U.S. patent application number 13/934062 was filed with the patent office on 2015-09-10 for high speed, high fidelity, high sensitivity nucleic acid detection.
The applicant listed for this patent is Arizona Board of Regents, a Body Corporate Acting for and on Behalf of Arizona State University. Invention is credited to Wayne D. Frasch, David Spetzler, Justin York.
Application Number | 20150252411 13/934062 |
Document ID | / |
Family ID | 39136840 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150252411 |
Kind Code |
A1 |
Frasch; Wayne D. ; et
al. |
September 10, 2015 |
HIGH SPEED, HIGH FIDELITY, HIGH SENSITIVITY NUCLEIC ACID
DETECTION
Abstract
Methods, compositions, and kits for nucleic acid detection.
Inventors: |
Frasch; Wayne D.; (Phoenix,
AZ) ; Spetzler; David; (Scottsdale, AZ) ;
York; Justin; (Phoenix, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arizona Board of Regents, a Body Corporate Acting for and on Behalf
of Arizona State University |
Scottsdale |
AZ |
US |
|
|
Family ID: |
39136840 |
Appl. No.: |
13/934062 |
Filed: |
July 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13299126 |
Nov 17, 2011 |
8530199 |
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13934062 |
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12439061 |
Aug 14, 2009 |
8084206 |
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PCT/US07/77128 |
Aug 29, 2007 |
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13299126 |
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60841007 |
Aug 30, 2006 |
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Current U.S.
Class: |
506/16 |
Current CPC
Class: |
C12Q 1/6832 20130101;
C12Q 1/6816 20130101; C12Q 1/6816 20130101; C12Q 1/6837 20130101;
C12Q 2521/325 20130101; C12Q 2561/125 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] Financial assistance for this project was provided by U.S.
Government, DARPA #N66001-03-C-XXXX; thus the United States
Government may have certain rights to this invention.
Claims
1. A kit for nucleic acid detection comprising: a plurality of
target-specific nucleic acid probes that are each complementary to
a target nucleic acid, including a first target-specific nucleic
acid probe, a nanorod detection probe and a second target-specific
nucleic acid probe, wherein upon hybridization to the target
nucleic acid the plurality of target-specific nucleic acid probes
will be directly adjacent to each other; a molecular post bound to
a solid support, where the molecular post binds to the first
target-specific nucleic acid probe, the nanorod detection probe to
the second target-specific nucleic acid probe, the first
target-specific nucleic acid probe and the second target-specific
nucleic acid probe are positioned at the 5' and 3' end,
respectively, of the series of target specific nucleic acid probes
and the plurality of target-specific nucleic acid probes ligate
together to produce a ligation product, and where the molecular
post is selected from the group consisting of F1-ATPases,
actomyosin, ciliary axonemes, bacteria flagellar posts,
kinesin/microtubules, and nucleic acid helicases; and exonuclease
or a denaturing agent or both for treating the ligation
product.
2-3. (canceled)
4. The kit of claim 1, wherein the solid support comprises a glass
coverslip.
5-9. (canceled)
10. The kit of claim 1, wherein the nanorod detection probe
comprises a gold nanorod.
11. The kit of claim 4, wherein the nanorod detection probe
comprises a gold nanorod.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to co-pending U.S.
application Ser. No. 13/299,126, filed Nov. 17, 2011 which, in turn
claims priority to U.S. application Ser. No. 12/439,061, filed Aug.
29, 2007 as International Application PCT/US07/77128, which, in
turn claims priority to U.S. Provisional Patent Application Ser.
No. 60/841,007 filed Aug. 30, 2006, all of which are incorporated
by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0003] Rapid and sensitive biosensing of nucleic acids or proteins
is vital for the identification of pathogenic agents of biomedical
and bioterrorist importance, providing forensic evidence, and for
diagnoses of genetic diseases, among other uses. Development of
methods that do not require target-amplification systems like
polymerase chain reaction (PCR) that increase the complexity of the
determination and the potential for error are a major challenge.
Surface-enhanced Raman scattering to detect a silver coating built
up on patches of several thousand immobilized target DNA molecules
bound to gold nanoparticles has been used to detect target DNA at
concentrations as low as 20 femtomolar, and is among the most
sensitive means to detect DNA (ref (3-6)). However, these methods
are limited by nonspecific binding, hybridization kinetics, and
extensive incubation times. These technologies all require the
binding of several thousand DNA-bound reporter groups as an
aggregate to obtain a detectable signal. The ultimate goal is to
achieve a detectable signal for each DNA molecule. Detection of a
molecule with a specific sequence necessarily depends upon
hybridization of the target with a probe DNA molecule, and upon the
target-dependent assembly of a molecular detection probe such as a
nanoparticle. Consequently, with single molecule biosensing, the
detection limit becomes dependent on the statistical difference
between target-specific and nonspecific binding events.
SUMMARY OF THE INVENTION
[0004] In a first aspect, the present invention provides methods
for detecting a target nucleic acid, comprising:
[0005] (a) contacting a plurality of target-specific nucleic acid
probes that are each complementary to a target nucleic acid, to a
sample under conditions whereby the plurality of target-specific
nucleic acid probes hybridize to the target nucleic acid if the
target nucleic acid is present in the sample, wherein upon
hybridization to the target nucleic acid, the target-specific
nucleic acid probes form a series of target-specific nucleic acid
probes directly adjacent to one another, wherein a first target
specific nucleic acid probe is capable of binding or is bound to a
molecular post and a second target-specific nucleic acid probe is
capable of binding or is bound to a detection probe, and wherein
the first target-specific nucleic acid probe and the second
target-specific nucleic acid probe are positioned at the 5' and 3'
end, respectively, of the series of target specific nucleic acid
probes;
[0006] (b) optionally binding the molecular post to the first
target-specific nucleic acid probe and/or binding the detection
probe to the second target-specific nucleic acid probe if the
molecular post and/or detection probe were not bound prior to
hybridization;
[0007] (c) ligating the series of target-specific nucleic acid
probes together to produce a ligation product;
[0008] (d) optionally binding the molecular post to the first
target-specific nucleic acid probe and/or binding the detection
probe to the second target-specific nucleic acid probe if the
molecular post and/or detection probe were not bound prior to
ligation;
[0009] (e) treating the ligation product with one or both of:
[0010] (i) exonuclease digestion; and
[0011] (ii) denaturation to create all single stranded bridges
after ligation;
[0012] (f) binding the molecular post to the first target-specific
nucleic acid probe and/or binding the detection probe to the second
target-specific nucleic acid probe if the molecular post and/or
detection probe were not bound prior to the step (e) treatment;
and
[0013] (g) detecting the ligation product.
[0014] In another aspect, the invention provides kits for nucleic
acid detection comprising a plurality of target-specific nucleic
acid probes that are each complementary to a target nucleic acid,
wherein upon hybridization to the target nucleic acid the plurality
of target-specific nucleic acid probes will be directly adjacent to
each other; wherein a first target specific nucleic acid probe is
capable of binding or is bound to a molecular post and a second
target-specific nucleic acid probe is capable of binding or is
bound to a detection probe, and wherein the first target-specific
nucleic acid probe and the second target-specific nucleic acid
probe are positioned at the 5' and 3' end, respectively, of the
series of target specific nucleic acid probes.
[0015] In another aspect, the present invention provide composition
comprising a plurality of target-specific nucleic acid probes that
are each complementary to a target nucleic acid, wherein upon
hybridization to the target nucleic acid the plurality of
target-specific nucleic acid probes will be directly adjacent to
each other; wherein a first target specific nucleic acid probe is
bound to a first affinity tag capable of binding to a molecular
post and a second target-specific nucleic acid probe is bound to a
second affinity tag capable of binding to a detection probe, and
wherein the first target-specific nucleic acid probe and the second
target-specific nucleic acid probe are positioned at the 5' and 3'
end, respectively, of the series of target specific nucleic acid
probes.
[0016] In a further aspect, the present invention provides
compositions comprising:
[0017] (a) a solid support; and
[0018] (b) a plurality of molecular posts attached to the solid
support, wherein the plurality of molecular posts comprise an
affinity target for binding to a specific affinity tag.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1. Stepwise LXR reactions and products when target and
probes are (i) complementary and (ii) contain a SNP.
[0020] FIG. 2. Single Nucleotide Polymorphism detection using the
LXR reaction.
[0021] FIG. 3A-3D. Schematic of embodiments using magnetic
particles.
[0022] FIG. 4. Schematic of additional embodiment using magnetic
particles.
[0023] FIG. 5A-B. HPLC results demonstrate the specificity of
ligase during the first step of LXR.
[0024] FIG. 6A-B. HPLC results demonstrate the specificity of
exonuclease during the second step of LXR.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In a first aspect, the present invention provides methods
for detecting a target nucleic acid, comprising:
[0026] (a) contacting a plurality of target-specific nucleic acid
probes that are each complementary to a target nucleic acid, to a
sample under conditions whereby the plurality of target-specific
nucleic acid probes hybridize to the target nucleic acid if the
target nucleic acid is present in the sample, wherein upon
hybridization to the target nucleic acid, the target-specific
nucleic acid probes form a series of target-specific nucleic acid
probes directly adjacent to one another, wherein a first target
specific nucleic acid probe is capable of binding or is bound to a
molecular post and a second target-specific nucleic acid probe is
capable of binding or is bound to a detection probe, and wherein
the first target-specific nucleic acid probe and the second
target-specific nucleic acid probe are positioned at the 5' and 3'
end, respectively, of the series of target specific nucleic acid
probes;
[0027] (b) optionally binding the molecular post to the first
target-specific nucleic acid probe and/or binding the detection
probe to the second target-specific nucleic acid probe if the
molecular post and/or detection probe were not bound prior to
hybridization;
[0028] (c) ligating the series of target-specific nucleic acid
probes together to produce a ligation product;
[0029] (d) optionally binding the molecular post to the first
target-specific nucleic acid probe and/or binding the detection
probe to the second target-specific nucleic acid probe if the
molecular post and/or detection probe were not bound prior to
ligation;
[0030] (e) treating the ligation product with one or both of:
[0031] (i) exonuclease digestion; and
[0032] (ii) denaturation to create all single stranded bridges
after ligation;
[0033] (f) binding the molecular post to the first target-specific
nucleic acid probe and/or binding the detection probe to the second
target-specific nucleic acid probe if the molecular post and/or
detection probe were not bound prior to the step (e) treatment;
and
[0034] (g) detecting the ligation product.
[0035] In a specific embodiment, step (e) comprises treating the
ligation product with exonuclease.
[0036] The methods disclosed herein can detect very small numbers
of individual molecules of a nucleic acid target via ligation
events with the target-specific nucleic acid probes, which are
ligated only in the presence of the nucleic acid target, producing
fully constructed ligated products comprising a molecular post and
a detection probe. The detection probe reveals the presence of the
bridging ligated product that is indicative of the nucleic acid
target. The exonuclease step ensures that only perfectly paired
target-specific nucleic acid probes are hybridized to the target
nucleic acid. As a result, amplification steps such as ligation
chain reaction (LCR) are not necessary, although may be useful, as
described below. When excluding an LCR step, ligation of the
target-specific nucleic acids can be carried out using any ligase
(as opposed to the requirement for thermal stable ligase, for
example Taq ligase, on LCR products), thus permitting a much wider
range of ligation conditions to be used. (See Para.
[0037] In the absence of an exonuclease digestion step, nonspecific
hybridization can produce an equal number of viable bridges as
perfectly complementary targets that get ligated. The inclusion of
an exonuclease digestion removes the ligand at the 3' end, thus
eliminating the ability for the DNA to assemble with both the post
and the detection probe. Thus, inclusion of an exonuclease
digestion step greatly improves the ability to detect target.
Additionally, when amplification is used the error rates of the
reaction increase as a function of the number of cycles (error rate
for one cycles*2.sup.number of cycles). The combination of a
ligation reaction followed by an exonuclease digestion is referred
to as LXR in the rest of this document. Thus the LXR reaction
taught here has an error rate that is 2.sup.x times better than the
error rate of LCR. The inclusion of the exonuclease step also
allows the number of complementary sequences to be compared to the
nonspecific background binding of the detection probe, where as
with LCR or PCR, the number of complementary probes must be
compared to the number of hybridized probes, which is orders of
magnitude greater than the nonspecific binding of the detection
probe alone.
[0038] Finally, since inclusion of the exonuclease step makes the
reaction much more specific, it facilitates conducting the
reactions in unpurified samples, including but not limited to crude
cell lysate.
[0039] For all of these reasons, the present methods, with the
inclusion of an exonuclease step, result in a far superior
detection method than prior art detection techniques.
[0040] In an alternative embodiment, the exonuclease step can be
replaced through or accompanied by denaturation using a denaturing
agent (including but not limited to heat and NaOH treatment) to
create all single stranded bridges after ligation, and thus to
ensures that only perfectly paired target-specific nucleic acid
probes are hybridized to the target nucleic acid.
[0041] The sample from which detection of the target nucleic acid
is made can be any sample of interest, including but not limited to
synthetic nucleic acids, genomic DNA, cell lysates, tissue
homogenates, forensic samples, environmental samples, and isolated
nucleic acid samples from cells, tissues, or complete
organisms.
[0042] The target nucleic acid can be any nucleic acid that can
serve as a bridge between a molecular post and a detection probe to
detect construction of the device and for which the means of
formation of that bridge is specific to that target nucleic acid.
Thus, the target nucleic acid can comprise DNA or RNA and can be
single stranded or double stranded. In a specific embodiment, the
target nucleic acid is double stranded. In a more specific
embodiment, the target nucleic acid is a double stranded DNA.
[0043] The plurality of target specific nucleic acid probes can be
any 2 or more nucleic acid sequences that are complementary to
directly adjacent sequences on the same target nucleic acid. There
is no other specific nucleic acid sequence requirement for the
plurality of target specific nucleic acids. The plurality of target
specific nucleic acid probes can independently comprise DNA or RNA
and can be single stranded or double stranded. In a specific
embodiment, the target specific nucleic acid probes are single
stranded. In a more specific embodiment, the target specific
nucleic acid probes are single stranded DNA. In a further specific
embodiment, the plurality of target-specific nucleic acids probes
comprise or consist of 3, 4, 5, 6, 7, 8, 9, 10, or more
target-specific nucleic acids probes.
[0044] There are significant benefits in using multiple
target-specific nucleic acid probes. Specifically, each
target-specific nucleic acid has an error rate that is associated
with it; say for example 10.sup.-4, since each target-specific
nucleic acid must engage in a successful ligation to avoid being
degraded by the exonuclease. The error rate for a
multi-target-specific nucleic acid probe ligation is the product of
the error rate for each individual target-specific nucleic acid.
For example, if 5 target-specific nucleic acids probes were used,
then the total error rate would be (10.sup.-4).sup.5=10.sup.-20.
Furthermore, the ligase reaction is most sensitive to mismatches
within 3 bases from the site of ligation in the 3' direction, thus
mismatches that are >3 bases away from the site of ligation are
unlikely to be detected. By using multiple target-specific nucleic
acid probes, the ligation reaction can be sensitive to longer
stretches of bases. For example, if each ligation site were 6 bases
away from each other, then all 6 bases in between would have to be
complementary for the probes to be ligated together. Thus
increasing the number of target-specific nucleic acid probes has
the additional effect of increasing specificity of binding to
longer target sequences. In the event that the target nucleic acid
is in a sample that contains similar nucleic acid sequences that
differ at a few non-adjacent nucleotides, it is possible that 2
target-specific nucleic acids probes would not be sufficient for
detection.
[0045] The preferred number of target-specific nucleic acids for
detection of a given specific nucleic acid target is dependent upon
the target nucleic acid that is being detected and can be
determined by those skilled in the art based upon the teachings
herein.
[0046] As used herein the term "directly adjacent" means juxtaposed
5' phosphate and 3' hydroxyl termini of two adjacent
target-specific nucleic acid probes hybridized to the complementary
target nucleic acid, which can be ligated together by the action of
a nucleic acid ligase.
[0047] Optimization of conditions for contacting the plurality of
target-specific nucleic acid probes to a sample under conditions
whereby the plurality of target-specific nucleic acid probes
hybridize to the target nucleic acid if the target nucleic acid is
present in the sample can be readily accomplished by those of skill
in the art. The hybridization conditions are thus optimized to
limit hybridization/ligation to those situations where the target
nucleic acid is present. Such optimization includes consideration
of the target-specific nucleic acid probe sequence, number, and
length, reaction buffer, reaction temperature, and reaction time.
The specific hybridization conditions used will depend on the
length of the target-specific nucleic acid probes employed, their
GC content, as well as various other factors as is well known to
those of skill in the art. Non-limiting exemplary conditions can be
found, for example, at the web site epicentre.com, by selecting
"technical resources-protocols", then accessing "SNP & Mutation
Discovery & Screening", then selecting the "Ampliqase
Thermostable DNA Ligase" pdf file. The inclusion of an exonuclease
digestion step in the methods of the invention to degrade
non-complementary hybridized nucleic acid sequences reduces the
stringency requirements for successful reactions.
[0048] As used herein, the term "molecular post" means any
biological or synthetic molecular structure capable of binding to
the first target-specific nucleic acid probes (directly or
indirectly), and that permits detection. The size of the molecular
post is not a critical feature of the invention, however it is
preferred that the post be of nanoscale dimensions. By keeping the
post on that scale, the detection probes will all be at
approximately the same z-axis position. Thus, when performing, for
example, optical detection, a single focal plane will encompass all
of the specifically bound detection probes. In one specific
embodiment, the molecular post comprises a biomolecule, including
but not limited to F.sub.1-ATPases, actomyosin, ciliary axonemes,
bacteria flagellar posts, kinesin/microtubules, and nucleic acid
helicases and polymerases. In another specific embodiment, the
molecular post comprises a magnetic particle. When using a magnetic
particle as the post, the post should be small enough to allow the
detection probe to be visible. Further non-limiting examples of
suitable molecular posts include synthetics materials, metals,
silicone based posts, plastics, carbon structures, and lipid
structures.
[0049] As used herein, the "detection probe" is anything that is
capable of binding to the second target-specific nucleic acid probe
(directly or indirectly), and which provides a means of detecting
the presence of the resulting ligation product, such as metallic
nanoparticles (rods, spheres, quantum dots, etc.) fluorescent dyes,
and nanoparticles labeled with fluorescent dyes. When the detection
probe comprises a metallic nanoparticle and the molecular post is a
magnetic particle, it is preferred that the metallic nanoparticle
detection probe be non-magnetic (for example, silver or gold). In a
specific embodiment, elemental metal nanorods are used as the
detection probe, including but not limited to gold, silver,
aluminum, platinum, copper, zinc, and nickel. In one example, gold
rod detection probes capable of visual observation by microscope
are attached to the second target-specific nucleic acid probe
through a biotin-avidin bond. In a further example, the gold
nanorod is coated with anti-DIG antibody (the affinity target),
which binds specifically to a DIG (Digoxigenin) second affinity
tag.
[0050] The molecular post and the detection probe can be bound to
the first and second target-specific nucleic acid probes either
directly or indirectly. In various specific embodiments, the
molecular post is indirectly bound to the first target-specific
nucleic acid probe via a first affinity tag and/or the detection
probe is indirectly bound to the second target-specific nucleic
acid probe via a second affinity tag. In these embodiments, the
first affinity tag and the second affinity tag may be the same or
different as is most suitable for their ultimate attachment to the
specific molecular post and the detection probe employed.
[0051] The first affinity tag can bind to the molecular post and
the second affinity tag can bind to the detection probe either
directly (for example by a covalent bond between the
target-specific nucleic acid probe and the affinity tag) or
indirectly through another molecule. In a specific embodiment, the
first and/or second affinity tags bind indirectly to the molecular
post and the detection probe, respectively. In this specific
embodiment, the affinity tag binds directly to the target-specific
nucleic acid probe and to an affinity target, wherein the affinity
target is bound to the molecular post or the detection probe.
Together, an affinity tag and affinity target make up a binding
pair. Either member of a binding pair can be used as an affinity
tag and either member can be used as an affinity target. An
affinity target includes both separate molecules and portions of
molecules, such as an epitope of a protein that interacts
specifically with an affinity tag. Antibodies, either member of a
receptor/ligand pair, and other molecules with specific binding
affinities can be used as affinity tags. Binding an affinity tag to
the target-specific nucleic acid probes thus permits an indirect
linkage between the target-specific nucleic acid probes and the
molecular post or the detection label. An affinity tag that
interacts specifically with a particular affinity target is said to
be specific for that affinity target. For example, an affinity tag
which is an antibody that binds to a particular antigen is said to
be specific for that antigen. Complementary nucleotide sequences
can also be used as binding pairs.
[0052] A non-limiting example of a binding pair is biotin/avidin.
Other non-limiting binding pair examples include digoxigenin
(DIG)/anti-digoxigenin antibody and other antigen/antibody pairs.
Epitope tags, such as his-tags, and antibodies directed against the
epitope tag (or fragments thereof) are further examples of binding
pairs for use with the methods of the present invention. Those of
skill in the art will understand that certain embodiments listed
herein as indirect binding of the affinity tag and the molecular
post or detection probe can also be used for direct binding
embodiments. For example, where the second affinity tag is an
epitope tag as described above, the detection probe can be a
labeled antibody against the epitope tag. Many further such
examples will be readily apparent to those of skill in the art.
[0053] The affinity tags are bound to the first and last
target-specific nucleic acid probes so as to not interfere with the
ability of the series of target-specific nucleic acid probes to be
ligated together after hybridization to the target nucleic acid. In
a specific embodiment, one of the affinity tags is bound at or near
the 5' end of one of the target specific nucleic acid probes, and
the other affinity tag is bound at or near the 3' end of the other
target-specific nucleic acid probe, so as to permit juxtaposition
of the 5' phosphate and 3' hydroxyl termini of the adjacent
target-specific nucleic acids at the desired sites of ligation
after hybridization of the target-specific nucleic acid probes to
the target nucleic acid. Such design of the target-specific nucleic
acid probes and the affinity tags is well within the level of skill
of those in the art.
[0054] Prior to hybridization to the target nucleic acid, the first
target specific nucleic acid probe is capable of binding or is
bound to a molecular post and a second target-specific nucleic acid
probe is capable of binding or is bound to a detection probe. Thus,
the hybridization may occur with (a) the first target-specific
nucleic acid probe being bound to the molecular post, (b) the
second target-specific nucleic acid probe bound to the detection
probe, (c) both being bound, or (d) neither being bound. In those
embodiments where the molecular post and/or detection probe are not
bound to the appropriate target-specific nucleic acid probe prior
to hybridization, they are bound at a later step, either after
hybridization but prior to ligation; after ligation; or after the
exonuclease digestion and/or denaturation step (see below). Based
on the teachings herein, it will be apparent to those of skill in
the art how to choose the appropriate stage of the method to carry
out binding of the molecular post to the first target-specific
nucleic acid probe and the detection probe to the second
target-specific nucleic acid probe for different experimental
designs. For example, when the molecular post comprises a magnetic
particle, it is preferable to bind the detection probe to the
second target-specific nucleic acid prior to hybridization. This
ensures that each side of the bridge binds to the appropriate
group, either the magnetic bead or the reporter group. In
embodiments where the molecular post and the detection probe are
both bound to the target-specific nucleic acid probes indirectly
via affinity tags, and the same affinity tag is used for both, then
it is preferred for at least one of the molecular post and the
detection probe to be bound to the target-specific nucleic acid
probe prior to hybridization. This is to ensure that there are not
any bridges that have reporter groups or magnetic beads on both
sides: the only possibility is that one side has a magnetic bead
and the other side has the reporter group. In embodiments where
different affinity tags are used, then the molecular post and the
detection probe can be bound at any appropriate step as noted
above.
[0055] When a non-magnetic particle is used as the molecular post,
it is preferably bound to the first target-specific nucleic acid
probe after ligation and exonuclease digestion and/or denaturation.
This is to minimize the interactions of the reporter group with the
enzymes, as such interactions can reduce the efficiency of the
enzymatic reactions. As will thus be apparent to those of skill in
the art, binding of the molecular post and the detection probe to
the target-specific nucleic acid probes can be done at any point of
the process (depending on how the procedure is designed), so long
as care is taken to ensure that only the desired binding
occurs.
[0056] As will be understood by those of skill in the art, the LXR
reactions are allowed to proceed as efficiently as possible, while
ensuring that the correct group is bound to each end of the DNA
bridge. If different moieties are used for the detection probe and
molecular post, then it is preferable to bind the two after the
exonuclease digestion and/or denaturation, so they do not interfere
with the enzymes. If the same moiety is used for both the detection
probe and molecular post, one of the two is preferably bound prior
to hybridization. In this case it is preferable to bind the most
inert group; while the linkage is the same, the groups that are
being linked to are different. (For example, avidin-biotin is used
to bind a gold nanoparticle on one side, and a magnetic bead on the
other), and thus one group is more inert than the other.
[0057] In a further non-limiting example, a "moiety" on an
accessible component of the molecular post can be designed, such as
a cysteine residue created by site-directed mutagenesis at a
specific position of a protein-based biomolecular post, such as the
.gamma. subunit of F.sub.1-ATPase. The first affinity tag can be
attached to the cysteine residue through linkage to its sulfhydryl
group. Alternatively, an affinity target can be used to coat the
molecular post, and can interact with the affinity tag. This
molecular post coated with affinity targets can then be linked
specifically to the affinity tag on the first target-specific
nucleic acid. As will be apparent to those of skill in the art,
site directed mutagenesis can be used to introduce a cysteine
residue (or other useful residues) to various protein-based
biomolecular posts so that they can be linked to affinity tags.
Furthermore, there are a variety of covalent modification reagents
that can modify specific amino acid side chains, as is known to
those of skill in the art.
[0058] In some cases it is preferred that the molecular post is
immobilized (i.e. secured in place) for detection. For example, it
may be preferred to immobilize the molecular post for some rotation
visualization techniques or if the detection depends on the
perturbation of the local environment, such as micro current or
impendence.
[0059] A series of molecular posts, either identical or two or more
different molecular posts, can be immobilized on a surface to
generate a molecular post array. If each post is coated with
different affinity targets and different first target-specific
nucleic acid probes (specific to the same or different target
nucleic acids) are labeled with different affinity tags, this
molecular post array can be used to detect multiple target nucleic
acids in a manner similar to use of a gene chip. As used herein, an
"array" comprises a solid surface, with molecular posts attached to
said surface. Arrays typically comprise a plurality of molecular
posts linked to different capture groups that are coupled to a
surface of a substrate in different, known locations. For example,
there are several silane derivatives to attach a variety of
functional groups to a glass surface. The term "solid surface" as
used herein refers to a material having a rigid or semi-rigid
surface. Such materials will preferably take the form of chips,
plates, slides, cover slips, small beads, pellets, disks or other
convenient forms, although other forms may be used. The surfaces
are generally coated with an affinity target. Such solid surfaces
can be coated in any way that improves desired binding to its
surface and/or minimizes non-specific binding to its surface. In a
specific embodiment, nickel-nitrilotriacetic acid (Ni-NTA) affinity
resin (Sigma-Aldrich product #P6611) is used. In a further
embodiment, acetylated BSA can be added to reduce non-specific
binding.
[0060] The ligation step of the methods of the invention can be
accomplished by techniques known to those of skill in the art using
commercially available nucleic acid ligases. Any DNA ligase is
suitable for use in the disclosed methods. Preferred ligases are
those that preferentially form phosphodiester bonds at nicks in
double-stranded DNA. That is, ligases that fail to ligate the free
ends of single-stranded DNA at a significant rate are preferred.
Thermostable ligases are especially preferred. Many suitable
ligases are known, such as T4 DNA ligase (Davis et al., Advanced
Bacterial Genetics--A Manual for Genetic Engineering (Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1980)), E. coli DNA
ligase (Panasnko et al., J Biol. Chem. 253:4590-4592 (1978)),
AMPLIGASE.RTM. (Kalin et al., Mutat Res., 283(2):119-123 (1992);
Winn-Deen et al., Mol Cell Probes (England) 7(3):179-186 (1993)),
Taq DNA ligase (Barany, Proc. Natl. Acad Sci. USA 88:189-193
(1991), Thermus thermophilus DNA ligase (Abbott Laboratories),
Thermus scotoductus DNA ligase and Rhodothermus marinus DNA ligase
(Thorbjamardottir et al., Gene 151:177-180 (1995)). T4 DNA ligase
is preferred for ligations involving RNA target sequences due to
its ability to ligate DNA ends involved in DNA:RNA hybrids (Hsuih
et al., Quantitative detection of HCV RNA using novel
ligation-dependent polymerase chain reaction, American Association
for the Study of Liver Diseases (Chicago, Ill., Nov. 3-7, 1995)).
In another embodiment, modified nucleic acid probes can be used
that allow adjacent nucleic acid probes to self ligate (see, for
example, U.S. Pat. No. 7,033,753; Silverman et al., Nucleic Acids
Research 2005, 33, 4978-4986).
[0061] While not required by the methods of the invention, the use
of LCR in connection with the methods can be useful. Specifically,
LCR can decrease the background and eliminate some false positives,
depending upon the initial amount of target. Thus, in a further
embodiment, ligation is accomplished by use of a ligation chain
reaction. The term "ligation chain reaction" ("LCR") describes the
process pioneered by Landegren et al. (1988 Science 241,
1077-1080). This process detects the presence of given DNA
sequences based on the ability of probes to anneal directly
adjacent to each other on a complementary target DNA molecule. The
probes are then joined covalently by the action of a DNA ligase,
provided that the nucleotides at the junction are correctly
base-paired. Thus multiple single nucleotide substitutions can be
distinguished. This strategy permits the rapid and standardized
identification of gene sequences in genomic DNA, using single
molecule FRET as a detection method (M. Wabuyele, H. Farquar, W.
Stryjewski et al., JACS 125, 6937-6945 (2003)). In this method, the
concentration of the solution is controlled so that only one
molecule can be present in the volume of the detection cell. Due to
its high specificity, LCR can be performed in crude samples,
without the need for purifying the nucleic acid target, which
significantly simplifies the assay process. The methods of the
present invention have as one advantage the ability to detect
multiple target nucleic acids simultaneously at single molecule
detection level.
[0062] In a further embodiment, the disclosed method may use target
dependent DNA ligation reactions (Cheng et al., 1996) to generate a
ligation product with affinity tags on both ends so that it can
serve as a bridge between the molecular post and the detection
probe (the "fully assembled" ligation product). Ligation reaction
requires the formation of juxtaposed 5' phosphate and 3' hydroxyl
termini of adjacent target-specific nucleic acid probes, which are
hybridized to a complementary nucleic acid target. The ligation
will occur only if the target-specific nucleic acid probes are
perfectly paired to the target nucleic acid and have no gaps
between them. In the event that the sequences are not perfectly
complimentary and ligation does not occur, there can still be a
detectable bridge that is held together by the hydrogen bonds
formed during hybridization, depending upon the initial starting
concentration of the target. The exonuclease and/or denaturation
step degrades this type of false positive, thereby increasing the
accuracy of the reaction and permitting the detection of single
nucleotides polymorphisms. Thus, the inclusion of the exonuclease
digestion and/or denaturation after the LCR changes the error rate
and the specificity of the reaction. Certain false positives will
be eliminated by inclusion of the exonuclease and/or denaturation
step. For example, if you have 1 target molecule and run enough
cycles to produce 1000 bridges, it would appear the same as if you
had 1000 targets that could hybridize but not ligate. The only way
to differentiate these two cases is using the exonuclease and/or
denaturation step.
[0063] In a non-limiting example, LCR conditions employed include
an initial hybridization step at 95.degree. C. for two minutes,
followed by 19 cycles of 1 minute at 95.degree. C. and 4 minutes at
65.degree. C. in the presence of a thermostable DNA ligase and
appropriate reaction components. Those of skill in the art are
well-versed in modifying such cycling conditions to provide optimal
hybridization and ligation based on the use of different nucleic
acid sequences or different buffer conditions.
[0064] Following ligation, the sample is treated with exonuclease
and/or denaturation to remove any imperfectly paired ligation
products. A non-limiting example of an exonuclease step is to
increase the temperature of the mixture of target and probe to
95.degree. C., and then cool it to 45.degree. C. The ligation
buffer, 330 mM Tris-acetate (pH 7.8), 660 mM potassium acetate, 100
mM magnesium acetate and 5 mM DTT, 1 mM ATP, and enzyme can then be
added to the sample to perform the ligation. The reaction time is a
function of the ratio of DNA to the amount of enzyme added and can
occur in a few minutes. The exonuclease reaction can then be run
using a reaction buffer of 50 mM Tris-HCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 10 mM MgCl.sub.2, 4 mM dithiothreitol,
and pH 7.5@25.degree. C. when using phi29 DNA polymerase (see, for
example, the web site neb.com/nebecomm/MSDSFiles/msdsM0269.pdf).
DNA Polymerase Phi29 is the preferred enzyme to perform the
exonuclease reaction as it has the largest strand displacement
activity that we are aware of, and therefore allows the longest
nucleotide sequence to be detected. However, any polymerase has the
potential to be used for this step. The amount of time required for
this step is a function of the ratio of DNA to enzyme and can be
accomplished in a few minutes.
[0065] The methods of the invention further comprise detecting the
ligation product following the product formation step. Such
detection can be by any means suitable for detecting the ligation
products, including but not limited to fluorescence microscopy,
surface plasmon resonance, gel electrophoresis, calorimetric
shifts, electric conductivity and autoradiography. Each of these
detection techniques are well within the level of skill in the
art.
[0066] In embodiments using a magnetic particle as the molecular
post, a magnetic field can be applied and varied, to differentiate
specifically bound detection probes from non-specifically bound
detection probes, resulting in movement of the detection probe
bound via the ligation product to the magnetic particle, thus
confirming target dependent association of the magnetic particle
with the detection probe. See, for example, FIG. 3. In one
embodiment, the ligation product has a magnetic bead bound to one
end and an affinity probe on the other (FIG. 3A). A first magnetic
field is induced to pull the ligation product to a surface that is
functionalized in such a manner as to bind to the affinity probe
(for example, via an avidin-biotin interaction) (FIG. 3B). In a
specific embodiment, the magnetic field is applied to the ligation
product in solution, to pull it to a surface. In a further
embodiment, the ligation product is formed on a surface, and the
magnetic field pulls the ligation product to (a) a separate region
of the surface; or (b) a second surface. The first magnetic field
is turned off and a second magnetic field is used to remove any
free magnetic particles (not bound to the surface) that did not
have the affinity probe to hold them to the functionalized surface
(FIG. 3C-D). The presence of the ligation product at the surface
can be detected by measuring the changes in the magnetic field due
to the presence of the remaining magnetic particles, or using
microscopy.
[0067] Ligation product detection includes, but is not limited to,
determining the number of ligation products present in the sample
(to provide a number of copies of the target nucleic acid in the
sample). When multiplex analysis is conducted, the detection step
preferably includes the separate detection of the different
detection probes (each specific for a different target nucleic
acid). For example, the color or type of the reporter group is
associated with a specific target. Thus multiple sets of target
specific nucleic acids can be used to assay different targets
simultaneously with the different colors seen under the microscope
(or via other detection methods) being associated with the presence
of the different targets.
[0068] In one further embodiment where molecular post binding
and/or detection probe binding occurs after ligation, the methods
of the invention further comprise forming a concentration gradient
of ligation products prior to contacting the ligation products with
the molecular post and/or detection probe. This embodiment
accelerates the binding of the molecular post and/or detection
probe to the ligation product. As used herein, the "concentration
gradient" results in decreasing the surface area in which the
ligation products are contained. This has the advantage that a
larger volume of a dilute sample can be examined with the result
that only a small area must be searched to find the evidence of the
ligation product. This results in concentrating the ligation
product in a dilute solution.
[0069] In a further embodiment a small area of the surface has a
high affinity to bind the ligation product. As the droplet of
sample is positioned over the surface, the affinity for the
ligation product removes the ligation product from the solution,
thereby creating a concentration gradient. The high affinity
binding surface may be surrounded by a low affinity surface, such
as a hydrophobic area, to enhance the concentrating effect by
restricting the size of the droplet to surface interaction.
[0070] In a specific embodiment, forming the concentration gradient
comprises providing a hydrophobic surface (including but not
limited to silane coated surface, lagmuir-blogett films, etc.), and
placing a small volume of a hydrophilic solvent (which can be any
solvent that has a hydrophobicity opposite that of the surface,
including but not limited to water, buffered solutions, saline,
etc.) containing the ligation products on the hydrophobic surface,
which creates a concentration gradient through surface tension
effects to direct the ligation products to the edge of the surface
and the bubble. This increases the amount of ligation product at
the surface edge, resulting in a decreased time of detection
through improved molecular post binding and/or detection probe
binding.
[0071] As will be understood by those of skill in the art, a
hydrophilic surface and a hydrophobic solvent can be used in an
alternative embodiment to accomplish the same goal of forming the
concentration gradient.
[0072] Any volume of solvent that is suitable for the intended
purpose can be used. The determination of a solvent volume
appropriate for a given application is well within the level of
those of skill in the art.
[0073] Other methods for forming the concentration gradient
include, but are not limited to, drying the volume of solvent on
the hydrophobic surface, and utilizing a semi-permeable
membrane.
[0074] FIG. 4 provides an example of forming a concentration
gradient.
[0075] Use of the concentration gradient provides more rapid
binding and a higher percentage of ligation products bound to the
detection probe/posts per unit time, thus improving the speed of
the overall methods. Furthermore, use of the concentration gradient
decreases the error rate significantly, by providing a localized
area of detection with a more controllable readout area.
[0076] The present invention offers significant improvements over
previous nucleic acid detection techniques. First of all, there is
no requirement for an enzyme or biomolecular motor which is
difficult to maintain. Second, the present invention provides much
more controlled conditions than are possible when using an enzyme
to generate movement. The invention herein allows a magnetic field
to induce movement of bound particles, thereby still
differentiating specifically bound probes by moving the particles
significant distances.
[0077] In another aspect, the present invention provides kits for
nucleic acid detection comprising a plurality of target-specific
nucleic acid probes that are each complementary to a target nucleic
acid, wherein upon hybridization to the target nucleic acid the
plurality of target-specific nucleic acid probes will be directly
adjacent to each other; wherein a first target specific nucleic
acid probe is capable of binding or is bound to a molecular post
and a second target-specific nucleic acid probe is capable of
binding or is bound to a detection probe, and wherein the first
target-specific nucleic acid probe and the second target-specific
nucleic acid probe are positioned at the 5' and 3' end,
respectively, of the series of target specific nucleic acid probes.
As used in this aspect of the invention, terms carry the same
meanings as for previous aspects of the invention.
[0078] In further specific embodiments, the first target-specific
nucleic acid probe is capable of binding to a molecular post,
and/or the second target-specific nucleic acid probe is capable of
binding to the detection probe, and the kit further comprises a
molecular post that binds to the first target-specific nucleic acid
probe and/or a detection probe that binds to the second
target-specific nucleic acid probe. In a further embodiment, the
molecular post is bound to a solid support, such as a glass
coverslip or other suitable support. The support can be derivatized
in any manner suitable for binding to the molecular post. In a
specific embodiment, the molecular post comprises a magnetic
particle.
[0079] The present invention also provides a composition comprising
a plurality of target-specific nucleic acid probes that are each
complementary to a target nucleic acid, wherein upon hybridization
to the target nucleic acid the plurality of target-specific nucleic
acid probes will be directly adjacent to each other; wherein a
first target specific nucleic acid probe is bound to a first
affinity tag capable of binding to a molecular post and a second
target-specific nucleic acid probe is bound to a second affinity
tag capable of binding to a detection probe, and wherein the first
target-specific nucleic acid probe and the second target-specific
nucleic acid probe are positioned at the 5' and 3' end,
respectively, of the series of target specific nucleic acid probes.
In a further embodiment, the plurality of target-specific nucleic
acid probes is ligated together.
[0080] The present invention also provides a composition
comprising:
[0081] (a) a nucleic acid complex comprising a plurality of
target-specific nucleic acid probes that are each complementary to
a target nucleic acid, wherein upon hybridization to the target
nucleic acid the plurality of target-specific nucleic acid probes
will be directly adjacent to each other; wherein a first target
specific nucleic acid probe is bound to a first affinity tag
capable of binding to a molecular post and a second target-specific
nucleic acid probe is bound to a second affinity tag capable of
binding to a detection probe, and wherein the first target-specific
nucleic acid probe and the second target-specific nucleic acid
probe are positioned at the 5' and 3' end, respectively, of the
series of target specific nucleic acid probes, and wherein the
series of target-specific nucleic acid probes are ligated
together;
[0082] (b) a molecular post bound to the first affinity tag;
and
[0083] (c) a detection probe bound to the second affinity tag.
[0084] The present invention further provides a composition
comprising:
[0085] (a) a solid support; and
[0086] (b) a plurality of molecular posts attached to the solid
support, wherein the plurality of molecular posts comprise an
affinity target for binding to a specific affinity tag. In a
specific embodiment, the molecular posts comprise magnetic
particles.
[0087] In a specific embodiment, the plurality of molecular posts
comprises more than one type of molecular post. In a further
specific embodiment, the different types of molecular posts on the
support comprise different affinity targets that are specific for
different affinity tags. In a further specific embodiment, the
composition further comprises a first target-specific nucleic acid
bound to a first affinity tag that binds to the affinity target on
the molecular post. In a further specific embodiment, the first
target specific nucleic acid probe is hybridized to a target
nucleic acid, and the target nucleic acid is further hybridized to
a second target-specific nucleic acid probe that is bound to a
second affinity tag, wherein the second affinity tag is bound to a
detection probe.
EXAMPLES
Example 1
[0088] Two different target nucleic acids, WT and MT, which differ
by one nucleotide, were tested with two different sets of
target-specific nucleic acid probes. For each of the two target
nucleic acids, the LXR reactions detailed in FIG. 1 were performed
with target-specific nucleic acid probes denoted with a + or with
target-specific nucleic acid probes containing an SNP denoted with
a -. The amount of bridge formed under each of the four conditions
was quantified by counting assembled devices (ligation products)
containing the DNA bridge with F1 (molecular post) and nanogold
(detection probe). The number of assembled devices is shown in FIG.
2 on the y-axis, where a comparison between the amount of binding
for the WT and MT targets with both complementary target-specific
nucleic acid probes and SNP target-specific nucleic acid probes is
shown as the percent increase in binding.
[0089] For experiments in which LXR products prepared from purified
target DNA were used for detection, target DNA, 3'-biotinylated
capture probe (target-specific nucleic acid), and 5'biotinylated
capture probe phosphorylated at the 3' end (target-specific nucleic
acid) were allowed to hybridize, and the capture probes were
ligated in the presence of 5,000 units of T4 ligase, T4 ligase
buffer (New England Biolabs), 4 mM ATP, and 4 mM DTT in a final
volume of 50 .mu.l. After ligation, 35 n1 of the product was
incubated with 5,000 units of Phi 29 DNA polymerase, which has
strong exonuclease activity, Phi 29 buffer (New England Biolabs), 2
mM dNTP, and BSA in a 50 n1 total volume. To bind DNA bridges to
the immobilized avidinated F.sub.1-ATPase, a 3 n1 droplet of either
LXR product, 3',5'-dibiotinylated-DNA, or 3'-biotinylated-DNA was
added to the cover slip at concentrations indicated such that the
droplet was within the surface area to which avidinated
F.sub.1-ATPase was bound, and incubated for 10 min followed by a
buffer wash.
[0090] Nanodevice assembly was completed by addition of 10 n1 of
nanorods, prepared as known in the art, in a droplet that covered
the entire surface to which avidinated F.sub.1-ATPase had become
bound, and incubated for 10 min, then washed thoroughly in
F.sub.1-ATPase buffer to minimize nonspecifically bound nanorods.
This allowed the avidin-coated gold nanorods to bind to the
F.sub.1-ATPase-immobilized, biotinylated DNA bridges. For samples
that were examined for rotation, the final buffer contained 0.5 mM
MgCl.sub.2 and 1 mM ATP.
[0091] FIG. 2 shows that the method is able to detect as few as 600
molecules of target DNA, and is able to distinguish target bound
detection probes from those that are non-specifically bound.
Example 2
[0092] The conditions used in this example are the same as in the
first example, but the data were analyzed via the HPLC rather than
on the microscope. Biotinylated 20-mer oligonucleotide probes were
mixed with a 40-mer target and allowed to ligate. Samples were then
viewed with high performance liquid chromatography (HPLC) where
each component corresponds to a distinct signal peak. When probes
were complementary with the target at the site of ligation, a
significant 3',5'-dibiotinylated 40-mer product peak formed as seen
in (A). When there was a single mismatch at the site of ligation,
no product was formed (B). Thus, HPLC results demonstrate the
specificity of ligase during the first step of LXR.
[0093] After ligation, samples were subjected to exonuclease
(phi-29 DNA polymerase) treatment. In the case of a mismatch during
ligation, the presence of a nick allowed exonuclease to bind and
extend the downstream probe, displacing the 3' biotin. This
resulted in the formation of a significant 5'-biotinylated 40-mer
product as seen by HPLC (A). If ligation was successful in the case
of a perfect match at the ligation site, no significant
5'-biotinylated 40-mer product was formed by exonuclease (B). Thus,
HPLC results demonstrate the specificity of exonuclease during the
second step of LXR.
Example 3
[0094] FIG. 4 shows an embodiment of the methods using magnetic
particles. A side view of the droplet on the surface is depicted at
the top of the figure; molecules at the edge of the droplet have
the fewest degrees of freedom, they may curve up along the edge of
the droplet, move along the surface, or move out into the middle of
the droplet. Those molecules that go to the surface are pulled out
of solution as they bind. This creates a concentration gradient
that pulls more molecules to the edge as shown in the top view.
Molecules in the middle of the droplet are able to move in any
direction; there is no preference to move toward or away from the
surface. Thus the concentration gradient formed at the edge of the
droplet and the surface is a dominating force, resulting in a
significant percentage of all the molecules binding at the edge.
Sequence CWU 1
1
6140DNAArtificialSynthetic 1aatgctaggc tcgacctagc atcggcgaat
cgcatcaggc 40220DNAArtificialSynthetic 2ttacgatccg agctggatcg
20320DNAArtificialSynthetic 3tagccgctta gcgtagtccg
20440DNAArtificialSynthetic 4ttacgatccg agctggatcg tagccgctta
gcgtagtccg 40540DNAArtificialSynthetic 5aatgctaggc tcgacctaga
atcggcgaat cgcatcaggc 40640DNAArtificialSynthetic 6ttacgatccg
agctggatct tagccgctta gcgtagtccg 40
* * * * *