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Claims  |
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I claim:
1. A method comprising:
(A) incorporating nucleotides of more than one nucleotide type into a
growing polynucleotide chain by a polymerase during a polymerase reaction
in which the order of incorporation is driven by base pair rules and a
delay occurs between each successive nucleotide incorporation;
(B) controlling at least one characteristic of the incorporation of at
least one nucleotide type of interest into the growing polynucleotide
chain such that the concentration of a nucleotide of interest is different
from the concentration of at least one other nucleotide and wherein the
incorporation delay of the nucleotide of interest is different from the
average incorporation delay for nucleotides of at least one other
nucleotide type;
(C) locating the polymerase with a local sensitive force detector;
(D) detecting by using the local sensitive force detector, a motion
associated with the nucleotide incorporation into the growing
polynucleotide chain; and
(E) producing data using a computer to transform detected incorporation
data into nucleofide data.
2. A method as defined in claim 1, wherein the locating step comprises,
(A) operating the detector in a scanning mode to locate a
polynucleotide/polymerase complex, and then
(B) switching from the scanning mode to a data acquisition mode in which a
probe tip of the detector is placed at the polynucleotide/polyinerase
complex.
3. A method as defined in claim 1, wherein the locating step comprises
operating the detector in an approaching mode to locate a
polynucleotide/polymerase complex,
and then
if a complex is found, then switching from the approaching mode to a data
acquisition mode in which a probe tip of the detector is placed at the
polynucleotide/polymerase complex, or
if a complex is not found, then switching from the approaching mode to a
scanning mode to locate a polynucleotide/polymerase complex.
4. A method as defined in claim 1, wherein the locating step comprises
operating the detector in an approaching mode to locate a
polynucleotide/polymerase complex,
and then
if a complex is found, then switching from the approaching mode to a data
acquisition mode in which a probe tip of the detector is placed at the
polynucleotide/polynerase complex, or
if a complex is not found, then re-engaging to locate a
polynucleotide/polymerase complex.
5. A method as defined in claim 1, wherein the detecting step comprises
using a probe of the detector to detect a motion occurring when a
nucleotide is incorporated into the growing polynucleotide chain and a
newly formed structure translocates through a reaction site of the
polynerase.
6. A method as defined in claim 5, wherein the detector comprises an atomic
force microscope (AFM) and wherein the motion generates a mechanical force
that is reflected by a motion of a cantilever of the AFM.
7. A method as defined in claim 1, wherein, during the incorporating step,
incorporation delays occur between successive nucleotide incorporations,
and wherein the controlling step comprises controlling an average
incorporation delay for nucleotides of the nucleotide type of interest to
differ from an average incorporation delay for nucleotides of at least one
other nucleotide type.
8. A method as defined in claim 7, wherein the controlling step comprises
controlling the average incorporation delays for nucleotides of all
nucleotide types other than the nucleotide type of interest to be at least
substantially the same as one another.
9. A method as defined in claim 8, further comprising controlling an
average incorporation delay for nucleotides of at least two nucleotide
types of interest to differ from an average incorporation delay for
nucleotides of at least one other nucleotide type.
10. A method as defined in claim 1, wherein the controlling step comprises
controlling an amplitude of a motion as measured by a probe detector
occurring when a nucleotide is incorporated into the growing
polynucleotide chain and a newly formed structure translocates through a
reaction site of the polymerase.
11. A method as defined in claim 1, wherein the controlling step comprises
conjugating a nucleotide of the nucleotide type of interest with a
molecule.
12. A method as defined in claim 1, wherein the controlling step comprises:
(A) performing a plurality of polymerization reactions, and
(B) during each of the polymerization reactions, controlling the at least
one characteristic of the incorporation of a different nucleotide type of
interest such that the incorporation of a nucleotide of the nucleotide
type of interest is distinguishable from the incorporation of nucleotides
of all other nucleotide types,
and further comprising determining, based on the controlling and detecting
steps, which nucleotide type was incorporated into the growing
polynucleotide chain during the incorporating step.
13. A method as defined in claim 12, wherein the determining step
comprises:
(A) examining each of the reactions separately, and, for each of the
reactions, (1) detecting whether or not a nucleotide of the nucleotide
type of interest for that reaction is incorporated and (2) generating a
set of data, and
(B) adding a plurality of sets of data together to generate nucleotide
sequence data for all of the data sets.
14. A method as defined in claim 1, wherein, during the controlling step,
at least one characteristic of the incorporation for the nucleotide type
of interest is unique when compared to the corresponding characteristics
of the incorporation for all other nucleotide types in the polymerization
reaction.
15. A method as defined in claim 14, further comprising determining, based
on the controlling and detecting steps, which nucleotide type was
incorporated into the growing polynucleotide chain during the detecting
step,
wherein the polymerization reaction is performed in a single reaction and a
plurality of nucleotide types each have a unique incorporation
characteristic when compared to corresponding incorporation
characteristics of other nucleotide types,
wherein the detecting step comprises detecting the unique incorporation
characteristic of each of the nucleotides types, and
wherein the determining step comprises determining, from the unique
incorporation characteristic, which nucleotide type was incorporated into
each location of interest of the growing polynucleotide chain.
16. A method as defined in claim 1, wherein the detecting steps further
comprises subtracting background noise from detected probe operating
parameters, the background noise arising from at least one of unintended
probe motion and enzyme motion.
17. A method as defined in claim 1, wherein the incorporating step
comprises controlling the polymerization reaction such that a rate of the
polymerization reaction permits detection of incorporations.
18. A method as defined in claim 1, wherein the incorporating step
comprises controlling the polymerization reaction such that a rate of the
polymerization reaction is outside of a range in which natural variations
in average incorporation delays would obscure acquired data.
19. A method as defined in claim 1, wherein the incorporating step
comprises using a polymerase that is attached to a substrate.
20. A method as defined in claim 1, wherein the incorporating step
comprises starting the incorporation at the beginning of the growing
polynucleotide chain.
21. A method as defined in claim 1, further comprising repeating the
incorporating, controlling, and detecting steps for a plurality of
polymerase reactions.
22. A method as defined in claim 1, wherein the incorporating step
comprises controlling the temperature of the polymerase reaction.
23. A method as defined in claim 1, further comprising detecting mutations
during the polymerase reaction by using the method to determine which
nucleotide type was incorporated into the growing polynucleotide chain for
a small number of nucleotides.
24. A method as defined in claim 1, wherein the detector comprises an
atomic force microscope (AFM).
25. A method as defined in claim 1, wherein the detecting step comprises
detecting a motion of the polymerase.
26. A method comprising:
(A) incorporating nucleotides of more than one nucleotide type into a
growing polynucleotide chain by a polymerase during a polymerase reaction
in which the order of incorporation is driven by base pair rules;
(B) controlling at least one characteristic of the incorporation of at
least one nucleotide type of interest into the growing polynucleotide
chain such that the incorporation of nucleotides of the nucleotide type of
interest is distinguishable from the incorporation of nucleotides of at
least one other nucleotide type;
(C) locating the polymerase with a local sensitive force detector;
(D) detecting using the local sensitive force detector, a motion associated
with the nucleotide incorporation into the growing polynucleotide chain;
(E) producing data using a computer to transform detected incorporation
data into nucleotide data; and
(F) withholding at least one reaction component until a time just prior to
a time at which data acquisition begins.
27. A method comprising:
(A) incorporating nucleotides of more than one nucleotide type into a
growing polynucleotide chain by a polymerase during a polyrnerase reaction
in which the order of incorporation is driven by base pair rules;
(B) controlling at least one characteristic of the incorporation of at
least one nucleotide type of interest into the growing polynucleotide
chain such that the incorporation of nucleotides of the nucleotide type of
interest is distinguishable from the incorporation of nucleotides of at
least one other nucleotide type;
(C) locating the polymerase with a local sensitive force detector in
Tapping Mode;
(D) detecting using the local sensitive force detector, a motion associated
with the nucleotide incorporation into the growing polynucleotide chain;
and
(E) producing data using a computer to transform detected incorporation
data into nucleotide data.
28. A method comprising:
(A) incorporating nucleotides of more than one nucleotide type into a
growing polynucleotide chain by a polymerase during a polymerase reaction
in which the order of incorporation is driven by base pair rules;
(B) controlling at least one characteristic of the incorporation of at
least one nucleotide type of interest into the growing polynucleotide
chain such that the incorporation of nucleotides of the nucleotide type of
interest is distinguishable from the incorporation of nucleotides of at
least one other nucleotide type and wherein secondary structure of a
polynucleotide template formed by the incorporation is minimized;
(C) locating the polymerase with a local sensitive force detector;
(D) detecting using the local sensitive force detector, a motion associated
with the nucleotide incorporation into the growing polynucleotide chain;
and
(E) producing data using a computer to transform detected incorporation
data into nucleotide data. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods and apparatus for
detecting nucleotide incorporation, and more particularly to a method and
apparatus for detecting nucleotide incorporation into a growing
polynucleotide chain. The invention is particularly well suited for rapid,
automated DNA sequencing.
2. Discussion of the Related Art
Deoxyribonucleic acid (DNA) is composed of four different types of bases:
adenine (A), guanine (G), cytosine (C), and thymine (T). A base together
with a phosphate and a sugar molecule form a nucleotide. In a DNA
molecule, the bases are arranged along a sugar-phosphate backbone to form
a chain. These chains are often referred to as "DNA strands." Two DNA
strands pair up to form a double-stranded DNA molecule. The strands pair
up due to hydrogen bonding between complementary bases. The nucleotide
composition and order of a given DNA strand are represented in a DNA
sequence. For example, "CCGAT" is a DNA sequence.
DNA sequencing is important for a variety of tasks, including basic
scientific research, medical studies, diagnostics, and genome projects.
For these tasks, rapid DNA sequencing methods are desirable. However, the
speed of previous DNA sequencing methods has been significantly limited by
several factors including time and labor for subcloning long DNA fragments
into sequencing vectors, sequencing chemistries, and DNA fragment
separation techniques, as well as sequence data reading.
Current DNA sequencing techniques are based on the generation of a
plurality of DNA fragments corresponding to the sequence of a DNA
template. One such DNA sequencing technique is disclosed in Maxam and
Gilbert, "A New Method for Sequencing DNA," Proc. Nati. Acad. Sci. USA,
Vol. 74(2), February 1977, where a DNA template of interest is chemically
degraded to produce a plurality of DNA fragments corresponding to the DNA
sequence. Another DNA sequencing technique is disclosed in Sanger et. al.,
"DNA Sequencing with Chain-terminating Inhibitors," Proc. Natl. Acad. Sci.
USA, Vol. 74(12), December 1977, where a plurality of terminated DNA
fragments complementary to a DNA template are synthesized. Both of these
techniques require labeling the DNA fragments with a reporter, such as a
radionucleotide label or a fluorescent label, and electrophoretically
separating the labeled DNA fragments on a sieving matrix, such as a
polyacrylamide. Sieving matrices are semi-porous materials that separate
DNA based on the size of the DNA molecule. Typically, the sieving matrices
are poured between two glass plates to form a gel. DNA is applied to one
end of the gel, an electrical current is applied, and the
negatively-charged DNA molecules travel through the gel toward the
cathode, with the smallest DNA molecules traveling the farthest.
This process, called gel electrophoresis, has several drawbacks including
(1) laborious gel pouring protocols and (2) variability in the gels due to
cleanliness of gel plates, fluctuations in ambient temperature, and
inconsistent qualities in gel reagents. This variability can alter the
quality of the sequence data and even render the sequence data unusable.
Furthermore, separating the DNA fragments requires electrophoresing the
fragments at a speed slow enough to (1) permit detection of labeled
fragments, (2) avoid decomposition of gel, and (3) allow for adequate
separation of fragments. These time constraints limit the speed at which
DNA sequencing can be performed using electrophoresis-based sequencing
methods.
In sum, limitations of previous DNA sequencing techniques include the need
for a reporter label, the time required for performing the techniques,
and/or the variability in gels. In light of these limitations, alternative
DNA sequencing methodologies are needed.
Atomic force microscopes (AFMs) recently have been used to study
biomolecules, as described below. An AFM has a tip that is end-mounted on
a flexible cantilever. Interactions between the tip and the sample
influence the motion of the cantilever, and one or more parameters of this
influence are measured to generate data representative of one or more
properties of the sample. AFMs can be operated in different modes
including contact mode, TappingMode, (Tapping and TappingMode are
trademarks of Digital Instruments, Inc.), and non-contact mode. In contact
mode, the cantilever is not oscillated, and cantilever deflection is
monitored as the probe tip is dragged over the sample surface. In
TappingMode, the cantilever is oscillated mechanically at or near its
resonant frequency so that the probe tip repeatedly taps the sample
surface, thus reducing the probe tip's oscillation amplitude. The
oscillation amplitude indicates proximity to the sample surface and may be
used as a signal for feedback. U.S. patents relating to Tapping and
TappingMode include U.S. Pat. Nos. 5,266,801, 5,412,980, and 5,519,212, by
Elings et al., all of which hereby are incorporated by reference. In the
non-contact mode, attractive interactions between the probe tip and the
sample (commonly thought to be due to Van der Waals' attractive forces)
shift the cantilever resonance frequency when the probe tip is brought
within a few nanometers of the sample surface. These shifts can be
detected as changes in cantilever oscillation resonant frequency, phase,
or amplitude, and used as a feedback signal for AFM control.
Whether operating in contact mode, TappingMode, or non-contact mode,
feedback is typically used during AFM scanning to adjust the vertical
position of the probe relative to the sample so as to keep the probe
tip-sample interaction constant. A measurement of surface topography or
another sample characteristic may then be obtained by monitoring a signal
such as the voltage used to control the vertical position of the scanner.
Alternatively, independent sensors may monitor the position of the tip
during scanning to obtain a map of surface topography or another measured
sample characteristic. Measurements can also be made without feedback by
monitoring variations in the cantilever deflection as the probe moves over
the surface. In this case, recording the cantilever motion while scanning
results in an image of the surface topography in which the height data is
quantitative. Additionally, the positioning of the AFM probe can be
enhanced by compensating for drift. U.S. patents relating to drift
compensation include U.S. Pat. Nos. 5,081,390 and 5,077,473 by Elings et
al., both of which are hereby incorporated by reference.
Proposals have been made to use AFMs to study biomolecules. For instance,
Radmacher et al., "Direct Observation of Enzyme Activity with the Atomic
Force Microscope," Science, Vol. 265, Sept. 9, 1994, (Radmacher) proposes
the use of an AFM to measure height fluctuations of an enzyme (lysozyme).
Radmacher believed that the measured height fluctuations probably
corresponded to motions of lysozyme during hydrolysis of a substrate
oligoglycoside.
Other proposed uses of AFM to study biomolecules are disclosed in Hansma,
"Atomic Force Microscopy of Biomolecules," J. Vac. Sci. Technol., Vol. B
14(2), March/April 1996 (Hansma). Hansma lists several DNA applications
including (1) calculation of persistence lengths for moving DNA molecules,
(2) imaging DNA molecules as a nuclease degrades DNA, and (3) monitoring
forces between DNA bases.
Still another proposal is disclosed in Kasas et. al., Escherichia coli RNA
Polymerase Activity Observed Using Atomic Force Microscopy," Biochemistry,
Vol. 36(3), Jan. 21, 1997 (Kasas). Kasas used an AFM to observe an RNA
polymerase transcribing DNA templates in sequential AFM images. Kasas also
noted that an RNA polymerase can maintain its biological activity when it
is adsorbed onto mica.
While Radmacher, Hansma, and Kasas all disclose use of an AFM to study
biomolecules, none of these publications disclose using an AFM to detect
incorporation of a nucleotide into a growing polynucleotide chain. That
is, these publications do not disclose using an AFM to determine the
sequence of a DNA molecule.
Determining the sequence of a DNA molecule is, however, contemplated in
U.S. Pat. No. 5,620,854 by Holzrichter. Holzrichter discloses use of an
AFM to determine the sequence of a DNA template. The Holzrichter patent
contains only a very limited discussion of varying concentrations of
nucleotides, and has several shortcomings. First, it lacks a sufficient
disclosure as to how to determine which of the four nucleotides is
incorporated into the growing nascent DNA strand. In particular,
Holzrichter proposes that each nucleotide addition reaction is different
based on fact that different nucleotide types (e.g., As, Cs, Gs, and Ts)
have different base pairing characteristics, and the method can
distinguish nucleotides then based on differences in the number of
hydrogen bonds. However, only two of four nucleotides have a different
number of hydrogen bonds (A and T have two hydrogen bonds and G and C have
three hydrogen bonds), and furthermore nucleotides cannot be easily
distinguished based on these differences alone. Second, Holzrichter's
process makes no correction for background noise. Third, the signal to
noise ratio of Holzrichter's process is not high enough to determine which
base was incorporated. Thus, Holzrichter does not enable a determination
of which nucleotide is incorporated into the DNA template. Therefore,
Holzrichter does not enable using an AFM for DNA sequencing or even for
distinguishing nucleotide incorporation from other events, such as
background movements of the polymerase not related to nucleotide
incorporation.
OBJECTS AND SUMMARY OF THE INVENTION
An object of the invention is to use a local sensitive force detector to
detect incorporation of a nucleotide of interest into a growing
polynucleotide chain. In accordance with a first aspect of the invention,
this object is achieved by (1) flagging at least one nucleotide type of
interest such that its incorporation can be distinguished from the
incorporation of other nucleotides and (2) detecting nucleotide
incorporations during the polymerase reaction. Determining which
nucleotide is incorporated permits sequencing of a growing polynucleotide
chain.
Another object of the invention is to provide a method for DNA sequencing
using a local sensitive force detector.
In accordance with another aspect of the invention, this process preferably
comprises using an atomic force microscope (AFM) as the detector and
operating the AFM in either contact mode, TappingMode, or non-contact
mode.
Still another object of the invention is to provide a method for rapid DNA
sequencing.
In accordance with still another aspect of the invention, this object is
achieved by using an AFM to obtain real-time detection of incorporation of
nucleotides into a growing polynucleotide chain during a polymerization
reaction. The AFM probe tip is placed at a polynucleotide/polymerase
complex. The probe tip detects motions that the polymerase undergoes when
a growing polynucleotide chain, the growth of which is governed by base
pair rules, translocates through a reaction site of the polymerase
following nucleotide incorporation. The motions of the polymerase generate
a force that is reflected, either directly of indirectly, by motion of the
AFM cantilever.
Still another object is to provide a method for DNA sequencing without
using a nucleotide label.
In accordance with still another aspect of the invention, this object is
achieved by using an AFM placed at a DNA/polymerase complex to detect
incorporation of nucleotides into a DNA template. Because motions that the
polymerase undergoes indicate that a nucleotide is incorporated, no
reporter label is required.
Another object of the invention is to provide a local sensitive force
detector capable of reliably detecting the incorporation of a nucleotide
of interest into a growing polynucleotide chain.
In accordance with still another aspect of the invention, this object is
achieved by providing a local sensitive force detector that includes a
probe and a detection device. The probe is configured to react to movement
of a polymerase during a polymerase reaction in which 1) the polymerase
reaction comprises nucleotides of more than one nucleotide type, 2) a
chemical bond forms between a newly incorporated nucleotide and previously
incorporated nucleotide, 3) the chemically bonded nucleotides extend a
growing polynucleotide chain, and 4) a characteristic of the incorporation
of at least one nucleotide type of interest is flagged during the reaction
such that the incorporation of nucleotides of the nucleotide type of
interest is distinguishable from incorporation of nucleotides of at least
one other nucleotide type. The detection device monitors operation of the
probe and is capable of detecting the movement of the growing
polynucleotide chain through the polymerase during nucleotide
incorporation.
The detection device preferably comprises a computer which is capable of
transforming detected incorporation data into nucleotide data.
The local sensitive force detector preferably comprises an atomic force
microscope (AFM).
BRIEF DESCRIPTION OF DRAWINGS
Preferred exemplary embodiments of the invention are illustrated in the
accompanying drawings in which like reference numerals represent like
parts throughout, and in which:
FIG. 1 is a schematic plan view of a local force detector for detecting
motions of a polymerase molecule during nucleotide incorporation into a
growing polynucleotide chain during a polymerization reaction;
FIG. 2 is a flowchart of a DNA sequencing method performed in accordance
with a first preferred embodiment of the invention in which a plurality of
polymerization reactions is performed and in which incorporations of
nucleotides of the nucleotide type of interest are flagged such that the
incorporation of those nucleotides are unique when compared to
incorporation of nucleotides of other types;
FIG. 3 is a graph illustrating the profile of data obtained from an AFM
detection of flagged incorporation of nucleotides into a growing DNA chain
during a polymerization reaction performed in accordance with the first
embodiment.
FIG. 4 is a flowchart of a DNA sequencing method performed in accordance
with a second preferred embodiment of the invention in which a
polymerization reaction is performed with the flagging of a plurality of
nucleotide types of interest such that each nucleotide incorporation type
is distinguishable from the incorporation of other nucleotide types; and
FIG. 5 is a graph illustrating the profile of data obtained from detection
of flagged incorporation of nucleotides into a growing DNA chain during a
polymerization reaction performed in accordance with the second
embodiment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Method Overview:
The present invention can be best understood by beginning with a brief
discussion of nucleic acid polymerization. Nucleic acids are a family of
macromolecules including DNA and RNA. Nucleic acid polymerization is an
enzymatic reaction in which a complementary chain (also referred to as a
strand) of nucleic acid is polymerized based on a polynucleotide template.
The polymerase forms a complex with the polynucleotide template
(polynucleotide/polymerase complex). This polynucleotide/polymerase
complex usually includes, but is not limited to, a polymerase molecule
plus at least one of the following: (1) a primed single-stranded DNA
template, (2) a polynucleotide duplex with gaps or single-stranded
protruding 5' termini, (3) a nicked double-stranded DNA, (4) an unprimed
single-stranded DNA (hairpin), or (5) a single-stranded RNA. The growing
polynucleotide chain usually (but not necessarily) comprises a DNA
molecule. Accordingly, this application will discuss polymerization
reactions in which the polynucleotide comprises a DNA molecule. However,
it should be understood that the invention also is applicable to other
polynucleotides, such as RNA.
During a polymerization reaction, at least the portion of the DNA molecule
that is serving as a DNA template is in a single-stranded state, normally
(but not necessarily) due to disassociation of a double-stranded DNA
molecule. Incorporation of nucleotides is performed under base pair rules,
where, in DNA, G and C always pair with one another, and A and T always
pair with one another. In RNA, G and C always pair with one another, and U
and A always pair with one another. The polymerization reaction is driven
by a type of enzyme, called a polymerase, which contains a reaction site.
The at least partially single-stranded DNA template rests in the reaction
site, with a single location of the DNA template available for nucleotide
incorporation. At the reaction site, the polymerase sequentially
incorporates nucleotides into a growing DNA chain. The order of the
nucleotides in the growing polynucleotide chain is governed by the DNA
template and base pair rules. Following the base pair rules, when an
appropriate nucleotide comes into contact with the template, the
polymerase incorporates the new nucleotide into the growing chain by
forming a 3', 5'-phosphodiester bond between the previously incorporated
nucleotide and the newly incorporating nucleotide. This bond formation
involves the hydrolysis of the phosphate bond on the newly incorporating
nucleotide and bond formation with the 3'-OH of the previously
incorporated nucleotide of the growing DNA chain. This growing chain is
hydrogen bonded to the DNA template to form a double-stranded DNA helix.
Next, the newly formed double-stranded DNA helix translocates through the
reaction site. This translocation moves the double-stranded DNA out of the
reaction site, shifting the single-stranded template over one nucleotide
and placing a subsequent location in the DNA template in the reaction
site. Thus, nucleotide incorporation is sequential.
Time necessarily elapses between the incorporation of one nucleotide and
the incorporation of the next nucleotide because, as described above,
incorporations do not occur simultaneously. This elapse of time between
incorporations can be characterized as a "delay." Assuming a particular
nucleotide concentration, delays in nucleotide incorporations are fairly
uniform, with some variability. Thus, it is appropriate to speak of an
"average incorporation delay." Therefore, depending upon reaction-specific
factors such as reaction temperature and the concentration of a given
nucleotide in the solution, a polymerization reaction for a particular
type of nucleotide (e.g., A, C, T, or G) could have an average
incorporation delay of a specific time.
The double-stranded DNA molecule additionally comprises two antiparallel
strands, where one strand is in the so-called 5'.fwdarw.3' orientation and
the other strand is in the so-called 3'.fwdarw.5' orientation. These two
antiparallel strands are said to be the reverse complement of each other.
That is, the strands have the same sequence when (1) one is read left to
right and the other is read right to left (i.e., one is reversed), and (2)
the complementary bases are substituted. DNA is polymerized in the
5'.fwdarw.3' direction, with nucleotides added to the 3'-OH end of the
strand. DNA nucleotides are generically abbreviated as dNTPs (or
deoxynucleoside triphosphates). The enzyme has two substrates: the growing
chain containing a free 3'-OH and a dNTP. It should be understood that
whenever this application refers to a DNA sequence, it is meant to be in
the 5'.fwdarw.3' orientation. Applying these rules to a DNA template of
5'-CCGAT-3', the newly synthesized, reverse complement strand will be
5'-ATCGG-3'. This DNA molecule can be schematically represented as
follows:
##STR1##
During a polymerization reaction many motions or movements occur, including
(but not limited to) those of the polymerase and those of the growing DNA
chain. The polymerase moves during polymerization due to events including
(but not limited to) (1) nucleotide incorporation and (2) translocation of
the newly-formed double-stranded DNA helix out of the reaction site.
Specifically, the polymerase moves when the nucleotide enters the reaction
site during the incorporation. Additionally, the polymerase moves to
accommodate ratcheting movement of the newly-formed, bulkier,
double-stranded DNA helix out of the reaction site. These polymerase
motions include (but are not limited to) conformational changes (e.g.,
changes in shape or size) that the polymerase undergoes during the
polymerase reaction. These motions are akin to the motions that a body
undergoes during breathing, where the chest expands and contracts to
accomodate the volume of air present in the lungs. Additionally, the DNA
itself is moving, including the ratcheting movement through the
polymerase. The motions indicate that a nucleotide has been incorporated
into the growing DNA chain.
The present invention involves using a local sensitive force detector to
detect motions of the polymerase during nucleotide incorporation into a
growing polynucleotide chain during a polymerization reaction. The present
invention is additionally concerned with determining the sequence of a DNA
template. The detector minimally requires a probe and a detection device
for detecting effects of polymerase movements at the probe. Such detectors
used in the art include scanning probe microscopes (SPMs) including atomic
force microscopes (AFMs) and instruments comprising a local force detector
plus a laser or other optical device, such as an interferometer, or other
devices such as piezoresistive or capacitive force/position sensors. The
invention will be described primarily in conjunction with an AFM, it being
understood that it is applicable to other local force detectors as well.
A preferred embodiment of an AFM usable in the invention is illustrated in
FIG. 1. The AFM includes a probe 10 controlled by a computer 14 to detect
motions of a polymerase 26 by intermittent or other contact with it, or by
using a "non-contact" technique. If the AFM operates in an oscillating
mode, the probe 10 may be oscillated by an oscillator 6, which can drive
the probe 10 appropriately, usually at or near the probe's resonant
frequency. The probe 10 includes 1) a cantilever 8 having a base fixed to
the oscillator 6 and a free end and 2) a probe tip 12 mounted on the
cantilever 8. An electronic signal is applied, under control of the
computer 14, from an AC signal source (not shown) to the oscillator 6 to
drive the probe tip 12 to oscillate at a free oscillation amplitude
A.sub.0 (assuming that the AFM is operating in TappingMode). The probe 10
can also be driven towards and away from the polymerase 26 using a
suitable actuator 16 also controlled by the computer 14. It should be
noted that rather than being configured for driving the probe 10 towards
the polymerase 26 as illustrated, the AFM could be configured for mounting
a glass cell 34 (detailed below) on a moveable XY stage (not shown) so
that the XY stage can be used to translate the sample relative to the
probe 10.
Probe movement is monitored by a suitable probe detector, such as a
displacement sensor 18 that may for example employ a laser and a
photodetector as well as other components. As is known in the art, the
signals from the sensor 18 can be used to determine probe oscillation
amplitude, frequency, and phase, as well as other parameters, and to
measure the probe-sample interaction based on the determined probe
parameters. The computer 14 can use this measurement as a feedback signal
to control the vertical probe-sample position via the actuator 16 so as to
keep the probe-sample interaction constant during data acquisition.
Finally, a suitable display device 24 is connected to the computer 14 and
displays a humanly-discernible image of the measurement results in a
visual image, such as a histogram.
The illustrated AFM is capable of operating in fluid so as to allow the AFM
to obtain data during biological reactions. Preferably, a DNA/polymerase
complex 50 is positioned in the AFM in a flow-through fluid chamber 32 of
a glass cell 34. The AFM cantilever 8 and a probe tip 12 are submerged
such that the height of the polymerase reaction fluid 36 is above the
maximum deflection point of the cantilever 8. A polymerase 26 is disposed
in the fluid chamber 32 for detection by the AFM. The polymerase 26
preferably is attached to a substrate 42 such as mica for reasons detailed
below.
In use, the AFM operator prepositions the AFM probe 10 over a
DNA/polymerase complex 50 to locate the complex. This positioning can be
accomplished by several techniques including, but not limited to, the
following. For instance, the operator can operate the AFM in scanning
mode, as described above, until a DNA/polymerase complex 50 is located.
Once the operator locates a complex, the operator switches the AFM from
scanning mode to a data acquisition mode which may, for instance, comprise
contact mode, TappingMode, or non-contact mode.
Alternatively, if a complex concentration in the cell is sufficiently high,
a complex can be located simply by lowering the AFM probe into position
with the expectation that there is a reasonable likelihood that the probe
will be lowered into a complex. If the probe happens to miss a complex,
the operator can simply raise the probe, move it slightly, and lower it
again. If a complex is not located after a reasonable number of
repetitions of this process, the probe 10 can be switched to a scanning
mode to locate a complex.
Next, the AFM is placed in data acquisition mode to detect motions in the
polymerase 26 during nucleotide incorporation into a growing
polynucleotide chain 38 at a reaction site 40 and subsequent movement of
the newly formed double-stranded DNA helix 48 or other motions associated
with incorporation. The polymerase's motions generate a mechanical force
that is reflected, either directly or indirectly, by motion of the AFM
cantilever 8. The changes in motion may, for instance, be reflected by
movement of a stationary probe, by reduction in oscillation amplitude of
an oscillating probe, and/or by phase changes of an oscillating probe. The
resulting changes in cantilever motion are detected by the AFM and
indicate that a nucleotide has been incorporated into the growing DNA
chain.
To determine which nucleotide is incorporated into the growing DNA chain
during the polymerization reaction, at least one characteristic of the
incorporation of nucleotides of the nucleotide type of interest is altered
(i.e., flagged) to distinguish incorporation of nucleotides of the
nucleotide type of interest (e.g., As) from nucleotides of the other types
(e.g., Ts, Cs, or Gs). The flagging alters a characteristic of the
detected motion such as (1) changes in average periods between motion
detections or (2) changes in the magnitude of the motion. For instance,
the average incorporation delay for nucleotides of the nucleotide type of
interest may be flagged such that the average incorporation delay for
nucleotides of the nucleotide type of interest is unique when compared to
the average incorporation delay for nucleotides of other types.
Flagging can be achieved by several techniques including (1) altering the
concentration of the nucleotides of the type of interest and (2) altering
the size of the nucleotides of the type of interest. An example of such a
flagging is using a one-half concentration of the nucleotide type of
interest when compared to concentrations of other nucleotide types.
Because it takes twice as long, on average, for a nucleotide of the
nucleotide type of interest at a one-half concentration to come into the
reaction site, this technique results in a doubling of the average
incorporation delay for nucleotides of the nucleotide type of interest
when compared to the other nucleotide types. Thus, flagging the nucleotide
types of interest provides a method for distinguishing which nucleotide is
incorporated into the growing DNA chain, and, hence, provides a basis for
a method for determining the sequence of a DNA molecule.
This flagging and subsequent detection method can be used for several
applications including (1) DNA sequencing and (2) mutation detection. For
DNA sequencing, the method is used | | |
