U.S. patent application number 14/108630 was filed with the patent office on 2014-05-15 for systems and methods for point-of-care amplification and detection of polynucleotides.
This patent application is currently assigned to Great Basin Scientific. The applicant listed for this patent is Great Basin Scientific. Invention is credited to Robert D. Jenison.
Application Number | 20140134619 14/108630 |
Document ID | / |
Family ID | 40637139 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134619 |
Kind Code |
A1 |
Jenison; Robert D. |
May 15, 2014 |
SYSTEMS AND METHODS FOR POINT-OF-CARE AMPLIFICATION AND DETECTION
OF POLYNUCLEOTIDES
Abstract
Composition and methods for amplifying and detecting
solution-state polynucleotide targets in a single device are
described. In one aspect, a method for a coupled isothermal
amplification and detection process utilizes a coated solid
support, including a solid substrate, a cationic layer, and a
plurality of target-specific probes attached to the coated solid
support. Polynucleotide targets in the sample are amplified by an
isothermal amplification process involving in situ hybridization
onto the coated solid support. The entire process can be carried
out with a high degree of specificity under low salt conditions in
less than one hour. Further aspects of the present invention
include methods for coupled hybridization/detection of
polynucleotide targets, coated silicon biosensors optimized for use
with the coupled detection systems to provide visual detection of
polynucleotide targets under visible light conditions, and kits for
practicing in the above described methods.
Inventors: |
Jenison; Robert D.;
(Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Great Basin Scientific |
West Valley |
UT |
US |
|
|
Assignee: |
Great Basin Scientific
West Valley
UT
|
Family ID: |
40637139 |
Appl. No.: |
14/108630 |
Filed: |
December 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12779397 |
May 13, 2010 |
8637250 |
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14108630 |
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12036048 |
Feb 22, 2008 |
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12779397 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
B01J 2219/00637
20130101; B01J 2219/00612 20130101; B01J 2219/00608 20130101; B01J
2219/00722 20130101; C12Q 1/6834 20130101; B01J 2219/00529
20130101; C12Q 1/6844 20130101; B01J 2219/00626 20130101; C12Q
1/6834 20130101; C12Q 2527/107 20130101; C12Q 2523/313 20130101;
C12Q 2521/513 20130101; C12Q 1/6834 20130101; C12Q 2527/101
20130101; C12Q 2527/125 20130101; C12Q 2537/101 20130101; C12Q
1/6844 20130101; C12Q 2527/101 20130101; C12Q 2527/125 20130101;
C12Q 2537/101 20130101; C12Q 2565/519 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-149. (canceled)
150. An isothermal amplification kit for detecting the presence or
absence of a polynucleotide target in a sample, comprising: a. a
coated, solid support comprising (i) a solid support, (ii) a
coating layer comprising at least one polycationic polymer, and
(iii) a plurality of target-specific probes attached to the coating
layer; b. one or more reaction medium components selected from a
helicase enzyme, DNA polymerase, target-specific DNA primers, and
dNTPs, wherein the reaction medium components are combined, the
monovalent cation concentration is between about 10 mM and about
100 mM.
151. The kit of claim 150, further comprising one or more labelling
agents.
152. The kit of claim 151, further comprising an anti-label,
antibody-enzyme conjugate capable of biding to a label from the one
or more labelling agents.
153. The kit of claim 150, wherein the solid support is formed from
a member of the group consisting of silicon, plastic, glass, and
membrane.
154. The kit of claim 150, wherein the solid support comprises a
silicon wafer or chip.
155. The kit of claim 150, wherein the polycationic polymer is
selected from one or more of the group consisting of polylysine,
poly(lys-phe), poly(lys-tyr), poly(lys-trp), poly(arg-trp), and
poly(arg-pro-tyr).
156. The kit of claim 150, wherein the polycationic polymer is
attached to the solid support.
157. The kit of claim 150, wherein the coating layer comprises a
compound selected from one or more of the group consisting of a
cationic silane, a cationic siloxane, or a cationic derivative
thereof.
158. The kit of claim 150, wherein the coating layer comprises or
is attached to a compound selected from one or more of the group
consisting of aminopropyltri-methoxysilane,
aminopropyltriethoxysilane, amino-functional organopolylsiloxanes,
and amino-functional siloxane alkoxylates.
159. The kit of claim 150, wherein the coating layer comprises an
attachment layer.
160. The kit of claim 150, wherein the plurality of target-specific
probes are attached to an attachment layer comprising a coating of
coupling agents deposited on a surface of the solid support.
161. The kit of claim 150, wherein the plurality of target-specific
probes comprise at least one probe having at least one terminus
blocked to reduce recognition by an isothermal amplification
enzyme.
162. The kit of claim 150, wherein the plurality of target-specific
probes comprise at least one probe modified or blocked to reduce
recognition by the helicase enzyme.
163. The kit of claim 150, wherein the plurality of target-specific
probes comprise at least one probe comprising a backbone having at
least one neutral charge.
164. The kit of claim 150, wherein the plurality of target-specific
probes comprise at least one probe comprising a methylphosphonate
backbone.
165. The kit of claim 150, wherein the plurality of target-specific
probes comprise at least one probe comprising a 2'-O-methyl
linkage.
166. The kit of claim 150, wherein the reaction medium components
include a single-stranded binding protein.
167. A coated biosensor for visual detection of a polynucleotide
target in a sample, comprising: a coated, solid support comprising
(i) a solid support, (ii) a coating layer comprising at least one
polycationic polymer, and (iii) a plurality of target-specific
probes attached to the coating layer; wherein the coated, solid
support does not comprise a layer that produces a thin-film effect
and is also configured to enable optical detection under visible
light of polynucleotide targets in the sample by immersion in a
reaction medium comprising a helicase enzyme, DNA polymerase,
target-specific DNA primers, and dNTPs having a monovalent cation
concentration between about 10 mM and about 100 mM.
168. The biosensor of claim 167, further comprising one or more
labels that associate with the target-specific probes.
169. The biosensor of claim 168, further comprising an anti-label,
antibody-enzyme conjugate capable of biding to the one or more
labels.
170. The biosensor of claim 167, wherein the solid support is
formed from a member of the group consisting of silicon, plastic,
glass, and membrane.
171. The biosensor of claim 167, wherein the solid support
comprises a silicon wafer or chip.
172. The biosensor of claim 167, wherein the polycationic polymer
is selected from one or more of the group consisting of polylysine,
poly(lys-phe), poly(lys-tyr), poly(lys-trp), poly(arg-trp), and
poly(arg-pro-tyr).
173. The biosensor of claim 167, wherein the polycationic polymer
is attached to the solid support.
174. The biosensor of claim 167, wherein the coating layer
comprises a compound selected from one or more of the group
consisting of a cationic silane, a cationic siloxane, or a cationic
derivative thereof.
175. The biosensor of claim 167, wherein the coating layer
comprises or is attached to a compound selected from one or more of
the group consisting of aminopropyltri-methoxysilane,
aminopropyltriethoxysilane, amino-functional organopolylsiloxanes,
and amino-functional siloxane alkoxylates.
176. The biosensor of claim 167, wherein the coating layer
comprises an attachment layer.
177. The biosensor of claim 167, wherein the plurality of
target-specific probes are attached to an attachment layer
comprising a coating of coupling agents deposited on a surface of
the solid support.
178. The biosensor of claim 167, wherein the plurality of
target-specific probes comprise at least one probe having at least
one terminus blocked to reduce recognition by an isothermal
amplification enzyme.
179. The biosensor of claim 167, wherein the plurality of
target-specific probes comprise at least one probe modified or
blocked to reduce recognition by the helicase enzyme.
180. The biosensor of claim 167, wherein the plurality of
target-specific probes comprise at least one probe comprising a
backbone having at least one neutral charge.
181. The biosensor of claim 167, wherein the plurality of
target-specific probes comprise at least one probe comprising a
methylphosphonate backbone.
182. The biosensor of claim 167, wherein the plurality of
target-specific probes comprise at least one probe comprising a
2'-O-methyl linkage.
Description
BACKGROUND
[0001] The incidence of antibiotic resistant bacteria has
dramatically increased over the past decade. If not diagnosed and
treated appropriately, these human pathogens can cause a range of
life-threatening illnesses including septicemia and toxic shock
syndrome. Early treatment of these infections has been associated
with improved outcomes in patients. Clinical outcome studies have
shown that reducing the time to diagnosis decreases the patient's
length of stay and morbidity and mortality, leading to significant
cost savings for the hospital. Standard microbiological methods in
practice today require 24-72 hours to identify the causative
pathogen in an infection. Therefore, there is an increasing need
for rapid, easy-to-perform tests to identify antibiotic-resistant
pathogens.
SUMMARY
[0002] The present invention provides systems and methods for
amplifying and detecting solution-state polynucleotide targets in a
single device. In one aspect, a method for detecting a
polynucleotide target in a sample includes applying the sample and
a reaction medium to a coated solid support comprised of a solid
substrate, a cationic layer, and a plurality of target-specific
probes attached to the coated solid support. Polynucleotide targets
in the sample are then amplified by an isothermal amplification
process. Alternatively, the polynucleotide targets are first
hybridized to the probes, eluted from the support, and then
amplified within the same device or applied to a second coated
solid support for amplification by an isothermal amplification
process. The amplification process is carried out under conditions
suitable for coupled amplification/hybridization in low salt
conditions. An additional, optional high salt hybridization step
may be included for enhanced hybridization and increased
sensitivity of detection.
[0003] Biotinylated primers may be utilized in the amplification
reaction to facilitate detection of bound polynucleotide targets
captured by the probes on the coated solid support. By way of
example, anti-biotin/horseradish peroxidase conjugates can be bound
to the biotinylated amplification products immobilized on the
surface of the coated solid support. The enzyme-catalyzed
conversion of a chromogenic substrate, such as tetramethylbenzidine
can result in production of precipitable matter on the surface of
the coated solid support creating a signal visually detected by the
naked eye under visible light conditions.
[0004] In another aspect, a method for detecting polynucleotide
targets in a sample includes applying the sample and a reaction
medium to a coated solid support comprised of silicon substrate, a
cationic layer, and a plurality of target-specific probes attached
to the cationic layer. Polynucleotide targets in the sample are
then amplified by an isothermal amplification process. The
amplification process is carried out under conditions suitable for
coupled amplification/hybridization in low salt conditions. An
additional, optional high salt hybridization step may be included
for increased sensitivity. Through the use of appropriate detection
reagents, polynucleotide targets captured by the probes may be
visually detected as described below.
[0005] Certain aspects of the Applicant's invention are predicated
on the unexpected discovery that a cationically configured solid
support containing immobilized target-specific capture probes can
reduce the negative effects of helicase-catalyzed unwinding and
release of surface-bound duplexes from the coated solid support
during a coupled isothermal amplification/hybridization
process.
[0006] In another aspect, a method for detecting the presence or
absence of a polynucleotide target in a sample includes applying
the sample and a reaction medium to a coated solid support
including a solid substrate and a plurality of target-specific
probes attached to the coated solid support. The sample is subject
to conditions and reagents suitable for denaturing polynucleotide
targets by an enzymatic process prior to hybridization and
detection of enzymatically denatured polynucleotide targets bound
to the target-specific probes.
[0007] In a particular embodiment, the coated solid support
includes a silicon substrate and a cationic layer attached thereto.
The coated silicon support does not comprise a coating layer (such
as an anti-reflective layer) capable of mediating eye-visible
detection of polynucleotides targets by destructive interference.
Target-specific probes attached to the cationic layer are then
hybridized to polynucleotide targets in the sample, which are then
detected using appropriate detection reagents.
[0008] In another aspect, the present invention provides a coated
silicon biosensor for direct, visual detection of polynucleotide
targets deposited on a coated silicon support comprised of a
silicon substrate, a cationic layer, and a plurality of
target-specific probes attached to the coated silicon support. The
coated silicon support can be configured to support visual
detection of polynucleotide targets in the sample by a process that
is not based on destructive interferences principles, but rather a
combination of light scattering and colorimetry. In particular,
Applicant has unexpectedly discovered a type of silicon support
that can facilitate rapid, visual detection of bound polynucleotide
targets under visible light conditions without the use of an
antireflective layer conventionally facilitating detection of
analytes, including nucleic acids, based on the principles of
destructive interference.
[0009] In a preferred embodiment, the present invention utilizes a
coupled helicase-dependent amplification (HDA)/hybridization system
carried out under isothermal, low salt conditions. Amplification
reactions are performed with the amplification primers in solution
phase in the presence of the probe-containing silicon support or
microchip under conditions providing solution-phase
amplification/hybridization reaction kinetics. Amplification
products are being continually denatured by helicase. As their
concentration(s) increase, the denatured polynucleotides can anneal
to oligonucleotide primers, re-anneal to complementary strands or
anneal to probes immobilized on the surface of a solid support,
such as a microchip. At the conclusion of the reaction, the support
or chip may be washed, and the presence of the target
polynucleotide detected. When used in conjunction with a
silicon-based support or microchip according to the present
invention, the coupled amplification/hybridization system is
capable of supporting direct visual detection of polynucleotide
targets under visible light conditions.
[0010] From a 10 .mu.L blood sample or nasal swab containing as few
as 10 target sequence copies, high concentrations of material can
be rapidly amplified (10.sup.5 to 10.sup.10 copies of DNA produced
depending on the approach). Advantageously, all signal generation
steps can be carried out rapidly under isothermal, low salt
conditions, generating highly sensitive signals visible to the
unaided eye. Depending on the amount of polynucleotide targets in
the sample, the entire process from extraction to detection can be
completed within 1 hour, requiring just 10 minutes of hands-on time
at low cost using one simple coated solid support without expensive
or sophisticated instrumentation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 depicts helicase-mediated denaturation and
hybridization of single-stranded polynucleotide targets from
aliquots of HDA amplified products diluted 1/5000 in 1.times.
hybridization buffer (high salt) or 1.times.HDA buffer (low
salt).
[0012] FIG. 2 depicts more stable binding of amplified
polynucleotide targets to the coated solid support in low salt
conditions (1.times. IsoAmp II) than in high salt conditions
(1.times.Hyb).
[0013] FIG. 3 depicts representative doubling times (FIG. 3A) and
doubling rates (FIG. 3B) for HDA amplicons produced using selected
primer sets (J, L, Q, and P) directed to the mecA gene specifying
methicilin resistance in Staphylococcus.
[0014] FIG. 4 depicts a calculation of predicted time to result
(TTR) on silicon chips based on the lower limit of detection (LLOD)
and amplicon doubling times.
[0015] FIG. 5 depicts a dose response analysis to calculate the
lower limit of detection (LLOD) of MRSA DNA amplified by HDA and
hybridized to coated silicon chips.
[0016] FIG. 6 depicts a time to result (TTR) analysis of MRSA DNA
amplified by HDA and hybridized to coated silicon chips.
[0017] FIG. 7 depicts an analysis of direct, on-chip HDA detection
to a thin film biosensor chip (On-chip).
[0018] FIG. 8 depicts detection of HDA amplicons from a blood
culture isolate on (left to right): an aged chip (Aged), an
un-blocked chip (Un-blocked), an N-hydroxysuccinimidyl (NHS)
acetate blocked chip (NHS--Ac block), and an acetic anhydride
blocked chip (AcAn block).
[0019] FIG. 9 depicts oligonucleotide sequences and/or structural
modifications used in the primers or probes described in the
Examples below.
DETAILED DESCRIPTION
[0020] In order to provide a clear and consistent understanding of
the specification and claims, the following definitions are
provided.
[0021] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation. Numeric ranges
recited herein are inclusive of the numbers defining the range and
include and are supportive of each integer within the defined
range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUBMB Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. Unless otherwise noted, the terms "a"
or "an" are to be construed as meaning "at least one of." The
section headings used herein are for organizational purposes only
and are not to be construed as limiting the subject matter
described. All documents, or portions of documents, cited in this
application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose. In the
case of any amino acid or nucleic sequence discrepancy within the
application, the figures control.
[0022] As used herein, the phrase "coated solid support" refers to
an assay configuration including a solid substrate; one or more
coating layers and a plurality of target-specific probes attached
to the coated solid support. Exemplary coating layers include but
not limited to cationic layers, silane layers, siloxane layers,
anti-reflective layers, probe density enhancing materials,
attachment layers and the like.
[0023] As used herein, the term "surface" generally refers to a
covalently-contiguous geometrical domain or a region of a
geometrical domain directly contactable by surrounding media and
having functional groups supporting chemical interactions between
polynucleotides, target-specific probes, and/or surface modified
chemical structures through electrostatic interactions, hydrogen
bonding, Van der Waals interactions, London interactions,
hydrophobic interactions or combinations thereof. In some
embodiments of the invention, a zwitter ionic surface may be used
to support biomolecular adsorption. A surface of the coated solid
support may be fabricated on the solid support or it may be an
intrinsic property of the solid support. A nonlimiting example of
surface fabrication is aminosilanization wherein cationic
functional groups are covalently linked to the solid support.
[0024] The term "solid substrate" refers to any material
intrinsically having a surface or any material that may be modified
to create a surface that can be configured for immobilization of
nucleic acid probes. The solid substrate provides a surface that is
transferable from solution to solution for detection of
polynucleotide targets, and includes but is not limited to slides,
sheets, strips, dipsticks, membranes, films, filters, beads,
nanoparticles, magnetic particles, microtiter wells, tubes,
strings, or any surface that can be configured for immobilization
of nucleic acid probes. A solid substrate may be formed from
silicon, glass, fiberglass, plastics, such as polycarbonate,
polystyrene or polyvinylchloride, complex carbohydrates, such as
agarose and Sepharose.TM., polymeric resins, including those formed
from acrylic, such as polyacrylamide, nitrocellulose filters or
other membranes, ceramic, latex beads, metals, such as gold,
organic and inorganic compounds, or combinations thereof.
[0025] The phrase "cationic layer" refers to a cationic layer in
the coated solid support, the cationic layer comprising a plurality
of cationic functional groups to which are attached target-specific
probes or secondary agents to which target-specific probes can be
attached.
[0026] The term "anti-reflective layer" refers to a thin-film
coating that is anti-reflective to specific wavelengths of light so
as to create characteristic surface color changes resulting from
destructive interference. More particularly, when reflected light
from the surface-thin film interface is out of phase with light
reflected from the air-thin film interface, specific wavelengths of
light are eliminated from the reflecting light by destructive
interference so as to create characteristic thin film surface color
changes.
[0027] The term "functional group" refers to the atom(s)
responsible for the characteristic reactions of a compound. For
example, the functional group of alcohols is --OH, the functional
group of aldehydes is --CHO, the functional group of carboxylic
acids is --COOH. A given functional group behaves in approximately
the same way in all molecules of which it is a part. A single
molecule may have a plurality of functional groups. Functional
groups may mediate, for example, a noncovalent interaction between
a surface and a polynucleotide. Exemplary functional groups may
include, but are not limited to biotin, N-hydroxysuccinimide,
vinylsulfone, metal ion chelates (e.g., Ni.sup.2+-NTA), glutathione
binding group, amino, aldehyde, epoxy, mercapto, maleimide,
heparin, methoxy, sulfonate, silane, azide, acrylate, aldehyde,
isocyanate, phosphonate, and epoxy.
[0028] The term "nucleic acid" refers to a polydeoxyribonucleotide
(DNA or an analog thereof) or polyribonucleotide (RNA or an analog
thereof) made up of at least two, and preferably ten or more bases
linked by a backbone structure. In DNA, the common bases are
adenine (A), guanine (G), thymine (T) and cytosine (C), whereas in
RNA, the common bases are A, G, C and uracil (U, in place of T),
although nucleic acids may include base analogs (e.g., inosine) and
abasic positions (i.e., a phosphodiester backbone that lacks a
nucleotide at one or more positions, U.S. Pat. No. 5,585,481).
Exemplary nucleic acids include single-stranded (ss),
double-stranded (ds), or triple-stranded polynucleotides or
oligonucleotides of DNA and RNA.
[0029] The term "polynucleotide" refers to nucleic acids containing
more than 10 nucleotides.
[0030] As used herein, the term "oligonucleotide" refers to a
single stranded nucleic acid containing between about 15 to about
100 nucleotides.
[0031] The term "nucleic acid backbone" refers to nucleic acids
groups or linkages, including but not limited to
sugar-phosphodiester linkages, 2'-O-methyl linkages, guanidine
linkers in DNA ("DNG"), S-methylthiourea linkers, methylphosphonate
linkages, phosphoramidate linkages, amide backbone modifications as
in polyamide or peptide nucleic acids (PNA), phosphorothioate
linkages, phosphonic ester nucleic acid linkages, pyranosyl
oligonucleotide linkages, bicyclo- and tricyclo-nucleic acid
linkages, formacetal and 3'-thioformacetal linkages, morpholino
linkages, or other modifications of the natural phosphodiester
internucleoside bond, or combinations of such linkages in a single
backbone. A nucleic acid backbone may include a mixture of linkages
in the same nucleic acid (e.g., sugar-phosphodiester and
2'-O-methyl linkages) or may have all of one type of linkages
(e.g., all 2'-O-methyl or all amide modification linkages).
[0032] The term "sample" refers to any biological sample source
containing or suspected of possibly containing a polynucleotide
target or polynucleotide target sequence. The test sample can be
derived from any biological source, such as for example, blood,
bronchial alveolar lavage, saliva, throat swabs, ocular lens fluid,
cerebral spinal fluid, sweat, sputa, urine, milk, ascites fluid,
mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissues,
fermentation broths, cell cultures, chemical reaction mixtures and
the like. The test sample can be used (i) as directly obtained from
a sample source or (ii) following a pre-treatment to modify the
character of the sample. Thus, the test sample can be pre-treated
prior to use by, for example, preparing plasma from blood,
disrupting cells, preparing liquids from solid materials, diluting
viscous fluids, filtering liquids, distilling liquids,
concentrating liquids, inactivating interfering components, adding
reagents, purifying nucleic acids, and the like.
[0033] The terms "polynucleotide target", "target sequence", and
"target nucleic acid" are used interchangeably and refer to a
nucleic acid in a test sample having a sequence of nucleotide bases
to which another sequence, such as target-specific probe binds via
standard complementary base pairing. The polynucleotide target is
directed to a nucleic acid sequence that can be detected,
amplified, or both amplified and detected. The polynucleotide
target is generally provided in a solution state, which is then
hybridized to a target-specific probe. Polynucleotide targets of
the invention may be single or double stranded and may comprise
DNA, RNA, as well as combinations or derivatives thereof. The
target sequence may be a relatively small part of a larger nucleic
acid, such as a specific subsequence contained in a genomic DNA or
messenger RNA (mRNA). Those skilled in the art will appreciate that
a target nucleic acid may exist in different forms, i.e.,
single-stranded, double-stranded, triple-stranded, or mixtures
thereof, such as in a partially double-stranded hairpin structure
or partially double-stranded duplex structure, and will further
appreciate that a target sequence may be present on any strand (+
or -) of the structure. For simplicity, a target nucleic acid may
be described as all or part of a single strand, but this is not
meant to limit the meaning of a target to one or a particular
nucleic acid strand. It is well known in the art that a
multi-stranded nucleic acid is readily converted to its
single-strand components by using standard methods, such as by
heating a nucleic acid above its melting temperature (Tm) and/or by
using chemical denaturants.
[0034] The term "target-specific probe" refers to a polynucleotide
bound to a surface that binds specifically to a polynucleotide
target sequence and which binding is capable of producing, directly
or indirectly, a detectable signal to indicate the presence of the
polynucleotide target sequence. Preferably, the target-specific
probe is a single stranded oligonucleotide having a length between
about 12 nucleotides to about 60 nucleotides. The target-specific
probe can be linked to a label in the detection step subsequent to
hybridization. Labeled target-specific probes may include a linkers
and/or a labels thereto (see e.g., U.S. Pat. Nos. 5,185,439,
5,283,174, 5,585,481 and 5,639,604).
[0035] The term "blocked" refers to a functional group in (in a
polynucleotide, polypeptide or non-polymeric agent) that is
chemically modified or derivatized to render the functional group
chemically inert or to reduce or eliminate enzymatic recognition as
a result of the modification or derivatization. By way of example,
a target-specific probe end may be "blocked" (by e.g., a "blocking
group") by reaction with or inclusion of a chemical group and/or
structural element rendering the probe less suitable for
recognition (or reaction) by an enzyme, such as helicase or DNA
polymerase, thereby preventing or substantially reducing e.g., the
enzyme from binding at, or near the blocked end, or catalyzing an
enzymatic reaction at or near the blocked end. A "blocked probe" or
"blocked oliognucleotide probe" may be created by structurally
modifying an existing oligonucleotide or by pre-engineering a
suitable modification or structural element into the original
design of, for example, a commercially obtainable oligonucleotide
probe. Free amino groups in cationic layers of the present
invention may be similarly "blocked" to provide a desirable level
of charge density/reactivity on a coated solid support surface.
[0036] The term "modified probe", "modified target-specific probe",
and "modified oligonucleotide" refers to a single stranded nucleic
acid having an unconventional structure, including at least one
nucleic acid structural element not found in human genomic DNA.
Generally, this refers to a purposeful variant from classical ribo-
and deoxyribonucleotides adenine, thymine, guanine, cytosine, or
uracil residues linked by phosphodiester bonds. The non-natural
structural element may be added to a conventional oligonucleotide
or it may be included in the design of the oligonucleotide during
its synthesis. When used in this context herein, the term will
generally mean: (1) a variant of the classical nucleotides leading
to a higher binding efficiency when a target-specific probe is
hybridized to a polynucleotide target as compared to an otherwise
identical target-specific probe containing the classical
nucleotides; and/or (2) a variant of the classical nucleotides
producing a substrate with structural features conferring a
reduction or elimination in the recognition by an isothermal
amplification enzyme of the target-specific probe bound to the
polynucleotide target or conferring a reduction or elimination in
enzymatic denaturation of the probe bound to the polynucleotide
target (as compared to an otherwise identical target-specific probe
containing the classical nucleotides).
[0037] The term "primer" refers to a single stranded nucleic acid
capable of binding to a single stranded region on a polynucleotide
target to facilitate polymerase dependent replication and/or
amplification of the polynucleotide target.
[0038] The term "polymer" refers to a chain of molecules consisting
of structural units and repeating units connected by a covalent
chemical bond.
[0039] The term "silane" or "silanizing reagent" refers to a
compound or reagent containing a silicon atom with or without a
polymeric chain of repeating subunits. Exemplary silanes include
but are not limited to organosilanes, aminosilanes, vinylsilanes,
epoxysilanes, methacrylsilanes, sulfursilanes, alkylsilanes,
polyalkylsilanes, (alkyl)alkoxysilanes, aminoalkylsilanes,
(aminoalkyl)alkoxysilanes (such as (3-aminopropyl)triethoxysilane),
and the like.
[0040] The term "siloxane" refers to a polymeric compound
containing a silicon-oxygen-silicon (Si--O--Si) molecular unit.
[0041] As applied to polynucleotides, the term "amplification"
refers to refers to any known in vitro procedure for producing
multiple copies of a polynucleotide target fragment (i.e.
"amplicons") in a linear or exponential fashion with temperature
cycling (e.g., polymerase chain reaction, PCR) or without
temperature cycling (e.g., isothermal amplication).
[0042] The term "isothermal amplification" refers to an
amplification process that does not require temperature cycling
between the polymerization and nucleic acid denaturation steps.
Accordingly, the polymerization and amplification steps in an
isothermal amplification process can be carried out at
substantially constant temperature conditions. Isothermal
temperatures for isothermal amplification reactions are generally
below the melting temperature (Tm, the temperature at which half of
the potentially double-stranded molecules in a mixture are in a
single-stranded, denatured state) of the predominant reaction
product, generally 90.degree. C. or below, and usually between
about 37.degree. C. to about 75.degree. C.
[0043] Although the polymerization reaction may occur in isothermal
conditions, an isothermal process may optionally include a
pre-amplification heat denaturation step to generate a
single-stranded target nucleic acid to be used in the isothermal
amplification step.
[0044] The term "isothermal amplification enzyme" refers to an
enzyme associated with an amplification process, including but not
limited to polymerases, enzymatic denaturation enzymes, such as
helicases, and denaturation accessory enzymes, including single
stranded binding proteins and the like. An isothermal amplification
enzyme may be thermophilic or mesophilic in nature.
[0045] The terms "helicase-dependent amplification" and "HDA" are
used interchangeably to describe an in vitro reaction process for
amplifying nucleic acids that uses a helicase preparation for
unwinding a double stranded nucleic acid to generate templates for
primer hybridization and subsequent primer-extension via one or
more polymerase enzyme(s). HDA utilizes two oligonucleotide
primers, a first primer hybridizing to a complementary sequence in
the sense strand of a polynucleotide target sequence and a second
primer hybridizing to a complementary sequence in the anti-sense
strand of a polynucleotide target sequence, whereby the two primers
define the outer boundaries of the amplified polynucleotide target.
The HDA reaction constitutes a general method for
helicase-dependent nucleic acid amplification.
[0046] The terms "hybridization" and "hybridize" are used
interchangeably to refer to the binding of a primer or
target-specific probe to a single stranded region of the
polynucleotide target under conditions in which primer or probe
binds specifically to its complementary sequence in the
polynucleotide target, but not other polynucleotide regions. The
specificity of hybridization may be influenced by the length of the
oligonucleotide primer, the temperature in which the hybridization
reaction is performed, the ionic strength, and the pH.
[0047] The terms "complementary" or "complementarity of" are used
in the context of nucleic acids to mean that a nucleotide sequence
in one nucleic acid strand capable of hydrogen bonding to another
sequence on an opposing nucleic acid strand due to the orientation
of the functional groups. The complementary bases typically are, in
DNA, A with T and C with G, and, in RNA, C with G, and U with A.
"Substantially complementary" means that a sequence in one strand
is not completely and/or perfectly complementary to a sequence in
an opposing strand, but that sufficient bonding occurs between
bases on the two strands to form a stable hybrid complex in set of
hybridization conditions (e.g., salt concentration and
temperature). Such conditions can be predicted by using the
sequences and standard mathematical calculations known to those
skilled in the art to predict the Tm of hybridized strands, or by
empirical determination of Tm by using routine methods. Tm refers
to the temperature at which a population of hybridization complexes
formed between two nucleic acid strands are 50% denatured. At a
temperature below the Tm, formation of a hybridization complex is
favored, whereas at a temperature above the Tm, melting or
separation of the strands in the hybridization complex is favored.
Tm may be estimated for a nucleic acid having a known G+C content
in an aqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41(%
G+C), although other Tm computations are known in the art which
take into account other nucleic acid structural
characteristics.
[0048] The phrase "conditions suitable for hybridization" refers to
the cumulative environmental conditions sufficient to facilitate
the specific binding of a first nucleic acid strand to a second
nucleic acid strand by complementary strand interactions and
hydrogen bonding so as to result in a stable hybridization complex
or hybrid. Such conditions include the chemical components and
their concentrations (e.g., salts, chelating agents, formamide) of
an aqueous or organic solution containing the nucleic acids, and
the temperature of the mixture. As is known to those skilled in the
art, other well known factors, such as the length of incubation
time or reaction chamber dimensions may contribute to the
environment.
[0049] The terms "high salt" or "high salt conditions" are used
herein with reference herein to hybridization conditions having a
monovalent cation concentration greater than about 0.5 M.
[0050] The terms "low salt" or "low salt conditions" are used
herein with reference to hybridization conditions in which the
monovalent cation concentration between about 10 mM to about 100
mM, preferably between about 25 mM to about 75 mM.
[0051] The terms "melting", "unwinding" or "denaturing" refer to
separating all or part of two complementary strands of a nucleic
acid duplex.
[0052] The term "helicase" refers to an enzyme necessary for
enzymatically unwinding or denaturing a nucleic acid alone or in
combination with at least one helicase accessory protein.
[0053] The term "helicase accessory protein" refers to an
additional protein that may be necessary for helicase activity or
for stimulating helicase activity. Helicase accessory proteins
include single strand DNA binding proteins (SSBs), which may be
necessary for unwinding nucleic acids when using mesophilic
helicases. E. coli MutL protein is an exemplary accessory protein
for enhancing UvrD helicase melting activity.
[0054] The term "helicase preparation" refers to a mixture of
helicase(s) and/or helicase accessory proteins necessary and
sufficient for enzymatically unwinding or denaturing a nucleic
acid. Where a thermostable helicase is utilized in a helicase
preparation, the presence of a single stranded binding protein may
be optional. A helicase preparation, when combined with a DNA
polymerase, a nucleic acid template, deoxynucleotide triphosphates,
and primers is generally capable of achieving isothermal nucleic
acid amplification in vitro.
[0055] The terms "attached" and "linked" are used interchangeably
to refer to any chemical connection between two components or
compounds, including both direct and indirect chemical connections.
The connection can be covalent or non-covalent in nature. Examples
of non-covalent attachments include, but are not limited to,
electrostatic interactions, ionic interactions, hydrogen bonding,
Van Der Waals interactions, dipole-dipole interactions, and
hydrophobic interactions.
[0056] The term "label" refers to a molecular moiety, compound or
conjugate that is directly or indirectly joined to a
target-specific probe or polynucleotide target for detection (e.g.,
an amplified polynucleotide), the label containing a physical or
chemical characteristic capable of eliciting a detectable and/or
measurable signal indicative of duplex formation between a
target-specific probe and a complementary polynucleotide target,
including but not limited to enzyme-catalyzed signals and the like.
Direct labeling can occur through bonds or interactions that link
the label to the polynucleotide (e.g., covalent bonds or
non-covalent interactions), whereas indirect labeling can occur
through use a "linker" or bridging moiety, such as an
oligonucleotide or antibody, which may be further directly or
indirectly labeled. Exemplary labels may include but are not
limited to chromophores; optically detectable dyes, particles, or
compounds, including colorimetric, luminescent, fluorescent,
bioluminescent, chemiluminescent, and phosphorescent compounds;
antibodies; haptenic or antigenic compounds used in combination
with a suitably labeled antibody; specific ligands or binding pair
members containing a ligand recognition site, such as such as
biotin or avidin; enzymes; enzyme cofactors or substrates;
complementary nucleotide sequences; enzymes; enzyme cofactors or
substrates; radioisotopes; and the like. Bridging moieties may
further be included to amplify a detectable signal. In addition,
the label may include a variety of different reactive groups or
chemical functionalities suitable for linkage to a variety of
biomolecule agents.
[0057] It will be understood that directly detectable labels may
require additional components such as, for example, substrates,
triggering reagents, light, and the like to enable detection of the
label. When indirectly detectable labels are used, they are
typically used in combination with a "conjugate". A conjugate is
typically a specific binding member which has been attached or
coupled to a directly detectable label. Coupling chemistries for
synthesizing a conjugate are well known in the art and can include,
for example, any chemical means and/or physical means that does not
destroy the specific binding property of the specific binding
member or the detectable property of the label.
[0058] The term "signal" refers to a property or characteristic of
a detectable label that permits it to be visually or instrumentally
detected and/or distinguished. Exemplary signals include but are
not limited to chromogenic signals, fluorescent signals,
chemiluminescent signals, radioactive signals, and the like.
[0059] The phrase "lower limit of detection" (or "LLOD") refers to
the lowest concentration of target that can be visually detected in
an assay using conventional laboratory equipment.
[0060] The present invention provides systems and methods for
amplifying and/or detecting solution-state polynucleotide targets.
In one aspect, a method for coupled
amplification/hybridization/detection of polynucleotide target(s)
in a sample includes applying the sample and a reaction medium to a
coated solid support comprised of solid support, a cationic layer,
and a plurality of target-specific probes attached to the coated
solid support. Polynucleotide targets in the sample may be
amplified by an isothermal amplification process and hybridized to
the target-specific probes under low salt conditions.
[0061] Advantageously, the present invention provides coated solid
supports and their use in methods for rapid, point-of-care
amplification detection of polynucleotide targets bound to the
coated solid supports of the present invention. Applicant has
unexpectedly discovered a method for amplifying and rapidly
detecting a polynucleotide target in a coupled isothermal
amplification/hybridization/detection process. Applicant has
determined that a cationically configured solid support containing
immobilized target-specific capture probes can reduce the negative
effects of unwinding and release of surface-bound duplexes from the
coated solid support during a coupled isothermal
amplification/hybridization process, which might otherwise reduce
detection of captured duplexes, particularly under low salt
conditions. The negative effects of unwinding and release of
duplexes during the coupled isothermal amplification/hybridization
process may be also reduced using target-specific capture probes
modified or incorporated with a structural element to reduce or
eliminate recognition by an isothermal amplification enzyme (such
as a helicase) when bound to a polynucleotide target.
[0062] Further, the coupled amplification/hybridization/detection
system of the present invention can be practiced without requiring
separate or extra steps directed to denaturation or annealing of
amplicon materials to surface-immobilized single-stranded DNA
capture probes.
[0063] The coated probe-containing supports of the present
invention provide more optimal isothermal
amplification/hybridization reaction kinetics. In one aspect, an
isothermal amplification reaction is performed in solution phase
onto the coated, probe-containing solid support using suitable
amplification enzymes, accessory products, and target-specific
primers. As the amplified polynucleotide target concentration(s)
increase, the polynucleotide targets anneal to oligonucleotide
primers, re-anneal to complementary strands, or anneal to probes
immobilized on the surface of a solid support, such as a microchip.
Target-specific probes may be further modified or incorporated with
a structural element to reduce or eliminate recognition by an
isothermal amplification enzyme (such as a helicase) when bound to
a polynucleotide target. In a preferred embodiment, the
amplification products are being continually denatured by helicase
in a helicase-dependent amplification reaction under conditions
supporting hybridization of single stranded reaction products to a
coated solid support in the form of a biosensor chip. At the
conclusion of the amplification/hybridization reactions, the
support or chip may be washed, and the presence of the
polynucleotide target detected.
I. Material Components of the Present Invention
[0064] 1. Sample and Sample Processing
[0065] A sample for use in the present method includes any nucleic
acid source containing or potentially containing a polynucleotide
target for detection. As such, the sample may include a
polynucleotide preparation or extract from any nucleic
acid-containing source, including animal cells, microbial cells,
such as bacteria and fungi, viruses, or combinations therefrom.
Nucleic acids may be extracted from any polynucleotide source
material using conventional extraction methodologies known to those
of skill in the art, including clinical samples from blood (e.g.,
10 .mu.l) or nasal swabs. A nucleic acid extract may be diluted
into a reaction buffer for direct amplification. In other
embodiments, the sample may include reaction products from an
isothermal nucleic acid amplification process or conventional
nucleic acid amplification process, such as polymerase chain
reaction (PCR).
[0066] 2. Coated Solid Support
[0067] In one embodiment, a coated solid support for polynucleotide
target detection includes a solid substrate, a cationic layer, and
a plurality of target-specific probes attached to the coated solid
support. In another embodiment, a coated solid support for
polynucleotide target detection includes a plurality of
target-specific probes attached to the coated solid support.
[0068] In another embodiment, a coated solid support for
polynucleotide target detection includes a silicon-based biosensor
for visual detection of a polynucleotide target in a sample. The
silicon-based biosensor includes a coated silicon support comprised
of a silicon substrate, a cationic layer, and a plurality of
target-specific probes attached to the coated silicon support,
whereby the coated silicon support is configured to support visual
detection of polynucleotide targets in the sample and does not
comprise a coating layer capable of mediating visual detection of
polynucleotide targets by destructive interference. In a particular
embodiment, the coated silicon support does not comprise an
antireflective layer used in thin film biosensors.
[0069] 2.1. Solid Substrate
[0070] A solid substrate according to the present invention can be
any material intrinsically having a surface or any material that
can be modified to create a surface to which a single-stranded
nucleic acid may be attached, either covalently or noncovalently. A
solid substrate may be formed from silicon, glass, plastic,
polypropylene, ceramic, metallic, organic, inorganic materials or
combinations thereof. A solid support may be in the form of a
slide, microchip, microarray, microtiter plate, dipstick, sheet,
membrane filter, film, bead, or any other suitable support forms
known in the art.
[0071] The solid substrate may be porous or nonporous. Exemplary
nonporous substrates include but are not limited to materials
commonly used for construction of nucleic acid microarrays, such as
glass slides, surface-derivatized glass slides, silicon wafers, or
any of a variety of laboratory-grade plastics. Plastic substrate
materials may polymethylacrylic, polyethylene, polypropylene,
polyacrylate, polymethylmethacrylate, polyvinylchloride,
polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,
polysulfone, cellulose acetate, cellulose nitrate, nitrocellulose,
and mixtures thereof.
[0072] Exemplary porous substrates include bibulous or nonbibulous
membrane filters. Filters for nucleic acid attachment and detection
are well known in the molecular biology, and include, for example,
filters made from nitrocellulose, nylon, or positively-charged
derivatized nylon.
[0073] In one embodiment, the solid substrate is in the form of
silicon wafer or chip. In a particular embodiment, a silicon wafer
for use in the present invention includes an un-polished "rough
side" surface used for application of cationic layers and/or
probes. A rough side silicon surface may be provided by use of a
silicon wafer having a polished side and a rough, un-polished side.
In one embodiment, the surface of the rough, un-polished side may
have a roughness characterized by a peak-to-valley range from about
1 .mu.M to about 10 .mu.M, from about 1 .mu.M to about 5 .mu.M, or
from about 0.5 .mu.M to about 2 .mu.M. The roughness of the surface
may be governed or modulated by the time of exposure to a caustic
bath (e.g., concentrated NaOH, 90.degree. C.). In a particularly
embodiment, the rough un-polished silicon surface is subjected to
the caustic bath for about 15 seconds.
[0074] Applicants have unexpectedly found that the rough side
surface of a silicon wafer can be employed without further
polishing to provide a visual detection medium without the need for
further polishing or thin film coatings for destructive
interference-based visual detection. While not wishing to be bound
by theory, it is believed that the rough side silicon surface for
use in the present invention can support visual detection of
polynucleotide targets by a process that is not based on
destructive interferences principles, but rather a combination of
light scattering and colorimetry.
[0075] In alternative embodiments, a thin film coated solid support
(or biosensor) may be utilized in which a polished side of a
silicon wafer is coated with an antireflective layer to provide a
medium for visual detection of target polynucleotides by
destructive interference. The antireflective layer may be formed as
a coating of silicon nitride as previously described (Jenison et
al., Expert Rev. Molec. Diagn., 2006). An additional attachment
layer comprised of T-structure polydimethylsiloxane may be formed
thereover to facilitate conjugation to the target-specific probes.
Exemplary thin film biosensors, including attachment layers for use
in a thin-film biosensor for use in the methods of the present
invention are described in U.S. Pat. No. 5,955,377 and U.S. patent
application Ser. No. 11/761,782, filed Jun. 12, 2007, the
disclosures of which are incorporated by reference herein.
[0076] Solid substrates for use in the present invention may be
molded into any of a variety of shapes and forms. Examples of such
shapes and forms include, but are not limited to, sheets, films,
slides, gels, foams, filaments, threads, membranes, beads, plates,
and the like. Substrates may be fabricated in the form of a planar
device having discrete isolated areas in the form of wells,
troughs, pedestals, hydrophobic or hydrophilic patches, die-cut
adhesive reservoirs or other physical barriers to fluid flow.
Examples of such substrates include, but are not limited to,
slides, microplates, sheets, films, dipsticks, and the like.
Because the devices of the present invention are particularly
useful in the preparation of polynucleotide arrays for detection of
polynucleotide targets, a cationic layer is preferably fabricated
on a device having at least one flat planar surface, such as a
slide.
[0077] The size of the coated solid support may vary, depending on
the final use of the immobilized polynucleotide targets. Those
skilled in the art will appreciate that arrays of polynucleotides
immobilized on miniaturized solid substrates have been under
development for many years. These solid substrates can be measured
in terms of mm.sup.2 planar surface area and can have numerous
different immobilized polynucleotide targets, each attached to a
different site-specific location on the miniaturized solid support.
Solid substrates in the form of slides or dipsticks are also within
the scope of the present invention. As known in the art, dipsticks
typically are rectangular in shape with each side measuring a few
centimeters.
[0078] In a particular embodiment, the solid substrates may include
a probe density enhancing material in the form a bead or pellet
containing a coated cationic surface. Beads can provide a means for
increasing probe density on the coated solid support. A bead may
constitute a solid substrate according to the present invention or
it may additionally constitute a probe density enhancing material
for linkage to another solid substrates or solid substrates
coating. The beads can provide a variety of surface chemistries or
functionalities (e.g., amine, carboxyl, hydroxy etc.) suitable for
rendering the bead cationic by e.g., amination, or by direct or
indirect linkage to cationic polymer according to the present
invention.
[0079] Suitable bead compositions include those used in peptide,
nucleic acid and organic moiety synthesis, and include, for
example, plastics, such as polystyrene, methylstyrene, acrylic
polymers, ceramics, glass, polymeric materials, such as
cross-linked dextrans, cellulose, nylon, and latex, paramagnetic
materials, titanium dioxide, latex. The beads may encompass any
type of solid or hollow sphere, ball, bearing, cylinder, or other
solid configuration, which may be porous or non-porous in nature.
The use of porous beads can increase the surface area of the bead
available for nucleic acid detection. Bead sizes generally range
from about 100 nm to about 5 mm, preferably from about 0.2 .mu.m to
about 200 .mu.m, more preferably from about 0.5 .mu.m to about 5
.mu.m. Methods for attaching target-specific probes to bead
surfaces are described in e.g., U.S. Pat. No. 5,514,785, the
disclosures of which are incorporated by reference herein.
[0080] A surface or layer on the solid support may be blocked or
aged to reduce surface passivation (or non-specific binding) of
reaction components so as to interfere with amplification and/or
detection of polynucleotides bound to the probes. For example,
easily accessible surface amines may be blocked by treatment with
NHS-acetate or acetic anhydride. In addition, solid supports,
including silicon substrates may be aged (for e.g., 3-12 months) to
reduce surface passivation of reaction components thereto.
[0081] 2.2. Cationic Layer.
[0082] Applicants have unexpectedly discovered that a cationic
layer can be used in conjunction with probes immobilized onto a
coated solid support to reduce the release of and/or increase the
detection of captured target sequences on the coated support
surface, thereby reducing the potentially negative effects of
helicase-catalyzed unwinding and release of bound duplexes from the
coated support surface. Moreover, contrary to conventional
expectations, Applicants have further discovered that use of a
cationic layer coating according to the present invention can
actually stabilize the binding and resultant detection of bound
polynucleotide duplexes on the surface of the coated solid support
under low salt conditions to a greater extent than in high salt
conditions typically used in facilitating hybridization.
[0083] In one embodiment the cationic layer is configured so that a
proportion of the cationic functional groups in the cationic layer
retain their cationic functionality following attachment to
target-specific probes or secondary agents promoting attachment to
the target-specific probes. In one aspect, between about 0.1 to
about 10% of the cationic functional groups are retained in the
coated solid support following attachment of target-specific probes
thereto. In another aspect, between about 0.5 to about 3% of the
cationic functional groups are retained in the coated solid support
following attachment of target-specific probes thereto.
[0084] In one embodiment, the cationic layer is formed from a
cationic polymer. Exemplary cationic polymers include polylysine,
poly(lys-phe), poly(lys-tyr), poly(lys-trp), poly(arg-trp), and
poly(arg-pro-tyr). Preferably, the cationic polymer substantially
covers at least a portion of the solid support.
[0085] The formation and/or incorporation of cationic layers onto
silicon surfaces can be especially facilitated using silane
chemistry-based methodologies and compounds well known to those of
skill in the art, including use of silane-modified polymers,
polysilanes, silazanes, polysilazanes, T-resins, and
polyalkosiloxane-polysilicates.
[0086] In one embodiment, the cationic layer may be formed from
polymeric or non-polymeric substances derivatized to form a
cationic layer. In one embodiment, the cationic layer may be
created by introducing amine groups on the surface of the solid
support. The amine groups may be introduced by cationic silanes,
cationic siloxanes, or cationic derivatives thereof, or they may be
added via a cationic polymer, for example, onto a coating layer
composed of various silanes, siloxanes, or derivatives thereof. By
way of example, a coating layer may be comprised of non-aminated
coatings, including hydrophobic coatings of polypeptides, silanes,
or siloxanes. Aminated layers may then be created thereon through
the use of various polycations, such as poly (lys-phe). Such an
approach can serve to increase the density of amines and create
stable, multilayer coatings.
[0087] Exemplary silanes include aminopropyltrimethoxysilane,
glycidoxypropyltrimethoxysilane, and aminopropyltriethoxysilane.
Exemplary siloxanes include amino-functional organopolylsiloxanes,
and amino-functional siloxane alkoxylates. U.S. Pat. No. 6,013,789
describes methods for introducing amine groups onto a polypropylene
surface, the relevant content of which is incorporated by reference
herein.
[0088] A cationic polymer, cationic probe density enhancing
material, or aminated surface of the coated support may be
chemically derivatized to facilitate linkage to a solid support or
to a target-specific capture probe. For example, amino groups may
be aldehyde-modified, hydrazide-modified, or sulfhydryl-modified to
facilitate chemical conjugation to a suitably modified capture
probe. Any crosslinking agent suitable for chemically linking or
conjugating functional groups, particularly amino groups in the
cationic layer to either the solid support and/or the
target-specific capture probes may be used, including but not
limited to heterobifunctional NHS esters such as SFB, SHNH, SIAB,
and NHS.
[0089] A solid support may be chemically modified to form a
cationic layer thereover. In particular, solid supports may be
modified by introducing a functionality selected from a group
consisting of: amino, carboxyl, thiol, and their derivatives. Amino
groups may be introduced onto the surface of a solid support by any
conventional method known to those of skill in the art, including
the use of plasma discharge in an ammonia- or
organic-amine-containing gas. The "plasma" is most preferably an
ionized gas, which gains sufficient ionization energy from an
electromagnetic field. Preferably, the ionization energy is applied
by a radio-frequency plasma discharge, a microwave frequency plasma
discharge, or a corona discharge. In a particularly preferred
embodiment of the invention, the amine is derived from an ammonia
gas and the elevated energy state is achieved via radio-frequency
plasma discharge.
[0090] 2.3. Additional Coating Layers.
[0091] Additional layers or coatings may be added over the cationic
layer, the solid support, or between the cationic layer and the
solid support. The additional layer(s) may be added in addition to
a cationic layer or as alternatives to the cationic layers. By way
of example, the additional coating(s) may be added to a solid
support directly. The cationic layer may be then created on the
additional coating layer. The additional coatings may include
polypeptides, polysaccharides, and/or silane chemistry-based
compound coatings, including silane-modified polymers, polysilanes,
silazanes, polysilazanes, T-resins, and
polyalkosiloxane-polysilicates.
[0092] Additional coating layers that may be included in the coated
solid support of the present invention include thin-film
anti-reflective layers, attachment layers, and probe density
enhancing materials.
[0093] For example, a coated solid support may further include a
thin film anti-reflective layer over the solid support. A thin film
anti-reflective layer is anti-reflective to specific wavelengths of
light so as to create characteristic surface color changes
resulting from destructive interference. More particularly, when
reflected light from the surface-thin film interface is out of
phase with light reflected from the air-thin film interface,
specific wavelengths of light are eliminated from the reflecting
light by destructive interference so as to create characteristic
thin film surface color changes. In one embodiment, the
anti-reflective layer of silicon nitride is deposited by
plasma-enhanced chemical deposition. Anti-reflective layers may be
deposited on a variety of different supports. Methods and supports
for depositing thin film anti-reflective layers in accordance with
the present invention are described in U.S. patent application Ser.
No. 11/761,782, filed Jun. 12, 2007, the disclosures of which are
incorporated by reference herein in their entirety.
[0094] An attachment layer may be incorporated into a coated solid
support as a layer serving as a chemical bridge connecting the
target-specific probes to any layer of the present invention.
Preferably, the attachment layer is physically adhered or otherwise
chemically attached to a surface of a support or coating layer so
as to minimize interference with biochemical processes occurring
over the coated supports (e.g., amplification, hybridization,
detection) and to provide sufficient durability against subsequent
processing steps.
[0095] In one embodiment the attachment layer constitutes a coating
of suitable coupling agents or cross-linking reagents deposited
over a coating layer or solid support surface. The choice of a
suitable coupling agent or cross-linking reagent will depend upon
the nature of reactive chemical groups and/or the agent chain
length which would minimize or avoid intra-unit interference within
or between polymers. See "Reagents For Organic Synthesis", L.
Fiezer, M. Fiezer, Vol. 1-8, Wiley & Son; "Cross Linking
Reagents" (1980 Ed.), Pierce Biochemical Reagent Catalog, Pierce
Chemical Co., Rockford Ill. and references therein, or "Advanced
Organic Chemistry" J. March, McGraw Hill (1968).
[0096] In one embodiment, probe density enhancing materials are
incorporated into the coated solid supports as an attachment layer
providing additional or alternative surface chemistries for
increasing the probe density on the surface of the cationic layer
or the solid support. As such, the probe density enhancing
materials are designed for attachment directly or indirectly (e.g.,
by way of cross-linking agents, etc.) to the target-specific probes
of the present invention. Exemplary probe density enhancing
materials include latex particles, silica particles, dextran,
dextran sulfate, dendrimers, acrylic acid, dextran sulfate,
polyvinyl pyrolidone, polyethylene glycol, polyvinyl sulfate,
polyvinyl alcohol, polyacrylic acid, poly(acrylamide) acrylic acid
copolymer, including chemical and polymeric derivatives
thereof.
[0097] Upon linking functional groups in any structural component
of the present invention, unreacted functional groups may be
blocked by chemically modifying or derivatizing the functional
group so as to render it chemically inert. By way of example, an
end of a target-specific probe may "blocked" (by e.g., a "blocking
group" or "capping compound") by reaction with another chemical
group rendering a terminus of the probe an unsuitable substrate for
an enzyme, such as helicase or DNA polymerase, thereby preventing
or substantially reducing the enzyme from binding at, or near the
blocked terminus, or catalyzing an enzymatic reaction at or near
the blocked terminus. Free amino groups in cationic layers of the
present invention may be similarly blocked for improved device
performance through optimization of surface charge
density/reactivity.
[0098] Exemplary blocking agents include, for example,
amine-reactive compounds capable of converting free amine groups
into amides or imides. By way of example, the blocking agent may be
an acetylating reagent. Amine-reactive compounds may include
compounds from one or more of the following chemical classes:
N-hydroxysuccinimidyl (NHS) esters, imidoesters, aryl halides, acyl
halides, isocyanates, isothiocyanates, nitrophenyl esters,
carbonyls, carboxylates, and acid anhydrides. Particular
amine-reactive compounds may include, for example, any one or more
compounds selected from the group consisting of NHS acetate,
disuccinimidyl suberate (DSS),
succinimidyl-3-(tri-N-butylstannyl)benzoate, methyl
N-succinimidyladipate (MSA),
mono(latosylamido)mono(succinimidyl)suberate, acetic anhydride,
aryl chlorides, acyl chlorides, 2,4-dinitrofluorobenzene (DFNB),
sulfonyl halides, aldehydes,
1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC) based
activation chemistries, maleic anhydride, succinic anhydride,
acetyl chlorides, benzoyl chlorides, propionyl chlorides, butyryl
chlorides, and penylethanoyl chlorides.
[0099] Suitable blocking agents may also be selected from
non-acetylating agents, such as diazoacetates, imidoesters,
carbodimides, maleimides, .alpha.-haloacetyls, aryl halides,
dicarbonyl compounds, sulfhydryls, and hydrazides. By way of
example, specific non-acetylating compounds may be selected from
the group consisting of, for example, N-ethylmaleimide,
N-.beta.-maleimidopropionic acid, N-.epsilon.-maleimidocaprioic
acid, iodoacetic acid, N-[iodoethyl](trifluoroacetamide),
3,4-difluoronitrobenzene (DFNB), sulfonyl halide, (ammonium
4-chloro-7-sulfobenzo-furazan)-chloride (SBF-chloride), glyoxal,
phenyglyoxal, 2,3-butanedione, 1,2-cyclohexanedione,
2-mercaptoethanol, dithiothreitol (DTT) followed by sulfhydryl
chemistries, (2,4,6-trinitrobenzene sulfonic acid (TNBSA), and
2-mercaptoethanol. The blocking agent may include or be modified to
include a detectable label.
[0100] 3. Target-Specific Capture Probes.
[0101] Target-specific capture probes may employ a variety of
different polynucleotides or oligonucleotides for detection of
polynucleotide target(s). The target-specific capture probes
contain sequences complementary to sequences in the polynucleotide
target(s), including a first end attached to the coated solid
support and an unattached second end.
[0102] Target-specific capture probes of the present invention are
generally configured as a single stranded polynucleotide attached
to the coated solid support, preferably an oligonucleotide between
about 12 and 60 nucleotides in length, more preferably 15 to about
40 nucleotides in length.
[0103] In one embodiment, the target-specific probes may employ
conventional oligonucleotide structures. In another embodiment, the
target-specific probe includes a modified oligonucleotide
(containing an unconventional nucleic acid structure) capable of
forming a duplex structure, whereby the modified oligonucleotide
includes a structural feature conferring a reduction or elimination
in the recognition of the oligonucleotide by an isothermal
amplification enzyme when bound to the polynucleotide target. In a
particular embodiment, a target-specific probe includes a modified
oligonucleotide incorporating a structural feature conferring a
reduction or elimination of enzymatic denaturation when bound to
the polynucleotide target
[0104] Unconventional nucleic acid structures for use in the
present invention include oligonucleotides with nonconventional
chemical or backbone additions or substitutions, including but not
limited to peptide nucleic acids (PNAs), locked nucleic acids
(LNAs), morpholino backboned nucleic acids, methylphosphonates,
duplex stabilizing stilbene or pyrenyl caps, phosphorothioates,
phosphoroamidates, phosphotriesters, and the like. By way of
example, the modified oligonucleotides may incorporate or
substitute one or more of the naturally occurring nucleotides with
an analog; internucleotide modifications incorporating, for
example, uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) or charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.);
modifications incorporating intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals,
boron, oxidative metals, etc.), or alkylators, and/or modified
linkages (e.g., alpha anomeric nucleic acids, etc.) (US
2007/0166741).
[0105] In one embodiment, the target-specific probe(s) are
internally modified to include at least one neutral charge in its
backbone. For example, the capture probe may include a
methylphosphonate backbone or peptide nucleic acid (PNA)
complementary to the target-specific sequence. These modifications
have been found to prevent or reduce helicase-mediated unwinding.
The use of uncharged probes may further increase the rate of
hybridization to polynucleotide targets in a sample by alleviating
the repulsion of negatively-charges nucleic acid strands in
classical hybridization (Nielsen et al., 1999, Curr. Issues Mol.
Biol., 1:89-104).
[0106] PNA oligonucleotides are uncharged nucleic acid analogs for
which the phosphodiester backbone has been replaced by a polyamide,
which makes PNAs a polymer of 2-aminoethyl-glycine units bound
together by an amide linkage. PNAs are synthesized using the same
Boc or Fmoc chemistry as are use in standard peptide synthesis.
Bases (adenine, guanine, cytosine and thymine) are linked to the
backbone by a methylene carboxyl linkage. Thus, PNAs are acyclic,
achiral, and neutral. Other properties of PNAs are increased
specificity and melting temperature as compared to nucleic acids,
capacity to form triple helices, stability at acid pH,
non-recognition by cellular enzymes like nucleases, polymerases,
etc. (Rey et al., 2000, FASEB J., 14:1041-1060; Nielsen et al.,
1999, Curr. Issues Mol. Biol., 1:89-104).
[0107] Methylphosphonate-containing oligonucleotides are neutral
DNA analogs containing a methyl group in place of one of the
non-bonding phosphoryl oxygens. Oligonucleotides with
methylphosphonate linkages were among the first reported to inhibit
protein synthesis via anti-sense blockade of translation. However,
the synthetic process yields chiral molecules that must be
separated to yield chirally pure monomers for custom production of
oligonucleotides (Reynolds et al., 1996, Nucleic Acids Res.,
24:4584-4591).
[0108] In one embodiment, the target-specific oligonucleotide
probes utilize a backbone of modified sugars joined by
phosphodiester internucleotide linkages. The modified sugars may
include furanose analogs, including but not limited to
2-deoxyribofuranosides, .alpha.-D-arabinofuranosides,
.alpha.-2'-deoxyribofuranosides, and
2',3'-dideoxy-3'-aminoribofuranosides. In alternative embodiments,
the 2-deoxy-.beta.-D-ribofuranose groups may be replaced with other
sugars, for example, .beta.-D-ribofuranose. In addition,
.beta.-D-ribofuranose may be present wherein the 2-OH of the ribose
moiety is alkylated with a C.sub.1-6 alkyl group (2-(O--C.sub.1-6
alkyl) ribose) or with a C.sub.2-6 alkenyl group (2-(O--C.sub.2-6
alkenyl) ribose), or is replaced by a fluoro group
(2-fluororibose).
[0109] Related oligomer-forming sugars include those used in
"locked nucleic acids" (LNA), which are bicyclic nucleic acids in
which a ribonucleoside (including e.g., a furanose ring) is linked
between the 2'-oxygen and the 4'-carbon atoms with a methylene
unit. LNAs were first described by Wengel and co-workers as a class
of conformationally restricted oligonucleotide analogues (Koshkin
et al., Tetrahedron, 54:3607-3630 (1998); Singh et al., Chem.
Comm., 4:455-456 (1998). Exemplary LNA nucleotides include modified
bicyclic monomeric units with a 2'-O-4'-C methylene bridge, such as
those described in U.S. Pat. No. 6,268,490, the disclosures of
which are incorporated by reference herein.
[0110] Oligonucleotides containing .alpha.-D-arabinofuranosides can
be prepared as described in U.S. Pat. No. 5,177,196.
Oligonucleotides containing 2',3'-dideoxy-3'-aminoribofuranosides
are described in Chen et al. Nucleic Acids Res. 23:2661-2668
(1995). Synthetic procedures for locked nucleic acids (Singh et al,
Chem. Comm., 455-456 (1998); Wengel J., Acc. Chem. Res., 32:301-310
(1998)) and oligonucleotides containing
2'-halogen-2'-deoxyribofuranosides (Palissa et al., Z. Chem. 27:216
(1987)) have also been described.
[0111] Duplex stabilizing stilbene or pyrenyl caps include
trimethoxystilbene and pyrenylmethylpyrrolindol caps (Glen
Research, Sterling, Va.).
[0112] Other sugar moieties compatible with hybridization of the
oligonucleotide can also be used, and are known to those of skill
in the art, including, but not limited to,
.alpha.-D-arabinofuranosides, .alpha.-2'-deoxyribofuranosides or
2',3'-dideoxy-3'-aminoribofuranosides. Oligonucleotides containing
.alpha.-D-arabinofuranosides can be prepared as described in U.S.
Pat. No. 5,177,196. Oligonucleotides containing
2',3'-dideoxy-3'-aminoribofuranosides are described in Chen et al.
Nucleic Acids Res. 23:2661-2668 (1995).
[0113] Chemically modified oligonucleotides may also include,
singly or in any combination, 2'-position sugar modifications,
5-position pyrimidine modifications (e.g,
5-(N-benzylcarboxyamide)-2'-deoxyuridine,
5-(N-isobutylcarboxyamide)-2'-deoxyuridine,
5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2'-deoxyuridine,
5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-napthylcarboxyamide)-2'-deoxyuridine, and
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine),
8-position purine modifications, modifications at exocyclic amines,
substitution of 4-thiouridine, substitution of 5-bromo- or
5-iodo-uracil, methylations, unusual base-pairing combinations,
such as the isobases isocytidine and isoguanidine, and the
like.
[0114] The phosphate backbone in the target-specific probe(s) may
employ oligonucleotides containing phosphorothioate linkages or
phosphoroamidates (Chen et al., Nucl. Acids Res., 23:2662-2668
(1995)). Combinations of such oligonucleotide linkages are also
within the scope of the present invention.
[0115] In another embodiment, the 5' or 3' probe end in the
target-specific probe may be blocked to reduce or eliminate
recognition by an isothermal amplification enzyme, such as a
polymerse and/or enzymatic denaturation by an isothermal
amplification enzyme, such as a helicase. In one embodiment, at
least one of the 5' or 3' probe ends is modified or blocked by
capping or by incorporation of a suitable terminus modifier or
spacer modifier as further described below (Glen Research,
Sterling, Va.). By way of example, an unattached 3'-OH probe end
may be modified or blocked to prevent incorporation of the probe
into a primer extension product by a polymerase during
amplification. This may be achieved by: (i) removing the 3'-OH;
(ii) incorporating a nucleotide lacking a 3'-OH, such as
dideoxynucleotide; (iii) incorporating cordycepin
(3'-deoxyadenosine) and other 3'-bases without a 3'-OH moiety; (iv)
incorporating a spacer lacking a 3'-OH, such as C3 propyl spacer;
(v) incorporating an amine or phosphate group; or (vi) any other
chemical modification rendering the probe inert as a primer for
primer extension as known to those of skill in the art.
Incorporating a biotin label onto the 3' hydroxyl of the last
nucleotide can serve a dual purpose by also acting as a label for
subsequent detection or capture of the nucleic acid attached to the
label.
[0116] In a further aspect, the probe may be modified or designed
with other functionalities increasing the utility of the
amplification/detection system. In a particular embodiment, the
probe includes or is chemically linked to a cleavable linker
facilitating the release of hybrid duplexes from the coated solid
support for further amplification and/or detection. The design and
use of cleavable linkers in oligonucleotide probes is described in
U.S. Pat. Nos. 5,380,833, 6,060,246, 6,027,879, and 7,291,471, and
U.S. Pat. Appl. No. 2005/0106576, the disclosures of which are
incorporated by reference herein. Photocleavable oligonucleotide
modifiers, including photocleavable spacer modifiers are
commercially available (Glen Research, Sterling, Va.).
[0117] In a further aspect, the probe may include or be chemically
linked to a terminus modifier or spacer modifier at the 5'-end,
3'-end or both. A terminus modifier or spacer modifier may be
incorporated to increase the distance between the capture probe
target sequences and the surface of the coated solid support and/or
to reduce or eliminate recognition of an isothermal amplification
enzyme or enzymatic denaturation by an isothermal amplification
enzyme when bound to a polynucleotide target. Exemplary terminus
modifiers and spacer modifiers include, for example, a variety of
commercially available 5'-amino modifiers, 3-amino modifiers, and
chemically derivatized modifiers thereof; spacer phosphoramidates
for inserting variable length spacer arms; spacers for introducing
abasic sites within an oligonucleotide; and photocleavable spacer
modifiers (Glen Research, Sterling, Va.).
[0118] The target-specific capture probes may be directly or
indirectly attached to any component of the coated solid support,
including the solid support, the cationic layer, a probe density
enhancing material, or a suitably modified solid support surface.
The probe may be covalently or non-covalently attached by its 5' or
3' end to the coated solid support. The probe may be covalently
attached through cationic functional groups directly or through
functional groups incorporated thereto for purposes of facilitating
this attachment. To facilitate attachment to any part of the coated
solid support, probes may be modified or designed to include at
their 5' or 3' terminal ends a variety of functional groups for
enhancing conjugation to other polymer- or nucleic acid-based
elements. Exemplary functional groups include amino, hydrazide,
aldehyde, and sulfhydryl groups.
[0119] A plurality of the same or different target-specific probes
may be directly or indirectly attached to the cationic layer or a
cation-modified solid support. Either end of the DNA molecule (5'
or 3' end) in the target-specific capture probe may be attached to
the solid support or to the cationic layer. In addition, as
described above, the unattached capture probe ends may be blocked
to reduce helicase recognition or primer extensions therefrom.
[0120] In one embodiment, the capture probe is attached to a
polycationic polymer in the cationic layer. In another embodiment,
the capture probe is attached to a cationic silane, cationic
siloxane, or cationic derivative in the cationic layer. In a
preferred embodiment, the capture probe is linked to a cationic
polymer (such as phe-lys) derivatized with an aldehyde
functionality.
[0121] A coated solid support may include one type of
target-specific probe of a single specificity or a plurality of
different target-specific probes with multiple specificities for
detecting a plurality of different target sequences from one
organism, a plurality of different organisms or combinations
thereof. The probe(s) may be attached to a discrete area in the
solid support as a spot or to a plurality of discrete areas in the
form of an array.
II. Methods for Amplifying and Detecting Polynucleotide Targets
[0122] 1. Amplification/Hybridization Detection System
[0123] The coated solid supports of the present invention are
configured to allow for isothermal amplification, hybridization and
detection on a single detection device. An isothermal amplification
process is particularly suited for use in such a device, since it
does not require high heat denaturation temperatures for DNA
amplification which may be incompatible with the materials used in
a coupled amplification/hybridization/detection device.
[0124] In one embodiment, a method for
amplification/hybridization/detection of polynucleotide target(s)
in a sample includes applying the sample and a reaction medium to a
coated solid support comprised of a solid substrate, a cationic
layer, and a plurality of target-specific probes attached to the
coated solid support. In an alternative embodiment, the
amplification/hybridization/detection includes a coated solid
support comprised of a solid substrate and a plurality of
target-specific probes attached to the coated solid support.
Polynucleotide targets in the sample are then amplified by an
isothermal amplification process and hybridized to the
target-specific probes. Upon amplification of nucleic acids in the
sample for a period of time sufficient for detection, the
polynucleotide targets may be hybridized to the target-specific
probes that are attached to the coated solid support.
[0125] In one embodiment, a coupled
amplification/hybridization/detection system includes an isothermal
amplification process utilizing an enzyme, such as helicase, or set
of enzymes capable of unwinding the synthesized amplification
products. The unwound single stranded amplification products can
anneal to amplification primers, re-anneal to complementary
strands, or anneal to probes immobilized on the surface of a coated
solid support. Polynucleotides may be amplified and hybridized to
the coated solid support under low salt conditions or be subjected
to an optional high salt hybridization step (for enhanced
sensitivity), followed by low salt washes and detection of the
bound complexes.
[0126] In another embodiment, an uncoupled
amplification/hybridization/detection system is employed whereupon
completion of the isothermal amplification process, the isothermal
amplification products are denatured by heat or a suitable
combination of unwinding enzymes to allow for their hybridization
to the target-specific probes that are attached to the coated solid
support. Inclusion of the terminal denaturation/unwinding step can
be applied to any isothermal amplification process carried out on
any one of the coated supports of the present invention.
[0127] In one aspect, polynucleotide targets in the sample are
initially purified by hybridization onto a coated solid support and
then transferred to a second coated solid support for the coupled
amplification/hybridization/detection process. In this case,
polynucleotide targets bound to cleavable, target-specific probes
are eluted from a first coated solid support, and then applied to a
second coated solid support for amplification by an isothermal
amplification process, such as HDA. Following conventional washes
to remove non-specifically bound polynucleotides and other
polynucleotides or reaction components, cleavable target-specific
probes attached to the coated support are cleaved, whereby the
cleaved probes, including those hybridized to the polynucleotide
targets are subjected to isothermal amplification over a second
coated solid support. Amplification products hybridized to the
second coated solid support may be directly detected under low salt
conditions or optionally subjected to a short (e.g. 15-30 minute)
high salt hybridization step (for enhanced sensitivity), followed
by low salt washes and detection of the bound complexes.
Alternatively, the bound complexes may be subjected to additional
cycles of elution, amplification, and/or hybridization prior to
detection.
[0128] 2. Hybridization/Detection System
[0129] In one embodiment, a method includes applying a sample and a
reaction medium to a coated solid support comprised of a solid
support and a plurality of target-specific probes attached to the
coated solid support. The coated solid support may further include
a cationic layer as described above. Conditions and reagents
suitable for denaturing polynucleotide targets in the sample by an
enzymatic process are provided, followed by hybridization of the
enzymatically denatured polynucleotide targets in the sample to the
target-specific probes. In a final step, the presence or absence of
polynucleotide targets bound to the probes is detected.
[0130] In another embodiment, the hybridization/detection system
includes applying a sample and a reaction medium to a coated
silicon support comprised of a solid support, a cationic layer, and
a plurality of target-specific probes attached to the coated solid
support. The coated silicon support does not comprise a coating
layer capable of mediating visual detection of polynucleotide
targets by destructive interference. In a particular embodiment,
the rough side of a silicon support is coated with the cationic
layer and the plurality of target-specific probes. Conditions and
reagents suitable for hybridizing polynucleotide targets in the
sample to the target-specific probes are provided. For example, the
polynucleotide targets may be enzymatically denatured prior to the
hybridization step as described above. In a final step, the
presence or absence of polynucleotide targets bound to the probes
is detected. In a particular embodiment, the hybridization and
detection steps may be carried out under low salt conditions as
described above. As described above, when used in conjunction with
a cationic layer, the hybridization and detection steps may be
carried out under low salt conditions, and may optionally include a
15-30 minute high salt hybridization step as well.
[0131] 3. Isothermal Amplification of Polynucleotide Targets
[0132] Amplification of polynucleotide targets necessarily requires
a suitable polymerase and a means for denaturing or unwinding
polynucleotides created during the process of isothermal
amplification. In one embodiment, all denaturation and
amplification steps are carried out enzymatically under isothermal
temperature conditions. In another embodiment, the polynucleotide
sample is initially denatured by heat denaturation before the
primer annealing step, and then enzymatically denatured during the
subsequent amplification/hybridization steps. Alternatively, or in
addition, the polynucleotide sample may be amplified in solution
phase first and then heat denatured prior to hybridization and
detection of amplification products on the coated solid
support.
[0133] Any isothermal amplification process known to those of skill
in the art may be adapted for use in the methods and kits of the
present invention. Exemplary isothermal amplification processes and
reagents for use in the present invention are incorporated by
reference with regard to helicase-dependent amplification (HDA) as
described in U.S. Pat. No. 7,282,328; strand-displacement
amplification (SDA) as described in U.S. Pat. Nos. 5,270,184,
5,422,252, 5,455,166, 5,470,723, 6,087,133, 6,531,302); multiple
displacement amplification (MDA) as described in U.S. Pat. No.
6,977,148; recombinase polymerase amplification (RPA) as described
in Piepenburg et al., PLoS Biology, 4(7):1115-1121, 2006, U.S. Pat.
No. 7,270,981, and U.S. Pat. Appl. No. 2005/0112631; loop-mediated
isothermal amplification (LAMP) as described in U.S. Pat. Nos.
6,410,278, 6,743,605, and U.S. Pat. Appl. No. 2007/0218464; rolling
circle amplification (RCA) as described in Fire et al., Proc. Natl.
Acad. Sci. USA, 92:4641-4645 (2002), Dean et al., Proc. Natl. Acad.
Sci. USA, 99:5261-5266 (2002), U.S. Pat. Nos. 5,714,320, 5,854,033,
6,235,502, and 6,344,329; nucleic acid sequence based amplification
(NASBA) as described in U.S. Pat. No. 5,130,238;
transcription-mediated RNA amplification (TMA) as described in U.S.
Pat. Appl. No. 2007/0178470; single primer isothermal amplification
(SPIA.TM. and Ribo-SPIA.TM., NuGen Technologies, San Carlos,
Calif.) as described in U.S. Pat. Nos. 6,251,639, 6,692,918, and
6,946,251 and U.S. Pat. Appl. No. 2007/0212695; and other
isothermal amplification methodologies known to those of skill in
the art, including those described in U.S. Pat. No. 6,929,915.
[0134] An HDA process requires a polymerase preparation for
synthesis of amplification products and a helicase preparation to
facilitate enzymatic denaturation and annealing of amplification
primers to the amplified polynucleotide targets. A suitable
helicase preparation includes at least one type of helicase alone
or in combination with a helicase accessory product, such as
single-stranded binding proteins (SSBs). Preferably, the helicase
is a thermostable helicase, which is capable of mediating HDA in
the absence of additional accessory products.
[0135] The term "helicase" refers to any enzyme capable of
enzymatically unwinding or denaturing a nucleic acid alone or in
combination with a helicase accessory protein. Helicases can unwind
double stranded nucleic acids in the '5 direction or 3' directions.
Helicases are found in all organisms and are utilized in enzymatic
processes involving nucleic acids, including replication,
recombination, repair, transcription, translation and RNA splicing.
Any helicase that translocates along DNA or RNA in a 5' to 3'
direction or in the opposite 3' to 5' direction may be used in
present embodiments of the invention. This includes helicases
obtained from prokaryotes, viruses, archaea, and eukaryotes or
recombinant forms of naturally occurring enzymes as well as
analogues or derivatives having the specified activity. Examples of
naturally occurring DNA helicases include E. coli helicase I, II,
Ill, & IV, UvrD helicase, Rep helicase, RecQ helicase, PcrA
helicase, RecBCD helicase, DnaB helicase, PriA, PcrA, T4
Gp41helicase, T4 Dda helicase, T7 Gp4 helicases, SV40 Large T
antigen, herpesvirus helicases, including HSV-1 helicase, yeast
RAD, yeast Sgs1 helicase, DEAH_ATP-dependent helicases, RecQ
helicase, thermostable UvrD helicases from T. tengcongensis and T.
thermophilus, thermostable DnaB helicase from T. aquaticus, MCM
helicase, as well as analogs, homologs, thermostable helicases,
genetically-modified helicase variants of the above, or any of the
helicases disclosed in U.S. Pat. No. 7,282,328, the disclosures of
which are incorporated by reference herein.
[0136] Under certain circumstances, helicases (especially
mesophilic helicases) require or exhibit improved activity in the
presence of single-strand binding proteins (SSB), a helicase
cofactor known to stabilize single stranded nucleic acids. In these
circumstances, the choice of SSB is generally not limited to a
specific protein. Examples of single strand binding proteins
include T4 gene 32 protein, E. coli SSB, T7 gp2.5 SSB, phage phi29
SSB (Kornberg and Baker, supra (1992)) and truncated forms of the
aforementioned. Compositions and methods for HDA are described in
U.S. Pat. No. 7,282,328, the disclosures of which are expressly
incorporated by reference herein.
[0137] A variety of different polymerases may be used for
amplifying polynucleotide targets. Use of these polymerases may be
selected on the basis of processivity and strand displacement
activity. Subsequent to melting and hybridization with a primer,
the nucleic acid is subjected to a polymerization step. A DNA
polymerase is selected if the nucleic acid to be amplified is DNA.
When the initial target is RNA, a reverse transcriptase is used
first to copy the RNA target into a cDNA molecule and the cDNA is
then further amplified in HDA by a selected DNA polymerase. The DNA
polymerase acts on the target nucleic acid to extend the primers
hybridized to the nucleic acid templates in the presence of four
dNTPs to form primer extension products complementary to the
nucleotide sequence on the nucleic acid template.
[0138] In one embodiment, the DNA polymerase is selected from a
group of polymerases lacking 5' to 3' exonuclease activity and
which additionally may lack 3'-5' exonuclease activity. Exemplary
DNA polymerases include an exonuclease-deficient Klenow fragment of
E. coli DNA polymerase I (New England Biolabs, Inc. (Beverly,
Mass.)), an exonuclease deficient T7 DNA polymerase (Sequenase;
USB, (Cleveland, Ohio)), Klenow fragment of E. coli DNA polymerase
I (New England Biolabs, Inc. (Beverly, Mass.)), Large fragment of
Bst DNA polymerase (New England Biolabs, Inc. (Beverly, Mass.)),
KlenTaq DNA polymerase (AB Peptides, (St Louis, Mo.)), T5 DNA
polymerase (U.S. Pat. No. 5,716,819), and Pol III DNA polymerase
(U.S. Pat. No. 6,555,349).
[0139] DNA polymerases possessing strand-displacement activity,
such as the exonuclease-deficient Klenow fragment of E. coli DNA
polymerase I, Bst DNA polymerase Large fragment, and Sequenase, are
preferred for Helicase-Dependent Amplification. T7 polymerase is a
high fidelity polymerase having an error rate of 3.5.times.10.sup.5
which is significantly less than Taq polymerase (Keohavong and
Thilly, Proc. Natl. Acad. Sci. USA 86, 9253 9257 (1989)). T7
polymerase is not thermostable, however, and therefore is not
optimal for use in amplification systems that require thermocycling
or that are enhanced at higher temperatures. T7 Sequenase is a
preferred polymerase for amplification of DNA by isothermal HDA
processes.
[0140] 4. Hybridization of Polynucleotide Targets
[0141] Conditions suitable for promoting formation of hybridization
complexes between the target-specific probe and its complementary
strand in the polynucleotide target to produce a hybridization
complex are well known in the art. As is known to those skilled in
the art, the specificity of hybridization may be influenced by the
length and composition of the oligonucleotide primer, the
temperature in which the hybridization reaction is performed, the
ionic strength, and the pH. Suitable conditions may be empirically
determined. Hybridization conditions may include the chemical
components and their concentrations (e.g., salts, chelating agents,
formamide) of an aqueous or organic solution containing the nucleic
acids, and the temperature of the mixture. Other well known
factors, such as the length of incubation time and physical nature
and dimensions of the solid support may contribute to an
appropriate environment for hybridization and may be also modified
as known to those of skill in the art. Bound complexes may be
washed to remove un-hybridized polynucleotides and other reaction
components prior to further amplification or detection of
polynucleotide targets.
[0142] 5. Detection of Polynucleotide Targets
[0143] Polynucleotide targets bound to complementary
target-specific probes on coated solid supports in the above
described coupled amplification/detection or
hybridization/detection systems may be detected using any suitable
detection methodology known to those of skill in the art. Detection
may be provided by such characteristics as color change,
luminescence, fluorescence, or radioactivity. Typically, the
detection of polynucleotide targets is facilitated by complexing to
a suitable label or reporter. A suitable label may include any
molecular moiety or group having a physical or chemical
characteristic capable of producing a response or signal that is
directly or indirectly detectable and/or measurable, for example,
by catalyzing a reaction that produces an optically detectable
signal. The label may be incorporated into the starting materials
before or during the completion of any one of the amplification,
hybridization, and/or detection steps.
[0144] Exemplary labels include but are not limited to chromogenic
dyes or substrates facilitating colorimetric detection; luminescent
moieties, including fluorescent, bioluminescent, phosphorescent, or
chemiluminescent compounds; haptenic or antigenic compounds used in
combination with a suitably labeled antibody; specific binding pair
members containing a ligand recognition site (e.g., biotin and
avidin); enzymes; enzyme substrates, radioisotopes; metal
complexes; magnetic particles; radio frequency transmitters, and
the like. In addition, the label may include a variety of different
reactive groups or chemical functionalities suitable for linkage to
a variety of biomolecule agents.
[0145] In one embodiment, the label includes an enzyme catalyzing
reaction of substrates to produce colored, fluorescent,
luminescent, electron dense or radioactive products. More
particularly, the label may be linked to bound target
polynucleotides to form a chromogenic, precipitable polynucleotide
label complex of a size and composition such that light scattered
from the complex on illumination with white light can be detected
by human eye, preferably without magnification.
[0146] Exemplary enzyme labels include peroxidases, such as
horseradish peroxidase (HRP), alkaline or acidic phosphatase,
galactosidase, glucose oxidase, NADPase, luciferase,
carboxypeptidase and the like. In some embodiments, direct visual
detection may be enhanced by using an enzyme catalyzing the
formation of precipitable, chromophore-containing products
producing a visible color change. Colored precipitates can be
monitored by e.g., spectrophotometry, flatbed scanning, microscopy,
or by the naked eye. Exemplary enzymes catalyzing formation of
precipitable, chromophore-containing products include horseradish
peroxidase (HRP), alkaline phosphatase, and glucose oxidase.
[0147] Enzyme labels may be supplied in the form of enzyme/binding
member conjugates or antibody-enzyme conjugates. Exemplary enzyme
conjugates include streptavidin/HRP- and anti-biotin antibody/HRP
conjugates. Exemplary chromogenic substrates for HRP include
3,3',5,5' tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB),
and 3-amino-9-ethyl carbazol (AEC). TMB is a non-precipitating
substrate that acts as electron donor for the conversion of
hydrogen peroxide into water. TMP is enzymatically converted to a
visually detectable colored complex by HRP. Exemplary chromogenic
substrates for alkaline phosphatase include BCIP/NBT, Fast Red and
AP-Orange. Avidin-linked enzymes and chromogenic substrates are
commercially available from, for example, Pierce Chemical Company
(Rockville, Ill.) and Sigma (St. Louis, Mo.). Enzymatic labeling
and detection are described, for example, in U.S. Pat. No.
4,789,630, the disclosures of which are incorporated by reference
herein.
[0148] Binding pair members containing a ligand recognition site,
such as biotin and avidin, may be incorporated into any of the
amplification primers or in the target-specific probes.
[0149] Fluorometric detection methods are based on emission of
photons or excitons of lesser energy or different wavelength from
certain molecules following excitation at a suitable wavelength
found in ultraviolet light, for example. There are a variety of
fluorescent molecules known to those of skill in the art which can
serve as reporters than can be detected and quantified, after
excitation at a suitable wavelength, with several apparatuses such
as fluorometers, confocal fluorescence scanners, microscopes, etc.
Detection of polynucleotide targets may be facilitated by
incorporating fluorescent labels or dyes into the amplification
primers as known to those of skill in the art.
[0150] Chemiluminescent detection relies on enzymes such as
alkaline phosphatase or horseradish peroxidase, which can convert a
substrate with concomitant emission of light that can be detected
by autoradiography (solid phase) or luminometry (liquid phase)
[0151] Electrochemical detection is generally performed at the
surface of electrodes, whereby oxydo-reduction reactions of
reporter molecules yield electrons that can be monitored using a
suitable apparatus, such as a potentiostat.
[0152] In another embodiment, cationic polymers may be utilized for
electrostatic-based detection of polynucleotide targets bound to
capture probes in the coated solid supports of the present
invention. Cationic polymer-based nucleic acid detection
methodologies are typically based on electrostatic interactions
between positively charged polymers and negatively charged nucleic
acids (Pending patent application PCT/CA02/00485; Ho et. al., 2002,
Angew. Chem. Int. Ed., 41:1548-1551; Ho et al., 2002, Polymer
Preprints, 43:133-134; Nilsson et al., 2003, Nat. Mater.
2:419-424). These approaches exploit a modification of the optical
or electrochemical properties of polymer biosensors upon
electrostatic binding to a single- or a double-stranded
negatively-charged nucleic acid molecule. These macromolecular
interactions are associated with conformational and solubility
changes which contribute to signal generation (Ho et. al., 2002,
Angew. Chem. Int. Ed., 41:1548-1551). These polymer-based detection
technologies do not require any chemical labeling of the probe or
of the target and can discriminate between specific and
non-specific hybridization of nucleic acids that differ by a single
nucleotide acid.
[0153] Exemplary cationic "reporter" polymers include various
polythiophene derivatives, including water-soluble fluorescent
zwitterionic polythiophene derivatives (Nilsson et al., 2003, Nat.
Mater. 2:419-424) and water soluble polyfluorene phenylene
conjugates (Gaylord et al., 2002, Proc. Natl. Acad. Sci. U.S.A.,
99:10954-10957), and poly(3,4-ethylenedioxythiophene)
(Krishnamoorthy et al., 2004, Chem. Commun., 2004:820-821).
Cationic polymer-based detection methodologies and reagents are
further described in U.S. Pat. Appl. Publ. Nos. 2004/0171001 and
2007/0178470, the disclosures of which are incorporated by
reference herein.
[0154] Detection of polynucleotide labels may be facilitated by the
use of an "amplification label," which is a molecule that can
amplify the number of detectable labels that can be bound to a
polynucleotide target(s). An amplification label may, for example,
comprise a polymer that specifically binds to a polynucleotide
target and has a plurality of binding sites to which labels can be
attached to generate multiple detectable signals relative to one or
more bound polynucleotide targets. By way of example, the
amplification label may be a polymer having a plurality of amine
groups facilitating covalent attachment to a plurality of biotin
molecules. The biotin molecules can then be used to generate a
detectable signal. Because each biotin molecule generates an
independent signal, there are multiple signals generated relative
to a bound polynucleotide target to which the polymer binds,
thereby amplifying the corresponding signal thereto. Amplification
labels and associated detection reagents are disclosed in U.S.
Provisional Pat. Appl. No. 60/990,755, filed Nov. 28, 2007, the
disclosures of which are incorporated by reference herein.
III. Kits for Amplification and/or Detection of Polynucleotide
Targets
[0155] In one embodiment, a kit for isothermal amplification
includes an above-described coated solid support and one or more
enzymes collectively sufficient for an isothermal amplification
process. In a particular embodiment, the coated solid support
includes a cationic layer. In another embodiment, the coated solid
support is attached to at least one target-specific probes formed
from an oligonucleotide modified or incorporated with a structural
element reducing or eliminate recognition by an isothermal
amplification enzyme when bound to a polynucleotide target as
described above.
[0156] When supplied as a kit, any of the various components or
reagents may be packaged in separate containers and admixed prior
to use with the solid supports of the present invention. Such
separate packaging of the components permits long-term storage.
Thus, for example, a kit may supply anhydrous amplification enzymes
and/or enzyme substrates, and buffers for reconstituting the
enzymes and/or enzyme substrates. Any buffers designed to maintain
a suitable pH under the reaction conditions of the present
invention are contemplated. The anhydrous preparations may be
lyophilized, in which water is removed under vacuum, freeze-dried,
crystallized, or prepared using any other method removing water so
as to preserve the activity of the anhydrous reagents. Excipients
may be added to these preparations to stabilize these reagents,
such as serum albumins or Prionex. In other embodiments, the
reagents may be suspended in an aqueous composition comprising, or
example, glycerol or other solvents in which the enzymes and/or
other reagents are stable.
[0157] The kit may further include cell lysing reagents (including
non-ionic detergents (such as from the Triton series), cationic
detergents, anionic detergents, zwitterionic detergents, and the
like); one or more reaction mediums for hybridization of
polynucleotide targets to the probes and for removal of
nonspecifically bound reaction components; neutralization buffers;
wash buffers, and any of the above-described detection reagents or
solutions for detection of polynucleotide targets bound to the
probes.
[0158] The kits may include reagents in separate containers to
facilitate the execution of a specific test, such as cell lysis or
solution phase amplification. Polynucleotides and primers pairs may
be supplied used as internal controls or positive controls with
respect to hybridization and/or amplification. The kit may supply a
sample gathering component such as a membrane, filter or swab.
[0159] The reagents included in the kits can be supplied in
containers of any sort such that the life of the different
components are preserved, and are not adsorbed or altered by the
materials of the container. For example, sealed glass ampules may
contain lyophilized enzymes or buffers that have been packaged
under a neutral, non-reacting gas, such as nitrogen. Ampules may
consist of any suitable material, such as glass, organic polymers,
such as polycarbonate, polystyrene, etc., ceramic, metal or any
other material typically employed to hold reagents. Other examples
of suitable containers include simple bottles that may be
fabricated from similar substances as ampules, and envelopes, that
may consist of foil-lined interiors, such as aluminum or an alloy.
Other containers include test tubes, vials, flasks, bottles,
syringes, or the like. Containers may have a sterile access port,
such as a bottle having a stopper that can be pierced by a
hypodermic injection needle. Other containers may have two
compartments that are separated by a readily removable membrane
that upon removal permits the components to mix. Removable
membranes may be glass, plastic, rubber, etc.
[0160] The kits may be further supplied with a set of instructions
for using the contents in a given kit. The instructions may be
printed on paper or other substrate, and/or may be supplied as an
electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM,
Zip disc, videotape, audio tape, etc. Detailed instructions may not
be physically associated with the kit; instead, a user may be
directed to an internet web site specified by the manufacturer or
distributor of the kit, or supplied as electronic mail. The
instructions may instruct the user of the kit in any aspect of the
above-described methods, method steps or methodologies relating to
the practice of the present invention.
[0161] The following Examples are provided to aid in the
understanding of the invention and not construed as a limitations
thereof
Example 1
[0162] Target-specific coated support preparation.
[0163] "Rough-side" silicon biosensors employed a silicon wafer
having a polished side and an un-polished side, whereby the
un-polished "rough" side surface was used for coatings and testing.
Thin film biosensors (Inverness Medical-Biostar; Jenison et al.,
Expert Rev. Molec. Diagn., 2006) employed silicon wafers coated on
a polished side with .about.475 angstroms of silicon nitride,
followed by coating with .about.135 angstroms of T-structure
aminoalkyl polydimethyl Isiloxane (United Chemical Technologies) as
an attachment layer. Surfaces were coated with 5 .mu.g/ml of poly
(lys-phe) (Sigma) in 1.times.PBS pH 6, 2 M NaCl over night at room
temperature with rotation. Surfaces were washed with water and then
coated with 10 .mu.M SFB (succinimidyl formyl benzoate (Solulink)
in 0.1 M Borate pH 8.5) to convert free amino groups to free
aldehydes for creating stable hydrazone linkages with
hydrazide-modified capture probes. After SFB coating, wafers were
extensively washed with water, dried with a stream of nitrogen, and
stored in a nitrogen purged dry box. DNA capture probes, modified
at the 5'-end with hydrazide linkers, were diluted to 50, 150, and
500 nM in Spotting buffer (100 mM Phosphate pH 8, 10% glycerol). 1
.mu.l of each dilution was spotted onto an SFB-modified surface.
The probe was incubated under a humid environment for 2-16 hours.
Surfaces were then washed with water and treated with 0.1% SDS at
>37.degree. C. for 2-16 hours to remove loosely adsorbed capture
probe. Surfaces were again washed with water, dried, and stored in
a nitrogen purged dry box protected from light.
Example 2
Primers and Probes
[0164] Primer and probe oligonucleotide sequences were designed
using Primer 3 software
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) with
input sequences downloaded from the GenBank genetic sequence
repository. The sequences used in these studies are shown in FIG.
9. Reverse primers were modified at their 5'-termini with biotinTEG
(5''-BT) (Glen Research, Sterling Va.) to facilitate detection of
amplified sequences. Target-specific capture probes (CP) were
modified on their 5'-termini with a 5'-l-linker (Integrated DNA
Technologies, Coralville, Iowa) and iS18 spacer (Glen Research,
Sterling Va.) to facilitate immobilization to surface aldehyde
groups with optimized spacing for DNA hybridization. Amplicon sizes
in the Examples below were 82, 84, or 116 base pairs for the L, J,
and Q mecA amplicons, respectively, and 95 base pairs for the ApoB
amplicon.
Example 3
On-Surface HDA Protocol
[0165] For on-surface HDA, surfaces were secured in wells of
microwell plate using double sided tape. A Master mix was prepared
using HDA Universal II kit reagents (BioHelix Corp., Beverly,
Mass.) by adding the following (1.times. concentration): 1.times.
annealing buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl), 40 mM NaCl, 4
mM MgSO.sub.4, 400 .mu.M dNTPs, 3 mM dATP, 0.1 .mu.m each primer
pair, 1.times. helicase enzyme mix and sterile water up to volume.
The Master mix was aliquoted (74 .mu.l) onto the surface of each
well. Purified or extracted MRSA genomic DNA (1 .mu.l) was added to
each reaction and the plate surface was covered with sealing tape
(Roche) and incubated at 65.degree. C. (hot plate or incubator) for
an appropriate amount of time, usually 45 minutes. Following
amplification, the supernatant was removed and surface(s) were
washed with wash A (0.1.times.SSC, 0.1% SDS) followed by wash B
(0.1.times.SSC). Next, a mouse monoclonal anti-biotin antibody
conjugated to HRP diluted 1/1000 in 1.times. hybridization buffer
(5.times.SSC, 0.1% SDS, 0.5% StabilCoat (Surmodics)) was added to
the coated support surface(s) and incubated at room temperature for
10 minutes. The surface(s) were then washed with wash B and
incubated with 125 .mu.l of TMB at room temperature for 5 minutes.
The surface(s) were then washed with water and methanol and allowed
to dry.
Example 4
[0166] Hybridization of double-stranded DNA targets to
surface-immobilized target-specific probes is made possible using
helicase to unwind the double-stranded target.
[0167] To demonstrate that helicase can sufficiently denature
double-stranded DNA templates to create single-stranded regions
available for hybridization to target-specific probes, mecA
polynucleotide targets from methicillin-resistant Staphylococcus
aureus DNA were amplified in a microtiter tube (25 .mu.L) for 60
minutes using the mecA L primer set as described in Example 3. The
HDA amplified products (or amplicons) were diluted 1/500 in water
and hybridized to a thin film biosensor chip (Inverness
Medical-Biostar, Inc.) adhered to the bottom of a microtiter plate
containing 90 .mu.L of 1.times. hybridization buffer or 1.times.
IsoAmp II buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 40 mM NaCl, 4
mM MgSO.sub.4). For a negative control (Chip A) 10 .mu.L of water
was added to a chip containing 1.times. Hybridization buffer. For a
heat denaturation control (chip B), a 10 .mu.l amplicon aliquot was
heated to 95.degree. C. for 5 minutes and then added to a chip
surface containing 1.times. Hybridization buffer. For a no
denaturation control (Chip C), 10 .mu.L of diluted amplicon was
added directly to a chip containing 1.times.HDA buffer along with 5
.mu.L of the helicase diluent buffer (10 mM KCl, 1 mM Tris-HCl, 0.1
mM DTT, 0.01 mM EDTA, 0.01% TX-100, 5% glycerol pH 7.4 final
concentration). For the helicase denatured target sample (Chip D),
5 .mu.L of helicase (150 ng in helicase diluent buffer) was added
to the 10 .mu.L of diluted amplicon, and placed onto a chip
containing 1.times.HDA buffer. All surfaces were incubated 15
minutes at 53.degree. C. and then washed with wash A followed by
wash B. To each chip, 125 .mu.L of anti-biotin antibody/HRP
conjugate diluted 1/1000 in 1.times. Hybridization buffer was added
and incubated at room temperature for 10 minutes. The chips were
washed with wash B and then 125 .mu.L of TMB was added. The chips
were incubated another 5 minutes, washed with water and methanol
and allowed to dry.
[0168] As shown in FIG. 1, hybridization of target DNA requires
denaturation thereof, as expected. In particular, addition of
helicase to the target yielded a positive signal, albeit not as
strong as the heat denaturation control. This suggests that
helicase may not be as effective as heat in denaturing the sample.
It should be noted, however, that the helicase was not incubated at
its optimal temperature (65.degree. C.) for this study.
Additionally, the sensitivity of target detection is lowered 2-4
fold in 1.times.HDA buffer compared with 1.times. Hybridization
buffer at 53.degree. C. (data not shown). Nonetheless, this study
illustrates that helicase is sufficient to unwind double-stranded
targets for hybridization to surface-immobilized probes.
Example 5
[0169] Dissociation and detection of hybridized polynucleotide
targets from the cationic surface is unexpectedly affected by the
ionic strength.
[0170] Sequences from the mecA gene in methicillin-resistant
Staphylococcus aureus were amplified in a microtiter tube (25
.mu.L) for 60 minutes using the mecA L primer set as described in
Example 3. Amplicons from an HDA reaction were diluted 1/5000 in
1.times. Hybridization buffer and annealed to a thin film biosensor
chip for 15 minutes at 53.degree. C. Surfaces were washed with wash
A and wash B. Then either 100 .mu.l of 1.times. IsoAmp II buffer or
1.times. Hybridization buffer was added and incubated at various
temperatures. Reactions were stopped at various times by washing
with wash A followed by wash B. To each chip, 125 .mu.L of
anti-biotin antibody/HRP conjugate diluted 1/1000 in 1.times.
Hybridization buffer was added and incubated at room temperature
for 10 minutes. The chips were washed with wash B and then 125
.mu.L of TMB was added. The chips were incubated another 5 minutes,
washed with water and methanol and allowed to dry.
[0171] As shown in FIG. 2, at 58.5.degree. C. surface-bound target
DNA rapidly dissociated from the chip surface (t.sub.1/2.about.30
minutes) in the presence of the high salt 1.times. Hybridization
buffer (containing 825 mM monovalent cation). In contrast, there
was still a significant fraction of target remaining in the
presence of 1.times. IsoAmpII buffer (containing 54 mM monovalent
cation), even after 4 hours at 58.5.degree. C. Additional
experiments conducted over a range of temperatures (53.degree. C.
to 64.degree. C.) confirmed that dissociation of target DNA from
the surface was more rapid at higher temperatures in 1.times.
Hybridization buffer, as expected for DNA duplexes
(t.sub.1/2.about.240 minutes at 53.degree. C. and <<30
minutes at 64.degree. C., data not shown). Rates of dissociation
from the coated support surface (as reflected by detectable
signal), while not quantitatively determined here, appear to be
similar to reported literature values. In contrast, dissociation of
target DNA from the surface was much slower in the lower ionic
strength IsoAmp II buffer under otherwise equal conditions
(t.sub.1/2>>360 minutes at 53.degree. C. and >>240
minutes at 58.5.degree. C., data not shown.
[0172] The conventional expectation would be for DNA duplexes to
dissociate faster in lower ionic strength buffers exhibiting less
charge stabilization. Therefore, the data above suggests that the
surface with a cationic character is playing a role in dissociation
of target DNA from the surface. While not wishing to be bound by
theory, it is believed that under high salt conditions, surface
cations are shielded from interacting with (and stabilizing) the
hybridized polynucleotide targets. Accordingly, the polynucleotide
targets can more freely dissociate from the surface. Under low salt
HDA reaction conditions, the cations may retain their ability for
electrostatic interaction with the backbone of the polynucleotide
target DNA, slowing or preventing its dissociation from the surface
even though the target may be no longer hybridized to the
surface-immobilized probe.
Example 6
Doubling Time for MRSA Primers in Solution
[0173] A series of HDA reactions were performed using a range of
genomic MRSA (ATCC) input DNAs (100-1,000,000 copies) in a reaction
mixture containing 1.times. annealing buffer (20 mM Tris-HCl, pH
8.8, 10 mM KCl) (BioHelix), 40 mM NaCl, 4 mM MgSO4, 400 .mu.m
dNTPs, 3 mM ATP, 0.1 .mu.m each primer, 1.times. helicase enzyme
mix (BioHelix), 0.2.times. EvaGreen (Biotium, Inc.). Samples were
placed in a Roche LightCycler 480 optical plate, covered with
sealing tape and placed into the Roche LightCycler 480. Plates were
incubated at 65.degree. C. and data was collected every 60 seconds.
EvaGreen is a double strand-specific dye conferring increases in
fluorescence as a function of amplification. Melt Curve analysis
was performed after the amplification to verify that a full-length
amplicon of expected Tm was produced. Amplification fidelity was
further confirmed by hybridization to target-specific probes
immobilized onto silicon chips as described above. Dye
incorporation was evaluated by plotting the time to detectable
signal (CP) against copy number of input genomic DNA. The slope of
the curves yields yield doubling times (time for primer pair to
create a copy) characteristic for each primer set. This analysis
provides a reasonable approximation of the expected amount of
amplicon present at any given time. Given that there is likely a
bit of a lag phase to overcome in the early stages of the HAD
reaction, actual doubling rates may be somewhat faster than the
averaged doubling times determined herein.
[0174] FIGS. 3A and 3B illustrate representative amplification data
for primer sets targeting the mecA gene in methicillin-resistance
in Staphylococcus. The tested primer pairs were found to exhibit
doubling times in solution ranging from about 1-3 minutes. Those
with the shortest doubling times (and found to be specific for
target amplification) were chosen for further assay
development.
Example 7
Determination of the Effect of the Chip on HDA Amplification
Efficiency
[0175] HDA reactions were set-up in 1.7 mL microcentrifuge tubes or
on a chip with 2000 copies of purified human DNA (Promega) as input
for amplification. Reactions were allowed to proceed for various
amounts of time and the reactions were stopped. Reactions from both
sample sets were applied to a 2% agarose gel stained with ethidium
bromide.
[0176] Both sets of reactions show clearly visible bands of the
correct size on the gel after 30 minutes amplification time. There
appears to be no adverse effect on amplification efficiency from
the chipl. On-chip amplicons are revealed as a single band on the
gel and the bands at equal time points are more intense.
Example 8
Calculation of Theoretical TTRs Based on Theoretical Chip LLODs and
Calculated Doubling Times
[0177] The calculated primer doubling times in solution (e.g., 1.1
minutes for the "L" primer set) were used to calculate a
theoretical time to result (TTR) for a chip not interfering with
amplification/detection based on chip LLODs measured at 10-30 pM
input DNA (amplicon) under HDA temperature (65.degree. C.) and
buffer conditions. A plot of the amount of amplicon present as a
function of amplification time is shown in FIG. 4 below. A bolded
box brackets the limit of sensitivity for a given target-specific
probe set and a dashed line shows the amount that is 10-fold above
the LLOD (lower limit of detection). A vertical line approximates
the time to detectable signal for 10 copies of target as input into
the HDA reaction. The results show that from as few as 10 copies of
input DNA, detectable levels of amplicon can be obtained by
.about.30 minutes, further producing 10-fold level of the detection
threshold by 38 minutes.
Example 9
Detection of HDA Amplification Products on Silicon Chips (LLOD)
[0178] To determine the lower limit of detection (LLOD)
corresponding to on-chip HDA amplification/detection of MRSA DNA, a
dose response analysis of MRSA DNA amplification was performed
using the protocol described in Example 3. Sequences in the mecA
gene from purified methicillin-resistant Staphylococcus aureus
genomic DNA (ATCC, strain #11632) were amplified using the "L"
primer set described in Example 2 and depicted in FIG. 9. The dose
response for genomic input DNA ranging from 0 to 10,000 copies was
tested. Amplification reactions were incubated for 45 minutes. at
64.degree. C. on "rough-side" silicon chips containing a surface
immobilized mecA-specific capture probe (mecA1703 CP, FIG. 9). The
representative data in FIG. 5 includes negative controls NTC A and
NTC B. The results show that the LLOD is 10 copies of target DNA on
the chip. The LLOD result was further reproduced in experiments
amplifying the human ApoB gene and the cfb gene of Streptococcus
agalactiae.
Example 10
Detection of HDA Amplification Products on Silicon Chips (TTR)
[0179] To determine the time necessary for amplifying MRSA genomic
DNA to detectable levels (time to result, TTR), HDA reactions were
performed using mecA-specific primers in the presence of varying
amounts (0-1000 sequence copies) of purified genomic DNA from
methicillin-resistant Staphylococcus aureus applied to the
"rough-side" silicon chip coated with mecA-specific capture probe
1703 as described in Example 3. Signals on the chip were measured
at various time points by washing the chip to stop the reaction
prior to addition of the detection reagents. Representative data is
shown in FIG. 6. With 100 sequence copies of MRSA target DNA, the
chip yielded a detectable signal after 30 minutes; 10 copies of
input yielded a detectable after 45 minutes. These results are
generally consistent with the theoretical model in Example 8
predicting detectability of 100 copies by 27-30 minutes and 10
copies by 30-33 minutes. (To more accurately assess HDA performance
for 10 copies relative to the theoretical model, more time points
may be required around 30 minutes). This data suggests that on-chip
HDA amplification can perform near its predicted limits under
conditions wherein the chip does not adversely affect amplification
efficiency. This data also suggests that HDA does not appear to
show an extended lag phase at the onset of amplification slowing
the time to detectable result. To the extent that there may be such
a lag phase, however, the primers would generate faster than
predicted doubling times.
Example 11
Detection of HDA Amplification Products on Thin Film Biosensor
Chips
[0180] To test HDA on-chip amplification/detection on another
visual detection medium, we utilized a thin film biosensor chip
containing immobilized mecA probes (1703) for direct visual
detection according to the method described in Examples 3 and 10.
On-chip detection times were similar to the rough side silicon chip
used in Example 10. As shown in FIG. 7, amplification of 100 copies
of genomic DNA produced detectable signals by 30 minutes,
consistent with the results observed using the rough-side chips.
Coated silicon supports may therefore provide a flexible platform
for on-chip HDA amplification and detection.
Example 12
Detection of mecA Gene Sequences from Blood Culture Isolates Using
on-Chip HDA Detection in Relation to Varying Surface Treatments
[0181] Blood was drawn (.about.10 mL) and placed into a BACTEC
blood culture bottle. The bottle was then seeded with
.about.1,000,000 CFU of methicillin-resistant Staphylococcus aureus
(ATCC strain #11632) plated onto blood agar and grown at 37.degree.
C. overnight. The blood culture bottle was incubated in a BACTEC
instrument until the alarm sounded, indicating that the bottle was
positive for bacterial growth. Dilution plating revealed the
presence of about 10.sup.8 to 10.sup.9 organisms per mL at the time
the alarm sounded. Aliquots (10 .mu.l) were removed from a
MRSA+BACTEC blood culture bottle and added to 3 .mu.l of 1M NaOH to
lyse erythrocytes. Next, 87 .mu.l of an extraction mix was added
(10 mM Tris pH 7, 50 units achromopeptidase (ACP, Sigma)) and
incubated for 10 minutes at room temperature prior to boiling the
treated sample to inactivate the ACP. From the lysed sample 1 .mu.l
was then subjected to on-chip HDA reaction on thin film biosensor
chips for 40 minutes essentially as described in Example 3.
Negative controls included lysis of 10 .mu.L bacteria-free blood
culture.
[0182] Four different thin film biosensor chips were used: (1) a
chip that was aged 6 months after attachment of the probe not
subsequently treated to block remaining reactive groups (Aged); (2)
a freshly made chip not subsequently treated to block remaining
reactive groups (Un-blocked); (3) a freshly made chip from the same
batch, treated with 100 .mu.m NHS-acetate (Pierce) to block easily
accessible surface amines NHS--Ac block); and (4) a freshly made
chip from the same batch, treated with 100 .mu.m acetic anhydride
(Aldrich) to block easily accessible surface amines (AcAn
block).
[0183] Representative results are shown in FIG. 8. Clearly
detectable signal was observed on all chips. The negative controls
were devoid of signal, indicating that the matrix is not
interfering with amplification/detection except in the case of the
un-blocked freshly prepared chip, which showed evidence of surface
passivation, or non-specific binding, by components in the blood
culture extract, including achromopeptidase extracted bacterial
cells, blood components and/or culture media. These data show that
performance on a coated cationic surface can be improved by aging
or other treatments serving to block at least a proportion of the
free amines.
[0184] It appears that surfaces may become more hydrophobic over
time with poly-(lys, phe) coatings on silicon or thin film
biosensors as measured by increased contact angle in a goniaometer
(data not shown). This suggests that significant diffusion occurs
over time, changing the accessibility of amines. While this doesn't
appear to harm the ability to immobilize capture probes, it is
beneficial to control non-specific interactions involving
components in blood culture samples, for example. As a method to
"accelerate" this diffusion process, accessible free amines can be
modified with NHS-acetate or acetic anhydride. Although these
methods can block significant amounts of accessible amines,
blockage of all amines is not complete, leaving others available to
interact locally with immobilized target sequences so as to slow
their diffusion from the surface under low ionic strength
conditions (<50 mM Na.sup.+).
[0185] It is intended that the foregoing detailed description be
regarded as illustrative rather than limiting, and that it be
understood that it is the following claims, including all
equivalents, that are intended to define the spirit and scope of
this invention.
Sequence CWU 1
1
11127DNAArtificialprimer; mecA L FWD 1tggatagacg tcatatgaag gtgtgct
27227DNAArtificialprimer; mecA L REV 2attatggctc aggtactgct atccacc
27327DNAArtificialprimer; mecA J FWD 3tggatagacg tcatatgaag gtgtgct
27426DNAArtificialprimer; mecA J REV 4tgattatggc tcaggtactg ctatcc
26525DNAArtificialprobe; mecA1703 CP 5caagtgctaa taattcacct gtttg
25627DNAArtificialprimer; mecA Q FWD 6caaactacgg taacattgat cgcaacg
27725DNAArtificialprimer; mecA Q REV 7atgctttggt ctttctgcat tcctg
25822DNAArtificialprobe; mecA1653 CP 8aaacaaacta cggtaacatt ga
22933DNAArtificialprimer; APOB4 rev 9cagtgtatct ggaaagccta
caggacacca aaa 331026DNAArtificialprimer; APOB FWD 3 10cttcatgtga
gccaaagatg ctgaac 261120DNAArtificialprobe; APOB-CPl 11aatttggcct
tcatgtgagc 20
* * * * *
References