U.S. patent application number 12/869199 was filed with the patent office on 2011-01-20 for systems, compositions and methods for nucleic acid detection.
Invention is credited to Dmitri Vezenov.
Application Number | 20110015380 12/869199 |
Document ID | / |
Family ID | 40027916 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110015380 |
Kind Code |
A1 |
Vezenov; Dmitri |
January 20, 2011 |
Systems, Compositions And Methods For Nucleic Acid Detection
Abstract
The invention relates to stretch measurements of nucleic acids
and correlating those measurements to the extent of double- and
single-stranded content of a nucleic acid of interest, and to
compositions, systems, and devices related thereto. In preferred
embodiments, one performs the stretch or elasticity measurements
under conditions such that one can determine a nucleic acid
sequence or the presence of an oligonucleotide in a sample.
Inventors: |
Vezenov; Dmitri; (Center
Valley, PA) |
Correspondence
Address: |
Peter G. Carroll;MEDLEN & CARROLL, LLP
Suite 350, 101 Howard Street
San Francisco
CA
94105
US
|
Family ID: |
40027916 |
Appl. No.: |
12/869199 |
Filed: |
August 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12057251 |
Mar 27, 2008 |
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12869199 |
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60920280 |
Mar 27, 2007 |
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Current U.S.
Class: |
536/23.1 ;
977/774 |
Current CPC
Class: |
G01Q 60/42 20130101;
G01N 21/6458 20130101; B82Y 35/00 20130101; C12Q 1/6816 20130101;
G01N 21/6489 20130101; Y10T 436/143333 20150115; G01N 21/648
20130101; G01N 21/552 20130101; C12Q 1/6816 20130101; C12Q 2563/155
20130101; C12Q 2525/204 20130101; C12Q 2523/303 20130101 |
Class at
Publication: |
536/23.1 ;
977/774 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1. A nucleic acid conjugate comprising: a) a single-stranded
nucleic acid having a first end and a second end wherein said first
end is immobilized on a substrate, said conjugate further
comprising a nucleotide sequence complementary to a portion of said
single-stranded nucleic acid and hybridized thereto, b) a distance
marker, and c) a molecular handle, wherein said second end of said
single-stranded nucleic acid is linked to said distance marker and
to said molecular handle.
2. The conjugate of claim 1, wherein said distance marker is
selected from the group consisting of a luminescent moiety, a
dielectric sphere, and a metallic particle.
3. The conjugate of claim 2, wherein said luminescent moiety is a
quantum dot.
4. The conjugate of claim 1, wherein said molecular handle is
selected from the group consisting of a ligand, a magnetic
particle, and a particle of a size between 0.1.times.10.sup.-9 m to
10.sup.-4 m.
5. The conjugate of claim 1, wherein said substrate comprises a
transparent surface.
Description
FIELD OF INVENTION
[0001] The invention relates to stretch measurements of nucleic
acids and correlating those measurements to the extent of double-
and single-stranded content of a nucleic acid of interest, and to
compositions, systems, and devices related thereto. In preferred
embodiments, one performs the stretch or elasticity measurements
under conditions such that one can determine a nucleic acid
sequence or the presence of an oligonucleotide in a sample.
BACKGROUND OF INVENTION
[0002] Nucleic acid sequencing is one of the most important
technologies in bioscience today. Whole-genome approaches and human
expressed sequence tag (EST) sequencing have started to exert
profound influences on biology and medicine. New applications, such
as population-based biodiversity projects and genotyping using
single-nucleotide polymorphism (a "brute-force" approach), make
such efforts even more urgent. Thus, there is a need for simple and
robust methods for sequencing nucleotide sequences suitable for
routine diagnostic applications.
SUMMARY OF INVENTION
[0003] The invention relates to stretch measurements of nucleic
acids and correlating those measurements to the extent of double-
and single-stranded content of a nucleic acid of interest, and to
compositions, systems, and devices related thereto. In preferred
embodiments, one performs the stretch or elasticity measurements
under conditions such that one can determine a nucleic acid
sequence or the presence of an oligonucleotide in a sample.
[0004] The invention relates to stretch measurements of nucleic
acids and correlating those measurements to the extent of double-
and single-stranded content of a nucleic acid of interest, and to
compositions, systems, and devices related thereto. In preferred
embodiments, one performs the stretch or elasticity measurements
under conditions such that one can determine a nucleic acid
sequence or the presence of an oligonucleotide in a sample.
[0005] In some embodiments, the invention provides a system for
stretching a nucleic acid. The system comprises a substrate that
supports a nucleic acid conjugate. The conjugate comprises a
nucleotide sequence having a first end and a second end, the first
end being immobilized on (i.e., attached to) the substrate. The
conjugate also comprises a distance marker and a molecular handle.
The second end of the nucleotide sequence is linked to the distance
marker and to the molecular handle.
[0006] Another element of the system is an instrument configured to
exert force on the molecular handle, to measure distances of the
distance marker from the substrate, and to measure forces on the
molecular handle during stretching.
[0007] In some embodiments, the molecular handle may be a ligand
such as biotin, in other embodiments, a magnetic particle serves as
a molecular handle. The distance marker is preferably a particle of
a size between 1.times.10.sup.-9 m to 10.sup.-4 m, and may comprise
an inorganic oxide, including without limitation silica or titania,
an organic polymer such as polystyrene, or a composite
material.
[0008] In some embodiments, the distance marker comprises quantum
dots dispersed in or on it as a luminescent moiety. Other
luminescent moieties are within the scope of the invention, as are
dielectric spheres or metallic particles.
[0009] In some embodiments, the instrumental element of the system
may be equipped with a proximity probe that interacts with the
molecular handle. In some embodiments, a cantilever tip of the type
used in atomic force microscopy may serve as a proximity probe. The
proximity probe may include a receptor such as streptavidin to
promote interaction with the molecular handle.
[0010] In some embodiments, the instrument interacts with the
molecular handle by means of a magnet. In other embodiments, the
molecular handle is manipulated by an optical trap fashioned into
the instrument.
[0011] In some embodiments, the invention provides a method of
determining the presence of an oligonucleotide in a sample.
According to the method a sample suspected of containing an
oligonucleotide (of known or unknown nucleotide sequence) is
contacted with a nucleic acid conjugate comprising a nucleic acid,
preferably single-stranded, having a first end and a second end and
further comprising a nucleotide sequence that is complementary to
the suspected oligonucleotide. The first end is immobilized on a
substrate. In addition to the nucleic acid, the conjugate comprises
a distance marker and a molecular handle, both of which are
attached to the second end of the nucleic acid.
[0012] Preferably, the method employs an instrument configured to
exert (and measure) force on the molecular handle. The instrument
is used to move the molecular handle such that the nucleic acid is
stretched and to measure the distance from the substrate the
distance marker traverses to establish a first distance, and the
forces applied.
[0013] According to the method, the sample is caused to contact the
nucleic acid in the conjugate under conditions such that the
oligonucleotide in the sample hybridizes to the nucleic acid to
create a nucleic acid conjugate such that at least a portion of the
nucleic acid is a double-stranded nucleic acid. A second distance
(and the forces applied) is measured by moving the molecular handle
with the instrument under conditions such that the double-stranded
nucleic acid is stretched. The correlation of the two distances
determines the presence of the oligonucleotide.
[0014] In one embodiment, the present invention contemplates a
nucleic acid conjugate. The conjugate, in this embodiment,
comprises a single-stranded nucleic acid having a first end and a
second end wherein said first end is immobilized on a substrate,
which may be transparent. The conjugate further comprises a
nucleotide sequence complementary to a portion of the
single-stranded nucleic acid, and hybridized thereto. The conjugate
further comprises a distance marker and a molecular handle, both of
which are linked to the second end of the single-stranded nucleic
acid element of the conjugate. The distance marker may be a
luminescent moiety such as a quantum dot, a dielectric sphere or a
metallic particle. The molecular handle may be a ligand, a magnetic
particle, or any particle of a size between 0.1.times.10.sup.-9 m
to 10.sup.-4 m.
[0015] The invention is further embodied in a method that comprises
providing a system for stretching a nucleic acid, the system
comprising a substrate, a nucleic acid conjugate, and an
instrument. The conjugate comprises a single-stranded nucleic acid
having a first end and a second end, the first end being
immobilized on the substrate. The conujugate further comprises a
nucleotide sequence complementary to the single-stranded nucleic
acid and hybridized thereto to create a partially double-stranded
nucleic acid, wherein the complementary sequence has a free 3' end.
The conjugate further comprises a distance marker, and a molecular
handle. The second end of single-stranded nucleic acid element of
the conjugate is linked to the distance marker and molecular
handle. An instrument configured to exert force on the molecular
handle further comprises the system. A sample comprising a
nucleotide, preferably of known structure, is also provided.
[0016] According to one embodiment of the method, the instrument is
employed to move the molecular handle under conditions such that
the partially double-stranded nucleic acid is stretched, a first
distance of said distance marker from the substrate is measured
(optionally as a function of the forces applied), the sample and
the conjugate are contacted under conditions such that the
nucleotide becomes ligated to the free 3' end of the complementary
sequence to create an extended double-stranded nucleic acid
(extended by one nucleotide), the molecular handle is moved under
conditions that stretch the extended nucleic acid, a second
distance of the distance marker from the substrate is measured
(optionally as a function of the forces applied), and the first and
second distances are correlated to determine the presence of the
complementary nucleotide. The distance measurements and ligation
steps may be repeated until the entire sequence of the nucleic acid
is determined. Thermal noise that may be acquired during the
measurements may be removed by averaging data or by averaging
differences between sets of force-length data. Further statistical
confidence may be reached by fitting averaged or individual
force-length measurements to a model of a stretched polymer
chain.
[0017] Again, the distance marker may be a luminescent moiety such
as a quantum dot, a dielectric sphere or a metallic particle; the
molecular handle may be a ligand (e.g., biotin) a magnetic
particle, or any particle (e.g., an inorganic oxide, an organic
polymer or a composite material) of a size between
0.1.times.10.sup.-9 m to 10.sup.-4 m. The distance marker may have
quantum dots dispersed in or on it as a luminescent moiety. Other
luminescent moieties are within the scope of the invention, as are
dielectric spheres or metallic particles.
[0018] Further, the instrumental element of the system may be
equipped with a proximity probe that interacts with the molecular
handle. In some embodiments, a cantilever tip of the type used in
atomic force microscopy may serve as a proximity probe. The
proximity probe may include a receptor such as streptavidin to
promote interaction with the molecular handle. Alternatively, the
instrument may be configured to interact with the molecular handle
by means of a magnet. In other embodiments, the molecular handle is
manipulated by an optical trap fashioned into the instrument.
[0019] In some embodiments, the systems and methods that embody the
invention comprise, in addition to a nucleic acid conjugate and an
instrument for stretching a nucleic acid and measuring the distance
traversed and the forces applied, a device comprising a plurality
of channels, each configured to direct a liquid to the nucleic acid
conjugate. In some embodiments, the channels have a width less than
about a millimeter and, preferably, less than a micrometer. In some
embodiments, the liquid comprises a nucleotide or an
oligonucleotide.
[0020] In some embodiments, the systems and methods that embody the
invention provide for processing a plurality of samples, wherein
each sample comprises an oligonucleotide containing at least six
contiguous nucleotides, and wherein the samples collectively
contain all possible nucleotide sequences in a predetermined set of
nucleotides. In these embodiments, force-strength measurements are
made for a single-stranded nucleic acid, one of the samples is
contacted with a nucleic acid conjugate of the system under
hybridizing conditions for the oligonucleotide in that sample,
force-length measurements are made for the hybridized nucleic acid,
another of the samples is contacted with the single-stranded
nucleic acid or, alternatively, with the hybridized nucleic acid,
force-length measurements are again made. Differences between
measurements are correlated to determine a presence of a
complementary oligonucleotide in the nucleic acid and the process
is repeated with other samples to identify all partial sequences in
the single-stranded nucleic acid that hybridize with complementary
oligonucleotides in the samples.
[0021] In some embodiments, the invention relates to a system for
stretching a nucleic acid comprising: a) a substrate comprising, a
nucleotide sequence conjugate comprising a distance marker, and a
molecular handle, wherein a first end of said nucleotide sequence
is immobilized to said substrate, and wherein a second end of said
nucleotide sequence is linked to said distance marker and molecular
handle, b) an instrument configured to exert force on said
molecular handle and measure the distance of said marker from said
substrate. In further embodiments, said molecular handle is
selected from the group consisting of a ligand, magnetic particle,
and particle of a size between 1.times.10.sup.-9 m to 10.sup.-4 m.
In further embodiments, said ligand is biotin. In further
embodiments, said particle is an inorganic oxide, an organic
polymer, or composite particle. In further embodiments, said
inorganic oxide is silica or titania. In further embodiments, said
organic polymer is polystyrene. In further embodiments, said
composite particle is a polystyrene dispersed with quantum dots or
silica dispersed with quantum dots. In further embodiments, said
distance marker is selected from the group consisting of a
luminescent moiety, a dielectric sphere, and a metallic particle.
In further embodiments, said luminescent moiety comprises a quantum
dot. In further embodiments, said instrument comprises a proximity
probe comprising a receptor. In further embodiments, said probe is
a cantilever tip. In further embodiments, said receptor is
streptavidin. In further embodiments, said instrument comprises a
magnet. In further embodiments, said instrument is configured to
create an optical trap.
[0022] In some embodiments, the invention relates to a method of
determining the presence of an oligonucleotide in a sample
comprising: a) providing, i) a sample suspected of containing an
oligonucleotide; ii) a substrate comprising a nucleic acid
conjugate comprising a single-stranded portion complimentary to
said oligonucleotide a distance marker, and a molecular handle;
wherein a first end of said single-strand is immobilized on a
substrate and wherein a second end of said single-strand is linked
to said distance marker and molecular handle; iii) an instrument
configured to exert force on said molecular handle; b) mixing said
sample and said substrate under conditions such that said
oligonucleotide hybridizes to said nucleic acid conjugate; c)
moving said molecular handle with said instrument under conditions
such that the nucleic acid is stretched; d) measuring a distance of
said distance marker from said substrate; and f) correlating said
measured distance to a presence of said oligonucleotide in said
sample.
[0023] In further embodiments, the invention relates to a substrate
comprising, a nucleic acid conjugate comprising: a single-stranded
portion, a double-stranded portion, a distance marker, and a
molecular handle wherein a first end of said single-strand is
immobilized on a substrate and wherein a second end of said
single-strand is linked to said distance marker and molecular
handle. In further embodiments, said distance marker is selected
from the group consisting of a luminescent moiety, dielectric
spheres, and metallic particles. In further embodiments, said
luminescent moiety is a quantum dot. In further embodiments, said
molecular handle is selected from the group consisting of a ligand,
magnetic particle, and particle of a size between
0.1.times.10.sup.-9 m to 10.sup.-4 m. In further embodiments, said
substrate comprises a transparent surface.
[0024] In some embodiments, the invention relates to a method
comprising: A) providing i) a system for stretching a nucleic acid
comprising a) a substrate comprising, a nucleic acid conjugate
comprising: a) a single-stranded portion, b) a double-stranded
portion comprising a free 3' end, c) a distance marker, and d) a
molecular handle, wherein a first end of said single-strand is
immobilized on a substrate and wherein a second end of said
single-strand is linked to said distance marker and molecular
handle; b) an instrument configured to exert force on said
molecular handle and measure the distance and force of said
distance marker from said substrate ii) a sample comprising a
nucleotide; B) mixing said sample and system under conditions such
that said nucleotide is ligated to said free 3' end, C) moving said
molecular handle with said instrument under conditions such that
the nucleic acid is stretched, D) measuring a distance of said
distance marker from said substrate and, F) correlating said
distance to the presence of a complimentary nucleotide in said
nucleic acid. In further embodiment, the method further comprises
repeating steps B-D to determine the sequence of said nucleic acid.
In further embodiments, said distance marker is selected from the
group consisting of a luminescent moiety, dielectric spheres, and
metallic particles. In further embodiments, said luminescent moiety
is a quantum dot. In further embodiments, said molecular handle is
selected from the group consisting of a ligand, magnetic particle,
and particle of a size between 1.times.10.sup.-9 m to 10.sup.-4 m.
In further embodiments, said instrument comprises a proximity probe
comprising a receptor. In further embodiments, said probe is a
cantilever tip. In further embodiments, said receptor is
streptavidin. In further embodiments, said instrument is a magnet.
In further embodiments, said instrument is configured to create an
optical trap.
[0025] In further embodiments, the invention relates to a method
comprising: A) providing i) a system for stretching a nucleic acid
comprising a) a substrate comprising, a nucleic acid conjugate
comprising a single-stranded portion, a double-stranded portion
comprising a free 3' end, a distance marker, and a molecular
handle, wherein a first end of said single-strand is immobilized on
a substrate and wherein a second end of said single-strand is
linked to said distance marker and molecular handle; b) an
instrument configured to exert force on said molecular handle and
measure said force applied to said molecular handle and measure
said a distance of said distance marker from said substrate; ii) a
sample comprising a nucleotide; B) mixing said sample and system
under conditions such that said nucleotide is ligated to said free
3' end, C) moving said molecular handle with said instrument under
conditions such that the nucleic acid is stretched, D) generating a
plurality of force and length data and, F) correlating said data to
the presence of a complimentary nucleotide in said nucleic acid. In
further embodiments, the method further comprises the step of
calculating a noise average of said data measurements. In further
embodiments, the method further comprises the step of fitting said
thermal noise average to a polymer model. In further embodiments,
the method further comprises the step of removing the thermal noise
by averaging said data measurements or by averaging differences
between two sets of said force-length data. In further embodiments,
the method further comprises the step of removing the effect of
said noise by fitting the averaged or individual force-length data
measurements to a model of a stretched polymer chain.
[0026] In some embodiments, the invention relates to a system
comprising: a) a device comprising a plurality of channels
configured to direct a liquid to a substrate, said substrate
comprising, a nucleic acid conjugate comprising: a single-stranded
portion, a distance marker, and a molecular handle, wherein a first
end of said single-strand is immobilized to said substrate, and
wherein a second end of said single-strand is linked to said
distance marker and molecular handle; b) an instrument configured
to exert force on said molecular handle. In further embodiments,
said channels have a width that is less that 1 millimeter or less
than 1 micrometer. In further embodiments, said liquid comprises a
nucleotide.
[0027] In some embodiments, the invention relates to a method
comprising: a) providing i) a plurality of oligonucleotides
containing 6 (six) or more contiguous nucleotides ii) a substrate
comprising a nucleic acid conjugate comprising a single-stranded
portion complimentary to one of said plurality of oligonucleotide,
a distance marker, and a molecular handle; wherein a first end of
said single-strand is immobilized to the substrate and wherein a
second end of said single-strand is linked to said distance marker
and molecular handle; iii) an instrument configured to exert force
on said molecular handle; b) mixing one of said oligonucleotide and
said substrate under conditions such that said oligonucleotide
hybridizes to said nucleic acid conjugate; c) moving said molecular
handle with said instrument under conditions such that the nucleic
acid is stretched; d) measuring a distance of said distance marker
from said substrate; and f) correlating said measured distance to a
presence of said complimentary oligonucleotide sequence in said
nucleic acid.
[0028] In additional embodiments, the invention relates to a method
of determining the presence of an oligonucleotide in a sample
comprising: a) providing i) a sample suspected of containing an
oligonucleotide; ii) a substrate comprising a nucleic acid
conjugate comprising a single-stranded portion complimentary to
said oligonucleotide, a distance marker, and a molecular handle;
wherein a first end of said single-strand is immobilized to the
substrate and wherein a second end of said single-strand is linked
to said distance marker and molecular handle; iii) an instrument
configured to exert force on said molecular handle; b) mixing said
sample and said substrate under conditions such that said
oligonucleotide hybridizes to said nucleic acid conjugate; c)
moving said molecular handle with said instrument under conditions
such that the nucleic acid is stretched; d) measuring a distance of
said distance marker from said substrate at varying or constant
force or measuring the elastic response of said distance marker;
and f) correlating said measured distance as a function of applied
force, i.e., elastic response, to a presence of said
oligonucleotide in said sample by detection of bonding of said
oligonucleotide to the nucleic acid.
[0029] In further embodiments, the invention relates to a method of
sequencing a nucleic acid comprising: a) providing i) a plurality
of oligonucleotides containing 2 (two), 3 (three), 4 (four), 5
(five), 6 (six), 7 (seven), or 8 (eight) or more contiguous
nucleotides; ii) a substrate comprising a nucleic acid conjugate
comprising a single-stranded portion complimentary to one of said
plurality of oligonucleotide, a distance marker, and a molecular
handle; wherein a first end of said single-strand is immobilized to
the substrate and wherein a second end of said single-strand is
linked to said distance marker and molecular handle; iii) an
instrument configured to exert force on said molecular handle; b)
mixing one of said oligonucleotide and said substrate under
conditions such that said oligonucleotide hybridizes to said
nucleic acid conjugate; c) moving said molecular handle with said
instrument under conditions such that the nucleic acid is
stretched; d) measuring a distance of said distance marker from
said substrate at varying or constant force or measuring the
elastic response of said distance marker; and f) correlating said
measured distance as a function of applied force, i.e., elastic
response, to a presence of said complimentary oligonucleotide
sequence in said nucleic acid.
[0030] In some embodiments, the invention relates to a substrate
comprising, a nucleic acid conjugate comprising: a single-stranded
portion, a double-stranded portion, a distance marker, and a
molecular handle. In further embodiments, said distance marker is
selected from the group consisting of a luminescent moiety,
dielectric spheres, and metallic particles. In further embodiments,
said luminescent moiety is a quantum dot. In further embodiments,
said molecular handle is selected from the group consisting of a
ligand, magnetic particle, and particle of a size between
1.times.10.sup.-9 m to 10.sup.-4 m. In further embodiments, said
substrate comprises a transparent surface.
[0031] In additional embodiments, the invention relates to a method
comprising: A) providing i) a system for stretching a nucleic acid
comprising a) a substrate comprising, a nucleic acid conjugate
comprising a single-stranded portion, a double-stranded portion
comprising a free 3' end, a distance marker, and a molecular
handle, b) an instrument configured to exert force on said
molecular handle, ii) a sample comprising a nucleotide; B) mixing
said sample and system under conditions such that said nucleotide
is ligated to said free 3' end C) moving said molecular handle with
said instrument under conditions such that the nucleic acid is
stretched, D) measuring a distance of said distance marker from
said substrate with a changing force and, F) correlating said
distance to the presence of a complimentary nucleotide in said
nucleic acid. Additional embodiments further comprise repeating
steps B through D to determine the sequence of said nucleic acid by
cycling through nucleotides of different type and inferring the
nucleotide sequence because of the corresponding Watson-Crick base
pairing rules. In further embodiments, said distance marker is
selected from the group consisting of a luminescent moiety,
dielectric spheres, and metallic particles. In further embodiments,
said luminescent moiety is a quantum dot. In further embodiments,
said molecular handle is selected from the group consisting of a
ligand, magnetic particle, and particle of a size between
0.1.times.10.sup.-9 m to 10.sup.-4 m. In further embodiments, said
instrument comprises a proximity probe comprising a receptor. In
further embodiments, said probe is a cantilever tip. In further
embodiments, said receptor is streptavidin. In further embodiments,
said instrument is a magnet. In further embodiments, said
instrument is configured to create an optical trap.
[0032] In some embodiments, the invention relates to a method of
measuring a hybridization of a nucleic acid comprising; a)
providing: i) a first single-stranded nucleic acid, i) a second
single-stranded nucleic acid comprising one or more nucleotides in
a predetermined sequence, iii) a solid support, and iv) a force
spectrometer; b) immobilizing said first nucleic acid to said solid
support; c) immobilizing said second nucleic acid to a tip of said
force spectrometer; d) slidably contacting said first nucleic acid
with said second nucleic acid under hybridizing conditions; and e)
measuring forces between said first and second nucleic acids.
[0033] In additional embodiments, the invention relates to a method
of measuring a degree of hybridization of a complimentary
nucleotide to a nucleic acid sequence comprising; a) providing: i)
a single-stranded nucleic acid sequence, ii) a nucleotide sequence
wherein a portion of the nucleotide sequence is complimentary to
said nucleic acid sequence, iii) a solid support, and iv) a force
spectrometer; b) immobilizing said first nucleic acid to said solid
support; c) measuring a first elasticity of said first nucleic acid
sequence using said force spectrometer; d) mixing said second
nucleic acid with said first nucleic acid under hybridizing
conditions; e) measuring a second elasticity of said first nucleic
acid; and f) correlating the difference in said first elasticity
and said second elasticity with the degree of hybridization.
[0034] In other embodiments the invention relates to a method of
detecting the addition of a single nucleotide to a nucleic acid
template comprising; a) providing: i) a single-stranded nucleic
acid sequence wherein a portion of the said sequence is
double-stranded, ii) a nucleotide, iii) a solid support, and iv) a
force spectrometer; b) immobilizing said single-stranded nucleic
acid sequences to said solid support; c) measuring the elasticity
of said single-stranded nucleic acid sequence using said force
spectrometer; d) mixing said single nucleotide phosphate with said
single-stranded nucleic acid sequence under conditions such that
said single nucleotide phosphate incorporates into said
double-stranded portion within said single-stranded nucleic acid
sequence; e) measuring the elastic response of said single-stranded
DNA sequence with said single nucleotide phosphate incorporated
into said double-stranded portion; and f) correlating the change in
said elastic response with the incorporation of said
nucleotide.
[0035] In some embodiments, the invention relates to a method
comprising: a) providing i) a plurality of oligonucleotides
containing 6 (six) or more contiguous nucleotides covering all
possible sequences for a given number of nucleotides; ii) a
substrate comprising a nucleic acid conjugate comprising a
single-stranded portion complimentary to one of said plurality of
oligonucleotide, a distance marker, and a molecular handle; wherein
a first end of said single-strand is immobilized to the substrate
and wherein a second end of said single-strand is linked to said
distance marker and molecular handle; iii) an instrument configured
to exert force on said molecular handle; b) mixing one of said
oligonucleotide and said substrate under conditions such that said
oligonucleotide hybridizes to said nucleic acid conjugate; c)
moving said molecular handle with said instrument under conditions
such that the nucleic acid is stretched; d) measuring a distance of
said distance marker from said substrate; f) correlating said
measured distance to a presence of said complimentary
oligonucleotide sequence in said nucleic acid; and g)
reconstructing the sequence of said nucleic acid conjugate of the
substrate from the partial sequences of hybridized complementary
nucleotides.
[0036] In additional embodiments, the invention relates to a
substrate comprising; a single-stranded nucleic acid sequence
comprising a first end and a second end wherein said
single-stranded nucleic acid comprises an optical probe and a
magnetic particle wherein said first end of said single-stranded
nucleic acid is immobilized to the substrate and wherein said
second end is linked to said optical probe and magnetic
particle.
[0037] In some embodiments, the invention relates to chemical and
enzymatic methods for attachment of nucleic acid fragments to the
surface and to the near-field probes.
[0038] In other embodiments, the invention relates to microfluidic
platforms for automated reagent delivery for single nucleotide
addition cycling preferably using a force spectroscopy setup.
[0039] In other embodiments, the invention relates to probes used
as markers and reporters of the distance from the surface of a
solid support. The probes are selected from i) dielectric spheres,
for instruments based on evanescent field scattering, ii)
semiconductor quantum dots (QD), for instruments based on total
internal reflectance fluorescence (TIRF), or iii) metal
nanoparticles, for instruments based on capacitance changes.
[0040] In some embodiments, the invention relates to magnetic
probes to exert force on single-stranded fragments anchored to a
surface wherein these magnetic particles either simultaneously
function as near-field probes or are used in tandem with near-field
probes.
[0041] In additional embodiments, the invention relates to
selective surface chemistry for attachment of DNA fragments to i)
the surface of the solid support; ii) nanometer sized dielectric
spheres, QD, and metal nanoparticles or composite probes
thereof.
[0042] In some embodiments, the invention relates to methods of
numerical modeling (e.g. 2D and 3D numerical solutions of Maxwell
equations) evanescent field scattering and fluorescence of
sub-wavelength (nanometer scale) particles and methods to use
modeling to provide guidance in optimizing electromagnetic response
of near-field probes in the vicinity of the surface.
[0043] In additional embodiments, the invention relates to methods
of using sequencing technology platforms by combining force
spectroscopy setup with microfluidic systems for efficient
automated cycling of single nucleotide addition.
[0044] In some embodiments, the invention relates to methods of
hybridization for distinct oligo tags, preferably with 8- to
20-mers, in a freely arrayed, preferably 100.times.100, matrix of
single molecules by photometry of dielectric spheres or TIR
fluorescence of QD preferably with random thermal forces only.
[0045] In additional embodiments, the invention relates to methods
of generating a full force-extension profile, using force
microscopy, and detecting, in a single synthetic DNA fragment
elongation of a double-stranded nucleic acid by a single nucleotide
by polymerase and combining force microscopy and near-field probes,
to correlate measurements of end-to-end distances.
[0046] In some embodiments, the invention relates to methods of
detecting changes in electromagnetic (photonic) response of
near-field probes upon single nucleotide addition to an unknown
nucleic acid sequence.
[0047] In additional embodiments, the invention relates to methods
of reading unknown sequences of individual nucleic acid fragments
in a, preferably 100.times.100 or larger, array using combined
force spectroscopy and microfluidics setups.
[0048] In some embodiments, the invention relates to fabricating
components of a massively parallel device using arrays of freely
arrayed single molecules preferably with nucleotide incorporation
rates preferably of up to 1 Mb/sec.
[0049] In some embodiments, the invention relates to methods of the
simultaneous use of magnetic handles to exert force and quantum
dots, or other distance markers, to read the molecular
distance.
[0050] In additional embodiment, the invention relates to
sequencing methods using magnifying tags for specific
nucleotides.
[0051] In other embodiments, the invention relates to
nanometer-sized near field probes for force spectroscopy of nucleic
acid fragments attached to the surface of the solid substrate,
preferably these probes: i) specifically bind to the nucleic acid
fragments; ii) exert mechanical force on the nucleic acid; iii)
provide reading of the end-to-end molecular extensions.
[0052] In other embodiments, the invention relates to elasticity
measurements based on young's modulus.
A BRIEF DESCRIPTION OF THE FIGURES
[0053] FIG. 1 illustrates a change in the conformation of the
surface-bound nucleic acid fragment in the course of replication of
a single-stranded nucleic acid by DNA polymerase.
[0054] FIG. 2 shows a force-extension diagram for 200-mer
undergoing change from ss form (blue curve) to ds form (black).
Intermediate stages (red) corresponding to 100 ds bp, 101 ds bp,
and 110 bps re also shown.
[0055] FIG. 3 illustrates embodiments for excitation of near-field
probes by the evanescent field: TIR microscopy using prism (A) or
objective (B) illumination; and (C) use of slab waveguides for the
excitation of the probes positioned within the evanescent field in
the cladding of the waveguide.
[0056] FIG. 4 illustrates embodiments of magnetic tweezers force
spectroscopy assay with near-field probes for detection of
molecular end-to-end distances.
[0057] FIG. 5 shows FE curves for 200-mer with 100, 110, 111
single-stranded bases (the rest are ds) and illustrates the ability
to resolve the change in two force-extension curves where
differences are an order of magnitude smaller than the expected
noise level. The curves in the figure correspond to noiseless
curves in the insert of FIG. 2.
[0058] FIG. 6 illustrates a schematic diagram of the microfluidic
device for automated delivery of stock solutions of dNTPs and wash
buffers (A). Individual reaction chambers suitable for FS on freely
arrayed DNA fragments can be arranged as an array of chambers for
parallel reactions of SNA (B). Freely arrayed single molecules
display different brightness of the scattered (fluorescent) light
depending on the distance from the surface. Individual FE curves
are reconstructed from intensity profiles (C).
[0059] FIG. 7 shows a schematic diagram for surface modification of
the microfluidic chip for force spectroscopy using near-field
probes: DNA is first fragmented, then ligated with adaptors
recognizing either surface or probe complimentary adaptors (or
ligands), followed by hybridization (or binding) with recognition
sites on the surfaces of the support and probe.
[0060] FIG. 8 shows a modification of DNA fragments for surface
attachment.
[0061] FIG. 9 illustrates a force spectroscopy experiment to test
the response of the optical near-field probes to changes in the
separation from the surface.
[0062] FIG. 10 shows an embodiment that can be used in the force
spectroscopy experiment illustrated in FIG. 9.
[0063] FIG. 11 shows an embodiment that can be used in the
experiments illustrated in FIG. 4.
[0064] FIG. 12 shows intermediates that can be used to prepare the
embodiment in FIG. 11 as describe in Seo et al. (2004) Proc. Natl.
Acad. Sci. USA 101, 5488-5493, and Seliger et al. (1991)
Nucleosides and Nucleotides 10, 303-306.
[0065] FIG. 13 shows nucleotides with 3' O-allyl protecting groups
used as reversible terminators during sequencing methods as
described in Ju et al. (2006) Proc. Natl. Acad. Sci. USA 103,
19635-19640.
[0066] FIG. 14 illustrations one way of creating an evanescent
field (1) by creating a zero-mode waveguide having an nucleic acid,
molecular handle, and distance marker (2) immobilized to a
transparent material (4). The transparent material is coated with
an opaque film (3), preferably aluminum.
DETAILED DESCRIPTION OF THE INVENTION
[0067] The invention relates to stretch measurements of nucleic
acids and correlating those measurements to the extent of double-
and single-stranded content of a nucleic acid of interest, and to
compositions and devices related thereto. In preferred embodiments,
one performs the stretch or elasticity measurements under
conditions such that one can determine a nucleic acid sequence or
the presence of an oligonucleotide with a known sequence for the
purpose of sequencing an unknown sequence in a sample.
TABLE-US-00001 TABLE 1 Key to the elements identified in the
Figures. Name of Element FIG. 1 100 substrate 110 single stranded
DNA 120 double stranded DNA 130 DNA polymerase 140 bead FIG. 3 150
objective lens for electron multiplying CCD camera 160 case 170
chamber 180 DNA with bead attached to substrate 190 bead 200 glass
slide 210 optical near-field dove prism 220 hv emitted from
illuminted bead (red) 230 hv emitted from light source (blue) 240
evanescent waves 250 total internal reflection fluorescence
microscope (TIRFM) objective (grey sphere) 260 yellow wave guide
FIG. 4 270 small circle dNTP 280 fluorescet bead 290 magnetic bead
300 magnet S 310 magnet N 320 magnetic waves 330 source 340
detector FIG. 5 350 inset graph avg = 0 for no SNA addition - red
curve 360 inset graph avg~0.19 nm for SNA - blue curve 370 red line
- 110 bases 380 blue line - 111 bases 390 black line 100 bases FIG.
7 400 double stranded DNA 410 restriction endonuclease 420 isolated
excised single stranded fragment 430 ligase 440 single stranded DNA
complementary to bead fragment 450 single stranded DNA
complementary to primer 460 complementary primer fragment to 450
470 complementary fragment for 440 attached to a bead 480 substrate
FIG. 9 490 Atomic Force Microscope Tip 500 n = number of dNTP 510
dNTP 520 biotin 530 Streptavidin
[0068] In preferred embodiments, the invention is related to
methods and systems based on concepts of mechanical properties of
single molecules, according to which one may detect changes in
molecular elasticity of a single nucleic acid fragment as one
converts it from a single-stranded to double-stranded form. While
applicant will not be bound by this or any other theory of the
technique, one can use the technique to detect the addition of a
single nucleotide to a nucleic acid molecule, catalyzed by DNA
polymerase, preferably under conditions in which the polymerase
activity is reduced or arrested. In preferred embodiments, the
nucleic acid sequence is reconstructed from the order of addition
of successively added deoxynucleoside triphosphates.
[0069] In another preferred embodiment of the invention, one uses
single molecule force spectroscopy to detect changes in the
conformation of the nucleic acid fragments attached to the surface
of a solid support. Upon addition of a single nucleotide, the
fraction of base pairs (bp) in a double-stranded (ds) nucleic acid
segment increases, while the corresponding single-stranded (ss)
nucleic acid fragment undergoes a complementary decrease in the
number of bases. One detects changes imparted to the mechanical
properties and dynamics of the nucleic acid segment upon conversion
from ss to ds forms. It is contemplated that one can acquire
stretch measurements by force spectroscopy in various formats
(scanning probe, magnetic or optical tweezers). Detecting a change
in elasticity of the nucleic acid is equivalent to detecting the
addition of a nucleotide, thus providing (if the identity of the
added nucleotide is known) the means of reading the unknown nucleic
acid sequence after successive additions are made. Runs of the same
nucleotides, e.g., AA, AAA, may be detected using nucleotides with
reverse terminators implied through the magnitude of the response
when exposed to a solution that is limited to one specific
nucleotide.
[0070] In other embodiments, it is contemplated that one may create
stock solutions of oligonucleotides with known sequences that cover
all possible sequences. For example, one may create 32 stock
solutions of 4-mers. If each stock solution contains only one
particular oligonucleotide sequence then the summation of stock
solutions contain every possible combination of an oligonucleotide
with 4 nucleotides. Similarly, one may create 65,000 stock
solutions of 8-mers. Thus applying the methods disclosed herein,
one may use these stock solutions to determine a particular
sequence when ligation or hybridization is occurs.
[0071] Force spectroscopy of single biomolecules is a tool for
unraveling mechanics of single biomolecules and polymers. A typical
experiment involves i) grabbing (e.g. by a biotin-avidin link) a
free end of the biomolecule or polymer that is attached at its
other end to a solid support, and ii) pulling by the grabbed end,
while recording the pulling force, to generate a force-extension
(FE) curve. While applicants will not be bound by any theoretical
explanation of any applications of the invention, statistical
mechanical models of polymer chains may describe the mechanical
response of the system.
[0072] The model description of the system has improved to the
level that it is possible to fit force-extension curves to
determine molecular end-to-end distances with sub-nanometer
precision. This analysis applies to experiments on both synthetic
polymers in good solvents and biological heteropolymers such as
proteins and nucleic acids. The ability to determine the end-to-end
distance to an accuracy of a single chemical bond (.about.0.1 nm)
provides a "molecular ruler" with which to study changes in
molecular conformation. For example, the change in contour length
when a region of green fluorescent protein is unfolded, as
determined from experimental FE curves, was used to assign
conformational changes in the protein with resolution at the level
of a single amino acid residue.
[0073] The behavior of nucleic acids in both single- and
double-stranded forms can be described by a set of statistical
models for polymer chains: from extensible free-jointed chain (FJC)
to extensible worm-like chain (WLC) models (see FIG. 1). According
to this description, when the chains (i.e. DNA backbone) are
stretched by the application of an external force, the length of a
single monomer unit (per base pair) can increase due to changes in
the bond angles and (to a lesser extent) bond lengths as they
adjust to accommodate the stress. Thus, the conventional models of
statistical mechanics of polymer chains account for bond elasticity
if one treats the chain as "extensible." More importantly, the
mechanical behavior of the polymer chain, by this description, is
primarily determined by the entropic elasticity, which is highly
responsive to the change in persistence length (lp) of the polymer
(the distance over which the correlations in the polymer chain are
lost--looking at the molecule on scales shorter than lp, the
molecule appears straight; looking from a distance>>lp, the
molecule will appear randomly coiled).
[0074] According to the model, the change experienced by the DNA
molecule is the change in its stiffness: in the ss-DNA, the are
free to orient in solution in any possible way, while in the dsDNA,
the by must remain hydrogen bonded in Watson-Crick pairing and
maintain this orientation even under external force. This
structural difference is responsible for two orders of magnitude
difference in persistence length for the two forms of DNA--0.75 nm
(.about.3 bp) for ssDNA versus 50 nm (.about.150 bp) for dsDNA.
Change in the stiffness of the nucleic acid upon conversion from
single-stranded to double-stranded form, as manifested by the
different lp, is many embodiments of the invention.
Surface Immobilization of Nucleic Acid Linked to Molecular Handles
and Distance Markers
[0075] In some embodiments, the invention exploits surface
chemistry to attach nucleic acid fragments to flat surfaces in a
well-defined orientation. One may use high-resolution microscopy to
observe freely arrayed nucleic acid fragments on the surface of
glass. For the detection of the distance between the free end and
the surface, one may set up optical evanescent fields in the
vicinity of the glass-aqueous solution interface. Such evanescent
fields (or optical near-fields) in certain embodiments, may be
generated using a total internal reflection (TIR) configuration for
the illumination of the glass-solution interface on slab waveguides
or zero-mode waveguides. In a TIR arrangement, radiation from an
optically dense medium is incident onto an optically less dense
medium at angles larger than the critical angle, i.e. the angle at
which all incident energy is reflected back into an optically dense
medium. In spite of the fact that no radiation propagates in the
medium (the solution containing DNA fragments), the electromagnetic
fields penetrate into the second medium a short distance from the
interface.
[0076] In some embodiments, in order to observe the distance
between the free end and the surface of the support, one attaches
optical probes to these free ends. These optical probes interact
with the near-field and generate some propagating fields whose
amplitude depends on the intensity of the evanescent field,
therefore, directly reporting the position of the probe within the
evanescent field (see FIG. 5).
[0077] For fluorescent probes one can use semiconductor
nanocrystals, such as quantum dots (QDs). Quantum dots have sizes
that are in the range where quantum confinement effects become
important, energy gap becomes size-dependent and, thus, the
fluorescence wavelength for the same semiconductor shifts with
changes in the diameter of the QD. The QDs also have a large
absorbance cross-section over a broad frequency range, and
therefore, fluorescence of QDs of different sizes can be excited by
the same light source. The small size (<<length of the DNA
fragment) and high quantum efficiency of QDs and reduced
photobleaching make them preferable as probes of the optical
near-field.
[0078] The function of these optical near-field probes is to
provide photonic response (scattering or fluorescence) whose
intensity is directly related to intensity of the near-field
(which, in turn, is a function of the separation from the surface).
While embodiments of the invention will typically use optical
response as the detection method, the approach is different from
the use of fluorescent dNTPs: i) Due to the size of the probes
preferred, the optical signal is orders of magnitude higher, thus,
eliminating the need for high concentration of DNA or secondary
amplification steps, and enabling an assay to be carried out in a
single molecule format; ii) QD optical probes do not bleach, thus
continuous monitoring and prolonged use of high intensity sources
(lasers) is possible; iii) The probes report on the parameter
describing the whole molecule (end-to-end distance) rather than the
specific base added, thus no separation step is needed for base
calling in a given step, and the whole assay will be accelerated
accordingly.
[0079] In preferred embodiments of the invention, probes are used
as markers and reporters of the distance from a substrate. As used
herein, a "distance marker" means any molecular arrangement that is
configured to indicate the distance or relative distance of the
arrangement in relation to the substrate on which the marker is
immobilized. In more preferred embodiments, the distance markers
are near-field probes selected from i) dielectric spheres, for
detection with instruments based on evanescent field scattering,
ii) semiconductor quantum dots (QDs), for detection with
instruments based on total internal reflectance fluorescence
(TIRF), or iii) metal nanoparticles, for detection with instruments
based on capacitance changes.
[0080] For dielectric probes, one can use polystyrene spheres,
because i) they are available commercially in a variety of sizes
(from 20 nm to microns); ii) their surface chemistry has been
well-characterized and numerous surface modifications have been
reported; iii) they provide an excellent refractive index contrast
(index of 1.59 versus 1.33 of water), and iv) they can be doped
with magnetic particles. If a higher refractive index contrast is
desired one can use titania dioxide (n.about.2.4-2.7) nanoparticles
(or commercially available submicron particles from DuPont). The
chemistry of titania dioxide surfaces is also readily modified
using, for example, siloxane-based surface attachment of reactive
organic functional groups.
[0081] As used herein, a "molecular handle" means any molecular
arrangement that is configured to move upon the action of a force.
In preferred embodiments, molecular handles are magnetic particles
that move under the force of a magnet, and polystyrene particles
that move under the force of light. In other preferred embodiments,
the molecular handles and distance markers (near-field probes, e.g.
semiconducting nanoparticles) are attached to the single stranded
nucleic acid fragments. It is specifically contemplated that the
molecular handle and distance marker are a part of the same
molecular arrangement or particle or composite particles such as
CdTe doped sodium silicate particles. As handles, they are used to
actuate the single molecule in an array of single molecules; while
as distance markers, they report marker locations. The synthesis of
composite particles such as 1) CdTe nanocrystals capped with
1-mercapto-2,3-propandiol, 2) CdSe nanocrystals capped with sodium
citrate, and 3) core-shell CdSe/CdS nanocrystals capped with sodium
citrate is described in Rogach et al., Chem. Mater. 2000, 12,
2676-2685. One may modify these surfaces with
3-mercaptopropyltrimethoxysilane (MPS) in water-ethanol mixtures.
By addition of sodium silicate, "raisin bun"-type composite
particles form, with either CdTe, CdSe, or CdSe/CdS nanocrystals
which are homogeneously incorporated as multiple cores into silica
spheres of 40-80 nm size. Further, growth of larger silica spheres
(100-700 nm) can be performed by using either MPS-modified
semiconductor nanocrystals or "raisin bun"-type composite particles
as seeds, which gives semiconductor-doped silica globules of
desirable sizes in the submicrometer range. One observes a shift of
the photonic band gap to the red in photonic crystals made of
nanoparticles-doped silica due to the refractive index of the
semiconductors.
[0082] A laser beam brought to a focus with a strongly converging
lens forms a type of optical trap widely known as an optical
tweezer. Multiple beams of light passing simultaneously through the
lens' input pupil may focus to multiple optical tweezers, each at a
location determined by the associated beam's angle of incidence and
degree of collimation as it enters the lens. Their intersection at
the input pupil yields an interference pattern whose amplitude and
phase corrugations characterize the downstream trapping pattern.
Imposing the same modulations on a single incident beam at the
input pupil would yield the same pattern of traps. Such wavefront
modification can be performed by a computer-designed diffractive
optical element (DOE), or hologram. Holographic optical trapping
(HOT) uses computer-generated holograms (CGHs) to project arbitrary
configurations of optical traps, and so provides control over
microscopic materials dispersed in fluid media. In some
embodiments, one uses optical traps to move particles linked to
nucleic acids, including quantum dots.
[0083] Quantum dots are semiconductor particles preferably with
diameters of the order of 2-10 nanometers, or roughly 200-10,000
atoms. As a semiconductor material, quantum dots have a
composition-dependent bandgap energy, which is the minimum energy
required to excite an electron to an energy level above its ground
state, commonly through the absorption of a photon of energy
greater than the bandgap energy. Relaxation of the excited electron
back to its ground state results in photon emission. Because the
bandgap energy is dependent on the particle size, the optical
characteristics of a quantum dot can be tuned by adjusting its
size. A synthetic method for quantum dots (<5% root-mean-square
in diameter) made from cadmium sulfide (CdS), cadmium selenide
(CdSe), or cadmium telluride (CdTe) is described in Murray et al.
(1993) J. Am. Chem. Soc. 115, 8706-8715. Quantum dots that can span
the visible spectrum are known, and CdSe has become the preferred
chemical composition for quantum dot synthesis. Many techniques are
possible for post-synthetically modified quantum dots, such as
coating with a protective inorganic shell (Dabbousi et al. (1997)
J. Phys. Chem. B 101, 9463-9475, and Hines et al. (1996) J. Phys.
Chem. 100, 468-471); surface modification (Gerion et al. (2001) J.
Phys. Chem. B 105, 8861-8871, and Gao et al. (2003) J. Am. Chem.
Soc. 125, 3901-3909) and direct linkage to active molecules
(Bruchez et al. (1998) Science 281, 2013-2016, and Chan et al.
(1998) Science 281, 2016-2018). CdSe quantum dots with diameters
between 2 and 8 nm have emission wavelengths from 450-650 nm,
spanning the entire visible spectrum. By also adjusting the quantum
dot composition (ZnS, CdS, CdSe, CdTe, PbS, PbSe, and their
alloys), it is possible to span the wavelength range 400-4000
nm.
[0084] Because quantum dots have high surface area to volume
ratios, a large fraction of the constituent atoms are exposed to
the surface, and therefore have atomic or molecular orbitals that
are not completely bonded. These "dangling" orbitals may form bonds
with organic ligands such as trioctylphosphine oxide (TOPO).
Strategies may be used to make hydrophobic quantum dots soluble in
aqueous solution. In one instance, a suspension of TOPO-coated
quantum dots is mixed with a solution containing an excess of a
heterobifunctional ligand, which has one functional group that
binds to the quantum dot surface and another functional group that
is hydrophilic. Thereby, hydrophobic TOPO ligands are displaced
from the quantum dot through mass action, as the new bifunctional
ligand adsorbs to render water solubility. Using this method,
(CdSe)ZnS quantum dots may be coated with mercaptoacetic acid and
(3-mercaptopropyl) trimethoxysilane, both of which contain basic
thiol groups to bind to the quantum dot surface atoms, yielding
quantum dots displaying carboxylic acids or silane monomers.
Quantum dots covered with carboxylic acid groups may interact
directly with molecules containing basic functional groups, such as
amines or thiols. Thus, nucleotides and nucleic acids
functionalized with amine or thiol groups, as disclosed herein, can
be linked to quantum dots.
[0085] The term "conjugate", as used herein, refers to any compound
that has been formed by the joining of two or more moieties. A
"moiety" is any type of molecular arrangement designated by
formula, e.g., chemical name or structure. Within the context of
certain embodiments, a conjugate is said to comprise one or more
moieties. This means that the formula of the moiety is substituted
at some place in order to be joined and be a part of the molecular
arrangement of the conjugate. In a preferred embodiment, we refer
to nucleic acid conjugates meaning that the nucleic acid is one
moiety. It is not intended that the joining of two or more moieties
must be directly to each other. A linking group, i.e., any
molecular arrangement that will connect the moieties by covalent
bonds such as, but not limited to, one or more amide group(s).
Alkyl groups and ethylene glycol units, may join the moieties,
i.e., covalent linking. Additionally, although the conjugate may be
unsubstituted, the conjugate may have a variety of additional
substituents connected to the linking groups and/or connected to
the moieties.
[0086] As used herein, the term "immobilization" refers to the
attachment or entrapment, either chemically or otherwise, of a
material to another entity (e.g., a solid support) in a manner that
restricts the movement of the material. For example, a nucleic acid
may be immobilized to a solid support by hybridizing to a
complimentary sequence or by directly linking the molecule to the
support through covalent bonds.
[0087] As used herein, the term "ligand" refers to any molecule
that binds to or can be bound by another molecule. Examples of
ligands include, but are not limited to, molecules such as biotin,
carbohydrates, peptide, antigens, nucleic acids and other substance
that binds to another entity to form a complex.
[0088] A "receptor" means a moiety utilized to selectively bind to
a ligand.
[0089] As used herein, the term "selective binding" refers to the
binding of one material to another in a manner dependent upon the
presence of a particular molecular structure (i.e., specific
binding). For example, an immunoglobulin will selectively bind an
antigen that contains the chemical structures complementary to the
ligand binding site(s) of the immunoglobulin. This is in contrast
to "non-selective binding," whereby interactions are arbitrary and
not based on structural compatibilities of the molecules.
[0090] As used herein, the term "substrate" refers to a solid
object or surface upon which another material is layered or
attached such as mesogens. Solid supports include, but are not
limited to, glass, metals, gels, and filter paper, among
others.
[0091] As used herein, the term "sample" is used in its broadest
sense. In one sense it can refer to a biopolymeric material. In
another sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from animals (including
humans) and encompass fluids, solids, tissues, and gases.
Biological samples include blood products, such as plasma, serum
and the like. Environmental samples include environmental material
such as surface matter, soil, water, crystals and industrial
samples. These examples are not to be construed as limiting the
sample types applicable to the present invention.
[0092] Sequencing-by-synthesis is based on the detection of
nucleotide incorporation, using a primer-directed polymerase
extension. The sequence can be deduced iteratively. Various
protocols are based on fluorescently labeled nucleotides. In some
embodiments, the present invention relates to methods of sequencing
by synthesis that utilize unlabeled or unmodified nucleotides by
detecting stretching properties of nucleic acids with a proximity
probe.
[0093] A proximity probe microscope works by measuring a local
property--such as height, optical absorption, or magnetism--with a
probe or "tip" placed close to the substrate. The small
probe-substrate separation makes it possible to take measurements
over a small area. One may acquire an image when the microscope
raster-scans the probe over the substrate while measuring the local
property in question. One can use a variety of tip types. The
"normal tip" is a 3 .mu.m tall pyramid with .about.30 nm end
radius. In some preferred embodiments, one uses tips with higher
aspect ratio (i.e., long and thin) such as electron-beam-deposited
(EBD) tips or those made by microlithography processes. In other
preferred embodiments, the tips are coated with a film of gold so
that thiol based linking groups can be attached directly to the
tip, or the entire probe.
[0094] An atomic force microscope, "AFM", operates by measuring
attractive or repulsive forces between a tip and the sample. In its
repulsive "contact" mode, the instrument lightly touches a tip at
the end of a leaf spring or "cantilever" to the sample. A detection
apparatus measures the vertical deflection of the cantilever. Thus,
in contact mode the AFM can measure repulsion forces between the
tip and sample. AFMs can image samples in air and under
liquids.
[0095] AFMs can generally measure the vertical deflection of the
cantilever with high resolution. To achieve this, one may use an
optical lever. The optical lever operates by reflecting a laser
beam off the cantilever. Angular deflection of the cantilever
causes several-fold larger angular deflection of the laser beam.
The reflected laser beam strikes a position-sensitive photodetector
consisting of photodiodes. The difference between the photodiode
signals indicates the position of the laser spot on the detector
and thus the angular deflection of the cantilever. AFM cantilevers
have high flexibility. A high flexibility stylus exerts lower
downward forces on the sample, resulting in less distortion.
[0096] Tube or stack piezoceramics position the tip or sample.
Piezoelectric ceramics are a class of materials that expand or
contract when in the presence of a voltage gradient or, conversely,
create a voltage gradient when forced to expand or contract. One
uses piezoceramics to create three-dimensional positioning devices
of desired high precision. One can, preferably, use tube-shaped
piezoceramics because they combine a simple one-piece construction
with high stability and large scan range. For example, one can
cover four electrodes on the outer surface of the tube, while a
single electrode covers the inner surface. Application of voltages
to one or more of the electrodes causes the tube to bend or
stretch, moving the sample in three dimensions.
[0097] AFMs can take measurements in a variety of ways, such as,
but not limited to, recording the feedback output or the cantilever
deflection. For example, an optical lever AFM can measure the
friction between tip and sample. If the scanner moves the sample
horizontal or perpendicular to the long axis of the cantilever,
friction between the tip and sample causes the cantilever to twist.
A position-sensitive photodetector can distinguish the resulting
left-and-right motion of the reflected laser beam from the
up-and-down motion caused by topographic variations. AFMs can image
sample elasticity by pressing or pulling the tip into or out of the
sample and measuring the resulting cantilever deflection. AFMs can
also image the softness of a sample by pressing the cantilever into
it at each point in a scan. The scanner raises the sample or lowers
the cantilever by a preset amount, the "modulation amplitude". In
response, the cantilever deflects an amount dependent on the
softness of the sample: the harder the sample, the more the
cantilever deflects.
[0098] As used herein, the term "ligate" in relation to nucleic
acids and nucleotides means the process of joining two or more
nucleic acids, nucleotides or combinations thereof by creating a
covalent phosphodiester bond between the 3' hydroxyl of one
nucleotide and the 5' phosphate of another. It is not intended to
be limited to the actions of a DNA ligase, but also includes the
actions of a DNA polymerase.
[0099] As used herein, the term "solid support" is used in
reference to any solid or stationary material to which reagents
such as antibodies, antigens, and other test components are
attached. For example, the wells of microtiter plates provide solid
supports. Other examples of solid supports include microscope
slides, coverslips, beads, particles, cell culture flasks, as well
as any other suitable item.
[0100] As used herein, a "bead" means a material with a periphery
of preferably less that 1 millimeter and even more preferably less
than one micrometer and greater than 100 nanometers in diameter.
Preferably the bead is substantially spherical. The bead could also
be shaped in a rod or cube, but it is not intended that the bead be
limited to these shapes. Preferably the bead is made of a material
that is stable to dissolution in the liquid in which it is to be
suspended. Preferably the bead is made of a polymer or metal or a
combination thereof, but it is not intended that the bead be
limited to these materials. It is contemplated that the exterior
surface of the bead may vary chemically from its internal chemical
constitution.
[0101] As used herein, a "nucleotide" is a chemical compound that
consists of a heterocyclic base, a sugar, and one or more phosphate
groups. Preferably, the base nucleotide is a derivative of purine
or pyrimidine, and the sugar is the pentose (five-carbon sugar)
deoxyribose or ribose. Nucleotides are the monomers of nucleic
acids, with three or more nucleotides covalently bonded together
forming a "nucleotide sequence." As used herein, the term
"nucleotide" is intended to include substituted nucleotides
including conjugates linked to fluorescent moieties and those with
protecting groups such as those illustrated in FIG. 13.
[0102] Nucleic acids are said to have a "5'-terminus" (5' end) and
a "3'-terminus" (3' end) because nucleic acid phosphodiester
linkages occur at the 5' carbon and 3' carbon of the pentose ring
of the substituent mononucleotides. The end of a polynucleotide at
which a new linkage would be to a 5' carbon is its 5' terminal
nucleotide. The end of a polynucleotide at which a new linkage
would be to a 3' carbon is its 3' terminal nucleotide. A terminal
nucleotide, as used herein, is the nucleotide at the end position
of the 3'- or 5'-terminus. A nucleic acid may be double-stranded or
single-stranded.
[0103] Hybridization means the coming together (annealing) of a
single-stranded nucleic acid with either another single-stranded
nucleic acid or a nucleotide by hydrogen bonding of complementary
base(s). Hybridization and the strength of hybridization (i.e., the
strength of the association between nucleic acid strands) is
impacted by many factors well known in the art including the degree
of complementarity of the respective nucleotide sequences,
stringency of the conditions such as the concentration of salts,
the T.sub.m (melting temperature) of the formed hybrid, the
presence of other components (e.g., the presence or absence of
polyethylene glycol), the molarity of the hybridizing strands and
the G:C content of the nucleic acid strands.
[0104] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally (e.g., as in a
purified restriction digest) or produced synthetically, capable of
acting as a point of initiation of nucleic acid synthesis when
placed under conditions in which synthesis of a primer extension
product complementary to a nucleic acid strand is induced (i.e., in
the presence of nucleotides, an inducing agent such as DNA
polymerase, and under suitable conditions of temperature and pH).
The primer is preferably single-stranded for maximum efficiency in
amplification, but may alternatively be double-stranded. If
double-stranded, the primer is first treated to separate its
strands before being used to prepare extension products. In
preferred embodiments, the primer is attached to the end of a
nucleic acid such that a hairpin forms from self-hybridization.
Preferably, the primer is an oligodeoxyribonucleotide. The primer
must be sufficiently long to prime the synthesis of extension
products in the presence of the inducing agent. The exact lengths
of the primers will depend on many factors, including temperature,
source of primer and use of the method. It is also contemplated
that primers can be used in PCR (see below) to artificially insert
desired nucleotide sequences at the ends of nucleic acid
sequences.
[0105] As used herein, the terms "complementary" or
"complementarity" are used in reference to a sequence of
nucleotides related by the base-pairing rules. For example, the
sequence 5' "A-G-T" 3', is complementary to the sequence 3' "T-C-A"
5'. Complementarity may be "partial," in which only some of the
nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as for
detection methods that depend upon hybridization of nucleic
acids.
[0106] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method described in U.S. Pat. Nos. 4,683,195,
4,889,818, and 4,683,202, all of which are hereby incorporated by
reference. These patents describe methods for increasing the
concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. This process for
amplifying the target sequence consists of introducing a large
excess of two oligonucleotide primers to the DNA mixture containing
the desired target sequence, followed by a precise sequence of
thermal cycling in the presence of a DNA polymerase (e.g., Taq).
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is a controllable parameter, determined by the relative
positions of the primers with respect to each other. By virtue of
the repeating aspect of the process, the method is referred to as
the "polymerase chain reaction" (hereinafter "PCR"). Because the
desired amplified segments of the target sequence become the
predominant sequences (in terms of concentration) in the mixture,
they are said to be "PCR amplified."
[0107] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (i.e., hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide sequence can be amplified with the appropriate set
of primer molecules. In particular, the amplified segments created
by the PCR process itself are themselves efficient templates for
subsequent PCR amplifications.
[0108] A "marker" is a compound or composition detectable from
background by its properties, including without limitation
spectroscopic, photochemical, biochemical, immunochemical, and
chemical. For example, useful markers include luminescent dyes,
quantum dots, fluorescent proteins such as green, yellow, red or
blue fluorescent proteins, radioactive elements, compounds enriched
in particular atom isotopes, electron-dense reagents, enzymes
(e.g., as commonly used in an ELISA), biotin, digoxigenin, or
haptens and proteins for which antisera or monoclonal antibodies
are available.
[0109] Luminescence is a property of certain materials that renders
them capable of absorbing electromagnetic energy of a given
wavelength and emitting at a different wavelength. Examples include
fluorescence, bioluminescence and phosphorescence. Luminescence can
be caused by chemical or biochemical changes, electrical energy,
subatomic motions, reactions in crystals, or other, generally
non-thermal, stimulation of the electronic state of an atomic
system.
[0110] A "luminescent marker" is a molecular construction capable
of emitting light, that is bound, either covalently, generally
through a linker, or through ionic, van der Waals, hydrogen bonds,
or any physical spatial constraint to another material, substance,
or molecule. Preferably a luminescent marker is a molecule with
aromaticity or a molecule with highly conjugated double bonds as
typically found in fluorescent dyes, or quantum dots or
combinations thereof. In preferred embodiments, dyes are linked to
a nucleotide by the formation of amide bonds that result from
coupling an amine group on the linker to a carboxylic acid group on
the dye. Dyes contemplated in some preferred embodiments include
rhodamine dyes, boron dipyrromethene dyes, and cyanine dyes.
[0111] Sequencing methods using magnifying tags are described in
U.S. Pat. No. 6,723,513, hereby incorporated by reference. The
method correlated the relationship between a target nucleic acid
and its design polymer counterpart that, for example, may consist
of 48 code units of 10 base pairs each. In this example, every base
in the target nucleic acid is represented by two binary code units
in the design polymer molecule. The A's have been converted to "0"
and "0", C's to "0" and "1", G's to "1" and "0" and T's to "1" and
"1". The original sequence information found in a target nucleic
acid has been maintained in a new and synthetic nucleic acid: a
design polymer of 480 base pairs. In preferred embodiments, the
conversion of a 24-base pair target nucleic acid is initiated by a
class IIS restriction enzyme that cleaves off the two first bases
to be converted. These two bases are then identified with a ligase
dependent recognition system and replaced with four code units of
design polymer that represent the information of the two target
bases. The design polymer block with code units are then attached
to the target nucleic acid on the opposite side of where the two
first bases were initially removed, allowing the process to be
repeated with the next two bases in the target nucleic acid. The
process is repeated thus building up a design polymer of 48 units
representing the original target nucleic acid sequence of 24 base
pairs.
EXAMPLES
Example 1
Detection of Changes in the End-to-End Distances of Single Nucleic
Fragments Undergoing Polymerization by Polymerase
[0112] The total number of bases in a given nucleic acid (e.g.,
DNA) molecule undergoing polymerization is distributed between
single-stranded (ss) and double-stranded (ds) forms (FIG. 1). The
distribution changes as the polymerase moves along the ss. The
end-to-end distance of the dsDNA-ssDNA construct (i.e.
single-stranded part plus double-stranded part) changes according
to the progress of the reaction:
n.sub.dsDNA-M.sub.ssDNA+kdNTP.fwdarw.(n+k).sub.dsDNA-(m-k).sub.ssDNA
FIG. 2 shows theoretical predictions of the response of the
dsDNA-ssDNA construct to external force. From the simulation data
presented in the FIG. 2, one can identify approaches to implement
the detection of the extent of polymerization, i.e., i) to observe
the free end of the nucleic acid under constant force (e.g. no
force or F.about.30 pN), or ii) to acquire a full force-extension
curve over a 50 pN range. In the first approach, one should
determine the position of a centroid of the fluctuating end; in the
second case, one provides a fitting routine describing molecular
elasticity in order to average out the noise of thermal
fluctuations.
[0113] One detects differences in the averaged response before and
after addition of the solution of dNTP to decide whether the base
addition has happened. The detection of changes in the end-to-end
distances of single nucleic fragments undergoing polymerization via
SNA by polymerase is a genome sequencing method that does not
require fluorescently labeled dNTPs. For the detection of the
distance between the free end and the surface, one may use an
optical evanescent field in the vicinity of the glass-aqueous
solution interface. One may generate such optical near-fields using
total internal reflection (TIR) configuration for the illumination
of the glass-solution interface (FIG. 3). In addition to the
near-field optical probes reporting end-to-end distances of DNA
fragments, one may use magnetic probes as i) either part of the
optical probe (for instance by doping dielectric spheres with
ferro- and superparamagnetic nanoparticles), or ii) in tandem with
near-field probes to pull on individual nucleic acid fragments. One
may use magnetic handles to provide for uniform application of
external force using permanent magnets or electromagnets (FIG.
4).
Example 2
Detecting Distance of Near-Field Probe from Surface
[0114] The sensitivity of the method in detecting the distances
from the interface has been demonstrated in the following
experiment, performed on a model system. We prepared a polymer
sample (polymethylmethacrylate, PMMA) that had steps of increasing
depth: 50, 100, 150, and 200 nm. These steps were then covered by a
thin polymer film (collodion) that served as a cover slip in
conventional oil immersion microscopy. We imaged these steps using
a high numerical aperture objective (N.A.=1.4). Because of the
varying air gap thickness between the two dielectrics, the TIR was
"frustrated" by a different amount and the image intensity reflects
different amounts of light totally reflected at the polymer-air TIR
interface. From this data, one can clearly distinguish positions of
the dielectric surfaces at 50 nm and 100 nm distances away from the
cover slip, i.e. exactly within the range of distances expected of
the transformation for a 200-mer between the single-stranded and
double-stranded forms.
[0115] One may use electron multiplying CCD cameras capable of
single photon detection to provide imaging with the sub-nm
precisions for detection of SNA through corresponding shifts in the
position of the optical probe.
[0116] One may observe arrays of single DNA fragments on the
surface with several configurations of the optical near-field (FIG.
3): i) dove prism with observation of the scattered or fluorescent
light using the compound microscope (use of low NA objectives is
possible in this case); ii) TIR microscope with the same objective
used for illumination and observation (illumination is done using
marginal rays o the objective); iii) evanescent field of the
cladding using slab waveguides and iv) evanescent field in
subwavelength aperature (zero mode waveguide, see FIG. 14). The
experimental configuration involving a TIR microscope is preferable
because it ensures the high degree of flexibility in the
manipulation of the parameters of the evanescent field (intensity,
angle of incidence, penetration depth, wavelength, etc.), ease of
observation of the surface attached single molecules, and provides
access (from the top) to reaction chambers for further
manipulation.
[0117] Wide field microscopy allows one to simultaneously analyze
multiple single DNA fragments. With a field of view for high NA
objective on the order of 100 .mu.m.times.100 .mu.m, at least
10.sup.4 molecules can be arranged in a single reaction chamber
(with .about.1 .mu.m average spacing between nearest neighbors).
For a typical 1 megapixel CCD, a single optical probe is imaged on
10.times.10 pixel area, enough for unambiguous identification and
tracking of individual probes. An additional 10-20 fold increase in
the number of single molecules tracked at the same time can be
accomplished by employing CCD chips with high pixel count
(.about.10 Mpixel).
Example 3
Microfluidics for Miniaturization and Automation of the Cycling of
the Single Nucleotide Addition
[0118] Use of a single molecule assay cuts down significantly on
the amount of reagents required for sequencing. Given the small
surface area of the reaction chamber used for microscopy on the
single DNA fragments (-100 .mu.m.times.100 .mu.m), minimizing the
reaction volume is also appropriate. One implements automated
delivery of stock dNTP solutions and buffer washes by using
microfluidic devices with the basic features outlined in FIG. 6.
After preparation of the glass slide that presents surface reactive
sites to single-stranded genomic DNA fragments in defined
orientations, one positions the glass slide in a microfluidic
device and incubates with the DNA solution. After the DNA binding
is complete, one flushes the device with buffer and introduces a
suspension of near-field probes. Preferably, each of the DNA
fragments binds only one such probe, and after incubation with the
probes, one flushes the devices with the buffer and prepares them
for SNA reactions.
[0119] One adds single dNTPs to the reaction in limiting amounts
together with polymerase. Upon addition of the complementary dNTP,
the DNA polymerase extends the primer and pauses when it encounters
a noncomplementary base. After washing, one measures elongation of
the single DNA strands and records and adds a different dNTP. One
reiterates DNA synthesis and a new cycle begins. One pauses the
reaction by closing the inlet/outlet valves in the linked array of
reaction chambers. One records the state of each reaction chamber
as a series of images by scanning the whole chip containing an
array of freely arrayed single DNA molecules (within the
confinement of each reaction chamber).
Example 4
Attachment of DNA Fragment to Flat Surfaces and Near-Field
Probes
[0120] One works with a known 200 bp DNA fragment (synthesis) and
generates sufficient quantities of it by a PCR reaction. One may
use DNA fragments with unknown sequences. One isolates genomic DNA
from a common bacterial laboratory strain Escherichia coli K-12
whose sequence has been published. One isolates genomic DNA
isolated using the DNeasy kit (Qiagen, CA) according to the
manufacturer's instruction. One digests the DNA with restriction
endonucleases AccII, HaeIII, and Sau3A (New England Biolabs, MA).
These endonucleases recognize 4-base pair stretches within the DNA
thereby increasing the cutting probability. One tests these enzymes
by themselves or in combination to generate a blunt-ended DNA
library pool with average lengths between 100-300 base pairs. One
obtains a homogeneous population of similar sized fragments digests
by separation on 6% PAA gels and extracting and purifying fragments
corresponding to 200 bp size over a Qiaquick PCR purification
column.
[0121] To the blunt-ended double-stranded DNA fragments of the
genomic DNA library, one ligates small double-stranded DNA linkers
using T4 DNA ligase (New England Biolabs, MA). Two classes of
linkers (adaptors) termed "A" and "B" will be used in each reaction
(see FIG. 7 and FIG. 8). Adaptor A carries a universal primer
recognition site (SP6). Adaptor B carries a 3'-thiol modification
site that enables one to purify fragments by S-S bridge formation
(negative selection), and a 5' biotin tag that complexes to a
paramagnetic streptavidin coated polystyrene bead during the
sequencing reaction. Each adaptor pair contains a 5' overhang and a
3' blunt end to ensure directional ligation to the genomic DNA
fragment.
[0122] A blunt-ended cloning strategy results in three possible
ligation events: DNA fragments with flanking A/A, A/B, or BB linker
combinations (see FIG. 8). To specifically enrich for MB containing
single-stranded DNA fragments, one passes the ligated reaction
mixture over activated maleimide (Mal) groups that have been
immobilized to polystyrene beads. B-fragments containing the SH
group bind to the activated Mal-coated beads, one washes away
unbound material lacking the SH adaptor. One denatures the
immobilized fragments using alkali treatment thereby releasing the
A/B fragments that one uses in the subsequent sequencing steps
after neutralization and concentration over a MinElute PCR
purification column (Qiagen, CA). One assesses the quality and
quantity of the resulting ssDNA library with the Agilent 2100
bioanalyzer using an RNA Pico Lab chip. To prepare for the
sequencing conditions one hybridizes the ssDNA library molecules
onto beads onto which SP6 universal primer that contains a spacer.
Subsequently, one incubates the immobilized fragments with
paramagnetic Streptavidin beads (Dynal), beads-QD constructs, or
iron nanoparticles-QD constructs, all of which one captures by the
biotin group introduced with the adaptor B at the 5' end of the
ssDNA fragments.
Example 5
Primer Design and Generation of Double-Stranded Linkers
[0123] The adaptor pairs are designed to allow directional ligation
to the blunt-ended fragmented genomic DNA (Adaptor pair A:
5'-TATAGCATTTAGGTGACACTATAGGC-3; 5'-GCCTATAGTGTCACCTAAATGC-3' (SP6
primer recognition site underlined); Adaptor pair B:
5'-GCTGACCTAGTCATTGCTAGC-(CH2)2-SH-3';
5'-GCTGACCTAGTCATTGCTAGC-(CH2)2-SH-3'). One mixes and places equal
volumes of both complementary oligos at equimolar concentrations in
a standard heatblock at 90.degree. C. and allows the mixture to
cool to room temperature within 45 to 60 minutes. One captures
eluted single-stranded DNA fragments at their 5' ends by binding to
SP6 oligonucleotides (5'-NH2-(CH2)6-TACGATTTAGGTGACACTATAG-3) that
have been covalently bound to the sequencing chamber surface via a
reactive amino group.
[0124] It is also contemplated that primers may be developed that
incorporate desirable sequences into the ends of a nucleic acid
during PCR since the primer ends are replicated. Examples such as
hairpin loops and poly A tails are illustrated in FIG. 11.
Example 6
Directional Binding of Freely Arrayed DNA Fragments in a Low
Density
[0125] One captures DNA fragments by complementary binding to SP6
primer oligonucleotides that have been immobilized at low density.
To generate surfaces presenting c-SP6 on the amine-terminated
surface (via silanization of glass using
aminopropyltriethoxysilane), one incubates the sequencing chamber
with either of two types of bifunctional PEG linkers--capped with
i) either N-hydroxysuccinimyl (NHS) and methoxy (OMe) groups or ii)
NHS and Mal groups. One allows the SH coupled c-SP6
oligonucleotides to link to the reactive Mal groups. The covalently
bound c-SP6 primer binds and captures the complementary binding
site introduced via the A-linker. Alternatively, one directly links
the A-linker with a reactive group to enable direct covalent
binding of single DNA fragments to the bottom surface of the
sequencing chamber. See FIGS. 10 and 11 for alternative
examples.
Example 7
Atomic Force Microscope Experiments
[0126] The surface of glass presents identical DNA fragments
(designed synthetic sequences) terminated in the near-field probe
(polystyrene sphere or 4-6 nm CdS quantum dot, FIG. 9). The
near-field probe and force microscope probe (AFM tip) have
long-chain polymer linkers (polyethylene glycol, or PEG)
terminating in the biotin and avidin, respectively. One forms a
biotin-avidin bond by sampling the surface of the glass with the
AFM tip. One detects bond formation by monitoring the force exerted
on the AFM tip. Once non-zero force is detected, one generates FE
curves for a single DNA fragment and records the photonic response
of the near-field probe. One calibrates the photonic response,
because one independently records DNA end-to-end distance in a FE
curve obtained with a scanning probe microscope. By changing the
nature of the optical probe from dielectric to QD, one compares the
sensitivity of the two approaches. Since one uses the same DNA
sequence in these model experiments, one records the changes in
mechanical properties of the single DNA fragments by repeating
force spectroscopy experiments after one or more cycles of dNTP
addition and corresponding elongation of the dsDNA part by
polymerase. One carries out the polymerization reaction in situ in
the liquid cell of the force microscope. One observes change in the
fraction of the dsDNA (by 10 or more bp), then decreases the
fractional change to a single dNTP addition. To aid the alignment
and positioning of the AFM probe, one attaches near-field probes
and concurrently observes the sample with light microscopy.
Example 8
Massively Parallel Force Spectroscopy with Magnetic Tweezers
[0127] One may use magnetic tweezers for 1) force induced
dissociation experiments (on-off binding); 2) extension under
constant force with micron sized steps; and 3) rotation of the
single molecule (torque application). One translates millimeter
level changes in the distance between the magnetic probe and the
magnets into picoNewton changes in force, and translates changes in
intensity of the optical signal form the near-field probes
(scattered light or fluorescence) into sub-nm changes in the length
of the molecules.
[0128] One may also use iron ferromagnetic nanoparticles as
magnetic probes in tandem with QDs as near-field probes. Methods
for the synthesis of fully dispersed nanoscale iron particles are
disclosed in Zhang (2003) Journal of Nanoparticle Research 5,
323-332. The particles have sizes in the range of 6-8 nm. Smaller
particles of 2-4 nm have also been prepared with a similar
method.
[0129] Iron nanoparticles were prepared by mixing equal volumes of
0.94 M NaBH.sub.4 and 0.18 M FeCl.sub.3. The borohydride solution
was slowly added into the iron chloride solution with vigorous
stirring (400 rpm). Polyvinyl alcohol-co-vinyl acetate-co-itaconic
acid (PV3A, Aldrich) was identified as the most promising substrate
for the stabilization of iron nanoparticles. Furthermore, PV3A is
nontoxic and, thus, compatible with health related applications.
PV3A possesses multiple functional groups including hydroxyl
(--OH), carbonyl (--C.dbd.O), and carboxylic acid (--COOH). One
obtains the estimate of the rate of sequencing with parallel FS
setup by assuming that a single FE experiment on a 100.times.100
array can take 1 sec (e.g., FE curve with 30 data points derived
from 30 frames taken at the standard 30 Hz video rate). If the
exchange of dNTP buffer in the microfluidic device takes another
1-3 seconds, the maximum rate of .about.10,000 bp/4 sec=500 bp/sec
(1 base out of 4 is added on average). One boosts this rate further
by scanning an array of reaction chambers (thus making reaction
time less important). A pathway to a several orders of magnitude
increase in speed lies in increasing the field of view and the
resolution of the imaging CCD. For example, a 1000.times.1000 array
with a 10 megapixel CCD increases the rate for a single chamber to
.about.50,000 bp/sec. In addition, the instrument does not have to
rely on scanning multiple reaction chambers by a single objective
CCD: an array of chambers each having a dedicated solid immersion
lens (i.e. an objective directly fused into the surface of the
chamber) projects a magnified image of each reaction chamber on
individual CCD imagers, recording SNA reactions on all single DNA
fragments at the same time.
Example 9
Hybridization of 8-mer Segments to a DNA Array
[0130] To simulate thermal noise, the random Gaussian noise with
standard deviation
x 2 = k B T / k molec ##EQU00001##
was added to theoretical curves on extension vs. applied force
(k.sub.molec is the stiffness of the DNA molecule). The traces in
FIG. 5 show FE curves for 200-mer with 100, 110, 111
single-stranded bases (the rest are ds). The signal for SNA is
swamped by noise; however, further analysis shows that it is
recoverable if data is reproducible. Two methods are suggested in
the figure: i) subtracting the two curves (with 110 and 111 ss) and
then comparing the averages (avg=0 for no SNA addition--red curve,
avg.about.0.19 nm for SNA--blue curve), or ii) fitting the FE
curves to theoretical models setting the number of ss bases as a
fit parameter (the two fits differ by 0.93.+-.0.23.about.1 base).
Thus, in spite of the noise level being on the order of 4 nm, the
average change in the elastic response is detectable.
[0131] Further, from the simulation one sees that the addition of
>5 bases can be detected even without special care devoted to
noise. This observation leads to an alternative approach of DNA
sequencing that uses the same force spectroscopy platform. One may
use hybridization of unique 8-mer segments to a DNA array. The
detection requirements are then much less stringent, but at the
expense of having 4.sup.8.about.65000 stock solutions instead of
only 4 solution of the dNTPs.
Example 10
Generation of Hetero-Bifunctionally Labeled DNA Fragments from
Lambda DNA as a Model System
[0132] Fragments of Lambda DNA were made by sonication, and sizes
of approximately 500 bp were selected by PAGE. The selected
fragments were "end-repaired" and then ligated to a bifunctional
adapter (carrying an amino- and a sulfhydryl group). The ligated
DNA was separated from unincorporated adapter by PAGE, eluted and
used directly to bind the DNA fragment to a glass surface.
[0133] Sonication was done on ice in continuous 10 sec pulses at 5
W output and analyzed on a 3% agarose gel. With increasing number
of pulses (1.times., 3.times., or 5.times.), the Lambda DNA was
disrupted into shorter fragments.
[0134] The End-Repair Kit.TM. by EpiCentre was used to "blunt" and
phosphorylate the ends of the fragments and the prepared fragments
(selected to be about 400 bp in length) were incubated with ligase
and the adapter.
[0135] The adapter was prepared by mixing equimolar ratios of
5'-SH-T15-GAGAATGAGGAACCCGGGGCAGTTCCA-3' and
3'-NH2-A5-CTCTTCCTCCTTGGGCCCCGTCAAGGT-5' in an annealing buffer,
heating to 80.degree. C. (10 min), then slowly cooling to room
temperature. The sequencing primer was
5'GAGAATGAGGAACCCGGGGCAG-3'.
[0136] DNA fragments incubated with ligase resulted in high
molecular weight DNA. When the ligation mixture contained a 25
molar excess of the adapter, however, fragments about 500 bp in
length resulted, the size shift being attributable to ligation of
the adapter.
Sequence CWU 1
1
9122DNAArtificial SequenceSynthetic 1gcctatagtg tcacctaaat gc
22221DNAArtificial SequenceSynthetic 2gctgacctag tcattgctag c
21321DNAArtificial SequenceSynthetic 3gctgacctag tcattgctag c
21422DNAArtificial SequenceSynthetic 4tacgatttag gtgacactat ag
22527DNAArtificial SequenceSynthetic 5gagaatgagg aacccggggc agttcca
27627DNAArtificial SequenceSynthetic 6tggaactgcc ccgggttcct ccttctc
27722DNAArtificial SequenceSynthetic 7gagaatgagg aacccggggc ag
22821DNAArtificial SequenceSynthetic 8taaaccagtg tgcgcgcgcg g
21913DNAArtificial SequenceSynthetic 9ggcagcaaaa aat 13
* * * * *