of ligation-PCR reaction
mix. The ligation-PCR reaction mix consists of 10 .mu.l the TaqMan
Protein Assay Fast Master Mix (Life Technologies, Inc.), 1 ul
Universal PCR Assay and the connector oligonucleotide 9+5 and the
SFL ligase, and 5 .mu.l di-water. The ligation reaction was allowed
to proceed for 10 minutes. The ligated product was then placed in a
real-time PCR instrument (Step1Plus) and utilized according to the
manufacturer's instructions.
[0102] As described above, in some embodiments, the improved
process (PLA2) carries more target molecules into the PCR step
(e.g., resulting in more amplicons being generated). The
improvement provided thereby is shown in FIG. 5. As shown therein,
the dCT of the improved process (PLA2) is much improved as compared
to the typical process (PLA1). In this exemplary embodiment, the
improved process provides at least about a one- to three-fold dCt
improvement over the typical process.
[0103] The improved process also provides improved assay
sensitivity. As shown by the exemplary embodiment in FIG. 6, the
improved process provides about a two- to ten-fold increase in
sensitivity over the typical process (FIG. 6). Sensitivity was
calculated as the relative quantification (RQ) fold change using
the results from the typical process as the calibrator. The dCt of
the improved process was calculated as fold improvement over the
typical process. Since the RQ is calculated from the dCt threshold
of 2, and the fold-change is therefor indicative of the improvement
in sensitivity. The data show that the sensitivity of the assay was
improved by at least 2-fold, as determined using five different
targets (GFP, hCSTB, hICAM1, hLIN28, and hOCT3/4). The GFP data was
generated using the typical (e.g., PLA1) and improved (e.g., PLA2)
processes as described above using a cell lysate into which rGFP
was added (e.g., a "spiked-in" cell lysate) and a GFP probe
used.
Example 4
[0104] In this example, two different splint lengths were tested at
varying concentrations.
[0105] PLA experiments were carried out using typical PLA
conditions ("TaqMan Protein Assay Open Kit from Life Technologies,
Inc.) according to the manufacturers instructions, using a T4
ligase, except that splint concentrations were varied within the
range of 3.1 nM to 1000 nM. Splints were also designed to have a
two different splint lengths of 18 (9+9; "99") or 16 (8+8; "88").
Cystatin B (CSTB) assay probes (from "TaqMan Protein Expression
Assay Kit (Human CSTB); Life Technologies, Inc.) were used to
detect either 1000 pM or 0 pM (no protein control; "NPC") of
recombinant CSTB protein in buffer. Ct values were plotted for each
splint concentration and delta Ct values (NPC Ct values minus CSTB
Ct values) and were plotted for each concentration used.
[0106] As shown in FIG. 7, a reduction in delta Ct was observed for
the 99 splint at a low concentration of 3.1 nM as compared to
higher concentrations used. There was also a delta Ct observed for
the 88 splint at a concentration of 25 nM compared to higher
concentrations. Collectively, these data demonstrate that ligated
products are reduced when the splint length is decreased when the
T4 ligase is used.
Example 5
[0107] In this example, five different splint lengths were tested
using a single concentration.
[0108] PLA experiments were carried out using similar methods as
described in Example 4, except that SF ligase instead of T4 ligase
was used. Briefly, splints were designed to have a different splint
lengths of 12 (3+9), 13 (4+9), 14 (5+9 or 7+7), 17 (8+9), or 18
(9+9). The concentration used for each of these splints was 100 nM.
Raji lysate ("Protein Expression Lysate Control Kit from Life
Technologies, Inc.) was prepared at 500 cells/reaction or 0
cells/reaction ("NPC") and CSTB assay probes (from "TaqMan Protein
Expression Assay Kit (Human CSTB); Life Technologies, Inc.) were
used according to the manufacturer's instructions. Ct values were
plotted for each splint type and delta Ct values (NPC Ct values
minus 500 cell input Ct values) and were plotted for each.
[0109] As shown in FIG. 8, increasing dCT was observed for splints
of 12 nucleotides in length up to 14 nucleotides in length
(including both asymmetrical and symmetrical splint types). This
demonstrates that SF ligase is capable of ligating both asymmetric
and symmetric splints of both shorter and longer lengths.
Example 6
[0110] In this example, T4 ligase was compared to two different SF
ligases (e.g., SF and DLxD).
[0111] PLA experiments were carried out using similar methods as
described in Example 5, using the indicated ligases and splints of
varying length, as indicated. Briefly, splints were designed to
have a two different splint lengths of 14 (5+9; "95") or 18 (9+9;
"99"). The concentration used for each of these splints was 100 nM.
Raji lysate ("Protein Expression Lysate Control Kit from Life
Technologies, Inc.) was prepared at 500 cells/reaction or 0
cells/reaction ("NPC") and CSTB assay probes (from "TaqMan Protein
Expression Assay Kit (Human CSTB); Life Technologies, Inc.) were
used according to the manufacturer's instructions. Ct values were
plotted for each ligase and splint type and delta Ct values (NPC Ct
values minus 500 cell input Ct values) and were plotted for
each.
[0112] As shown in FIG. 9, the T4 ligase resulted no noticeable dCt
using the 5+9 splint. However, both SF ligases, SF and DLxD, were
capable of ligating the target DNA using shorter splint types. In
this experiment, SF used with the 5+9 splint resulted in the
highest dCt.
[0113] The improved processes described herein, and exemplified
throughout the Examples above, provide faster times from process
start to results (fast), reduce hands-on time (simpler and
cheaper), reduce lab plasticware usage (cheaper and greener), and
increased signals and sensitivities. These improved processes
provide simplified work flow by combining ligation and PCR steps,
reduced dilution factor from binding to ligation step, reduced
binding probe concentration to enable reduced dilution factor, use
of shorter connector oligo to control background signal, use lower
connector oligo concentration to control background signal, use of
SF ligase to enable use of shorter connector oligo length, ATP
enriched SF ligase purification scheme to omit ATP in ligation-PCR
step, and enabling use of the entire reaction volume to improve the
PLA signal and sensitivity.
[0114] While certain embodiments have been described in terms of
the preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations that come within the scope of the following claims.
Sequence CWU 1
1
8419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ala Thr Pro Lys Ile Asp Gly Ile Arg1
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Glu Gly Ser Asp Gly Glu Ile Ser1
535PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Tyr Trp Phe Asp Tyr1 546PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Glu
Gly Val Met Ile Arg1 555PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Leu Leu Lys Met Lys1
569PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Cys Glu Leu Lys Leu Asp Gly Leu Ala1
579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Val Glu His Lys Val Asp Gly Leu Ser1
589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Cys Glu Pro Lys Leu Asp Gly Leu Ala1
599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Cys Glu Leu Lys Leu Asp Gly Val Ala1
5109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Ala Glu Ile Lys Tyr Asp Gly Val Arg1
5119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Cys Glu Tyr Lys Tyr Asp Gly Gln Arg1
5129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Val Asp Tyr Lys Tyr Asp Gly Glu Arg1
5139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Phe Glu Ile Lys Tyr Asp Gly Ala Arg1
5149PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Phe Glu Gly Lys Trp Asp Gly Tyr Arg1
5159PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ala Arg Glu Lys Ile His Gly Thr Asn1
5169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Ala Cys Glu Lys Val His Gly Thr Asn1
5179PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ile Leu Thr Lys Glu Asp Gly Ser Leu1
5189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Val Glu Glu Lys Val Asp Gly Tyr Asn1
5198PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Leu Glu Val Arg Gly Glu Val Phe1
5208PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Val Glu Val Arg Gly Glu Cys Tyr1
5218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Leu Glu Val Arg Gly Glu Val Tyr1
5228PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Leu Glu Ala Arg Gly Glu Ala Phe1
5238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Phe Met Leu Asp Gly Glu Leu Met1
5248PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Glu Gly Ser Asp Gly Glu Ile Ser1
5258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Phe Ile Leu Asp Thr Glu Ala Val1
5268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Phe Ile Ile Glu Gly Glu Ile Val1
5278PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ala Ile Val Glu Gly Glu Leu Val1
5288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Val Val Leu Asp Gly Glu Ala Val1
5298PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Tyr Gln Val Phe Gly Glu Phe Ala1
5308PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Leu Val Leu Asn Gly Glu Leu Phe1
5318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Phe Thr Ala Asn Phe Glu Phe Val1
5328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Leu Ile Leu Val Gly Glu Met Ala1
5335PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Phe Cys Tyr Gly Val1 5345PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Phe
Leu Tyr Thr Val1 5355PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 35Thr Phe Tyr Ala Leu1
5365PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Ile Cys His Gly Leu1 5375PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Asn
Ala Tyr Gly Ile1 5385PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Phe Val Tyr Gly Leu1
5395PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Lys Leu Tyr Ala Ile1 5405PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Tyr
Trp Phe Asp Tyr1 5415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Tyr Ala Phe Asp Ile1
5425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Phe Leu Phe Asp Leu1 5435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Asn
Leu Phe Asp Val1 5445PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 44Trp Ala Phe Asp Leu1
5455PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Tyr Val Phe Asp Ile1 5465PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Phe
Ala Phe Asp Ile1 5475PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 47Ile Leu Leu Asn Ala1
5485PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Phe Leu Phe Asp Val1 5496PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Asp
Gly Val Val Ile Lys1 5506PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Asp Gly Ile Val Ile Lys1
5516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Asp Gly Val Val Val Lys1 5526PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Asp
Gly Thr Val Leu Lys1 5536PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 53Glu Gly Leu Ile Val Lys1
5546PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Glu Gly Val Met Ile Arg1 5556PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Glu
Gly Leu Met Val Lys1 5566PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 56Glu Gly Val Met Val Lys1
5576PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Glu Gly Leu Met Ala Lys1 5586PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Glu
Gly Val Ile Ala Lys1 5596PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 59Glu Gly Tyr Val Leu Lys1
5606PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Glu Gly Val Val Ile Arg1 5616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Glu
Gly Tyr Val Ala Val1 5626PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 62Glu Gly Ile Ile Met Lys1
5635PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Ala Val Ala Phe Lys1 5645PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Ala
Ile Ala Tyr Lys1 5655PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 65Ala Leu Ala Tyr Lys1
5665PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Ala Ile Ala Tyr Lys1 5675PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Trp
Trp Lys Met Lys1 5685PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Leu Leu Lys Met Lys1
5695PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 69Trp Leu Lys Leu Lys1 5705PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Trp
Ile Lys Leu Lys1 5715PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Trp Leu Lys Ile Lys1
5725PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Trp Val Lys Asp Lys1 5735PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Ala
Ile Lys Cys Lys1 5745PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 74Ile Ile Lys Leu Arg1
5755PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75His Phe Lys Ile Lys1 5765PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Ile
Val Lys Tyr Val1 577298PRTParamecium bursaria Chlorella virus 1
77Met Ala Ile Thr Lys Pro Leu Leu Ala Ala Thr Leu Glu Asn Ile Glu1
5 10 15Asp Val Gln Phe Pro Cys Leu Ala Thr Pro Lys Ile Asp Gly Ile
Arg 20 25 30Ser Val Lys Gln Thr Gln Met Leu Ser Arg Thr Phe Lys Pro
Ile Arg 35 40 45Asn Ser Val Met Asn Arg Leu Leu Thr Glu Leu Leu Pro
Glu Gly Ser 50 55 60Asp Gly Glu Ile Ser Ile Glu Gly Ala Thr Phe Gln
Asp Thr Thr Ser65 70 75 80Ala Val Met Thr Gly His Lys Met Tyr Asn
Ala Lys Phe Ser Tyr Tyr 85 90 95Trp Phe Asp Tyr Val Thr Asp Asp Pro
Leu Lys Lys Tyr Ile Asp Arg 100 105 110Val Glu Asp Met Lys Asn Tyr
Ile Thr Val His Pro His Ile Leu Glu 115 120 125His Ala Gln Val Lys
Ile Ile Pro Leu Ile Pro Val Glu Ile Asn Asn 130 135 140Ile Thr Glu
Leu Leu Gln Tyr Glu Arg Asp Val Leu Ser Lys Gly Phe145 150 155
160Glu Gly Val Met Ile Arg Lys Pro Asp Gly Lys Tyr Lys Phe Gly Arg
165 170 175Ser Thr Leu Lys Glu Gly Ile Leu Leu Lys Met Lys Gln Phe
Lys Asp 180 185 190Ala Glu Ala Thr Ile Ile Ser Met Thr Ala Leu Phe
Lys Asn Thr Asn 195 200 205Thr Lys Thr Lys Asp Asn Phe Gly Tyr Ser
Lys Arg Ser Thr His Lys 210 215 220Ser Gly Lys Val Glu Glu Asp Val
Met Gly Ser Ile Glu Val Asp Tyr225 230 235 240Asp Gly Val Val Phe
Ser Ile Gly Thr Gly Phe Asp Ala Asp Gln Arg 245 250 255Arg Asp Phe
Trp Gln Asn Lys Glu Ser Tyr Ile Gly Lys Met Val Lys 260 265 270Phe
Lys Tyr Phe Glu Met Gly Ser Lys Asp Cys Pro Arg Phe Pro Val 275 280
285Phe Ile Gly Ile Arg His Glu Glu Asp Arg 290
29578306PRTBurkholderia pseudomallei 78Met Ser Gly Val Pro Tyr Gly
Phe Lys Pro Asn Leu Ala Ala Thr Leu1 5 10 15Thr Lys Pro Glu Leu Ile
Lys Phe Pro Val Trp Ala Ser Pro Lys Ile 20 25 30Asp Gly Ile Arg Cys
Val Phe Phe Gly Gly Val Ala Tyr Ser Arg Ser 35 40 45Leu Lys Pro Ile
Pro Asn Pro Val Val Gln Glu Phe Ala Lys Ala Tyr 50 55 60Ala Asn Leu
Leu Glu Gly Leu Asp Gly Glu Leu Thr Val Gly Ser Pro65 70 75 80Thr
Asp Ala Asn Cys Met Gln Asn Ser Met Ala Val Met Ser Lys Ala 85 90
95Ala Ala Pro Asp Phe Thr Phe His Val Phe Asp Trp Phe His Pro Ala
100 105 110Gln Ala His Ile Glu Phe Trp Gln Arg Ser Asp Val Val Glu
Asp Arg 115 120 125Ile Val Gln Phe Tyr Asp Arg Tyr Pro Glu Val Asp
Ile Arg Ala Ala 130 135 140Pro Gln Val Leu Cys Thr Ser Leu Ala His
Leu Asp Thr Asn Glu Ala145 150 155 160Arg Trp Leu Ala Asp Gly Tyr
Glu Gly Met Met Ile Arg Asp His Cys 165 170 175Gly Arg Tyr Lys Phe
Gly Arg Ser Thr Glu Arg Glu Gly Gly Leu Val 180 185 190Lys Val Lys
Arg Phe Thr Asp Ala Glu Ala Ile Val Ile Gly Phe Glu 195 200 205Glu
Glu Met His Asn Ala Asn Glu Ala Lys Arg Asp Ala Thr Gly Arg 210 215
220Thr Glu Arg Ser Thr Ser Lys Ala Gly Leu His Gly Lys Gly Thr
Leu225 230 235 240Gly Ala Leu Val Val Lys Asn Glu Arg Gly Ile Val
Phe Asn Ile Gly 245 250 255Thr Gly Phe Thr Ala Ala Gln Arg Ala Asp
Tyr Trp Ala Asn His Pro 260 265 270Ser Leu Phe Gly Lys Met Val Lys
Phe Lys His Phe Asp His Gly Thr 275 280 285Val Asp Ala Pro Arg His
Pro Val Phe Ile Gly Phe Arg His Pro Glu 290 295 300Asp
Met30579268PRTHaemophilus influenza 79Met Lys Phe Tyr Arg Thr Leu
Leu Leu Phe Phe Ala Ser Ser Phe Ala1 5 10 15Phe Ala Asn Ser Asp Leu
Met Leu Leu His Thr Tyr Asn Asn Gln Pro 20 25 30Ile Glu Gly Trp Val
Met Ser Glu Lys Leu Asp Gly Val Arg Gly Tyr 35 40 45Trp Asn Gly Lys
Gln Leu Leu Thr Arg Gln Gly Gln Arg Leu Ser Pro 50 55 60Pro Ala Tyr
Phe Ile Lys Asp Phe Pro Pro Phe Ala Ile Asp Gly Glu65 70 75 80Leu
Phe Ser Glu Arg Asn His Phe Glu Glu Ile Ser Thr Ile Thr Lys 85 90
95Ser Phe Lys Gly Asp Gly Trp Glu Lys Leu Lys Leu Tyr Val Phe Asp
100 105 110Val Pro Asp Ala Glu Gly Asn Leu Phe Glu Arg Leu Ala Lys
Leu Lys 115 120 125Ala His Leu Leu Glu His Pro Thr Thr Tyr Ile Glu
Ile Ile Glu Gln 130 135 140Ile Pro Val Lys Asp Lys Thr His Leu Tyr
Gln Phe Leu Ala Gln Val145 150 155 160Glu Asn Leu Gln Gly Glu Gly
Val Val Val Arg Asn Pro Asn Ala Pro 165 170 175Tyr Glu Arg Lys Arg
Ser Ser Gln Ile Leu Lys Leu Lys Thr Ala Arg 180 185 190Gly Glu Glu
Cys Thr Val Ile Ala His His Lys Gly Lys Gly Gln Phe 195 200 205Glu
Asn Val Met Gly Ala Leu Thr Cys Lys Asn His Arg Gly Glu Phe 210 215
220Lys Ile Gly Ser Gly Phe Asn Leu Asn Glu Arg Glu Asn Pro Pro
Pro225 230 235 240Ile Gly Ser Val Ile Thr Tyr Lys Tyr Arg Gly Ile
Thr Asn Ser Gly 245 250 255Lys Pro Arg Phe Ala Thr Tyr Trp Arg Glu
Lys Lys 260 26580268PRTHaemophilus influenza 80Met Lys Phe Tyr Arg
Thr Leu Leu Leu Phe Phe Ala Ser Ser Phe Ala1 5 10 15Phe Ala Asn Ser
Asp Leu Met Leu Leu His Thr Tyr Asn Asn Gln Pro 20 25 30Ile Glu Gly
Trp Val Met Ser Glu Lys Leu Asp Gly Val Arg Gly Tyr 35 40 45Trp Asn
Gly Lys Gln Leu Leu Thr Arg Gln Gly Gln Arg Leu Ser Pro 50 55 60Pro
Ala Tyr Phe Ile Lys Asp Phe Pro Pro Phe Ala Ile Asp Gly Glu65 70 75
80Leu Phe Ser Glu Arg Asn His Phe Glu Glu Ile Ser Ser Ile Thr Lys
85 90 95Ser Phe Lys Gly Asp Gly Trp Glu Lys Leu Lys Leu Tyr Val Phe
Asp 100 105 110Val Pro Asp Ala Glu Gly Asn Leu Phe Glu Arg Leu Ala
Lys Leu Lys 115 120 125Ala His Leu Leu Glu His Pro Thr Thr Tyr Ile
Glu Ile Ile Glu Gln 130 135 140Ile Pro Val Lys Asp Lys Thr His Leu
Tyr Gln Phe Leu Ala Gln Val145 150 155 160Glu Asn Leu Gln Gly Glu
Gly Val Val Val Arg Asn Pro Asn Ala Pro 165 170 175Tyr Glu Arg Lys
Arg Ser Ser Gln Ile Leu Lys Leu Lys Thr Ala Arg 180 185 190Gly Glu
Glu Cys Thr Val Ile Ala His His Lys Gly Lys Gly Gln Phe 195
200 205Glu Asn Val Met Gly Ala Leu Thr Cys Lys Asn His Arg Gly Glu
Phe 210 215 220Lys Ile Gly Ser Gly Phe Asn Leu Asn Glu Arg Glu Asn
Pro Pro Pro225 230 235 240Ile Gly Ser Val Ile Thr Tyr Lys Tyr Arg
Gly Ile Thr Asn Ser Gly 245 250 255Lys Pro Arg Phe Ala Thr Tyr Trp
Arg Glu Lys Lys 260 26581268PRTHaemophilus influenza 81Met Lys Phe
Tyr Arg Thr Leu Leu Leu Phe Phe Ala Ser Ser Phe Ala1 5 10 15Phe Ala
Asn Ser Asp Leu Met Leu Leu His Thr Tyr Asn Asn Gln Pro 20 25 30Ile
Glu Gly Trp Val Met Ser Glu Lys Leu Asp Gly Val Arg Gly Tyr 35 40
45Trp Asn Gly Lys Gln Leu Leu Thr Arg Gln Gly Gln Arg Leu Ser Pro
50 55 60Pro Ala Tyr Phe Ile Lys Asp Phe Pro Pro Phe Ala Ile Asp Gly
Glu65 70 75 80Leu Phe Ser Glu Arg Asn His Phe Glu Glu Ile Ser Ser
Ile Thr Lys 85 90 95Ser Phe Lys Gly Asp Gly Trp Glu Lys Leu Lys Leu
Tyr Val Phe Asp 100 105 110Val Pro Asp Ala Glu Gly Asn Leu Phe Glu
Arg Leu Ala Lys Leu Lys 115 120 125Ala His Leu Leu Glu His Pro Thr
Thr Tyr Ile Glu Ile Ile Glu Gln 130 135 140Ile Pro Val Lys Asp Lys
Thr His Leu Tyr Gln Phe Leu Ala Gln Val145 150 155 160Glu Asn Leu
Gln Gly Glu Gly Val Val Val Arg Asn Pro Asn Ala Pro 165 170 175Tyr
Glu Arg Lys Arg Ser Ser Gln Ile Leu Lys Leu Lys Thr Ala Arg 180 185
190Asp Glu Glu Cys Thr Val Ile Ala His His Lys Gly Lys Gly Gln Phe
195 200 205Glu Asn Val Met Gly Ala Leu Thr Cys Lys Asn His Arg Gly
Glu Phe 210 215 220Lys Ile Gly Ser Gly Phe Asn Leu Asn Glu Arg Glu
Asn Pro Pro Pro225 230 235 240Ile Gly Ser Val Ile Thr Tyr Lys Tyr
Arg Gly Ile Thr Asn Ser Gly 245 250 255Lys Pro Arg Phe Ala Thr Tyr
Trp Arg Glu Lys Lys 260 26582246PRTHaemophilus influenza 82Met Leu
Leu His Thr Tyr Asn Asn Gln Pro Ile Glu Gly Trp Val Met1 5 10 15Ser
Glu Lys Leu Asp Gly Val Arg Gly Tyr Trp Asn Gly Lys Gln Leu 20 25
30Leu Thr Arg Gln Gly Gln Arg Leu Ser Pro Pro Ala Tyr Phe Ile Lys
35 40 45Asp Phe Pro Pro Phe Ala Ile Asp Gly Glu Leu Phe Ser Glu Arg
Asn 50 55 60His Phe Glu Glu Ile Ser Ser Ile Thr Lys Ser Phe Lys Gly
Asp Gly65 70 75 80Trp Glu Lys Leu Lys Leu Tyr Val Phe Asp Val Pro
Asp Ala Glu Gly 85 90 95Asn Leu Phe Glu Arg Leu Ala Lys Leu Lys Ala
His Leu Leu Glu His 100 105 110Pro Thr Thr Tyr Ile Glu Ile Ile Glu
Gln Ile Pro Val Lys Asp Lys 115 120 125Thr His Leu Tyr Gln Phe Leu
Ala Gln Val Glu Asn Leu Gln Gly Glu 130 135 140Gly Val Val Val Arg
Asn Pro Asn Ala Pro Tyr Glu Arg Lys Arg Ser145 150 155 160Ser Gln
Ile Leu Lys Leu Lys Thr Ala Arg Asp Glu Glu Cys Thr Val 165 170
175Ile Ala His His Lys Gly Lys Gly Gln Phe Glu Asn Val Met Gly Ala
180 185 190Leu Thr Cys Lys Asn His Arg Gly Glu Phe Lys Ile Gly Ser
Gly Phe 195 200 205Asn Leu Asn Glu Arg Glu Asn Pro Pro Pro Ile Gly
Ser Val Ile Thr 210 215 220Tyr Lys Tyr Arg Gly Ile Thr Asn Ser Gly
Lys Pro Arg Phe Ala Thr225 230 235 240Tyr Trp Arg Glu Lys Lys
2458312DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 83aaaaaaaaaa aa 12846PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Lys
Xaa Asp Gly Xaa Arg1 5
* * * * *