U.S. patent application number 15/006962 was filed with the patent office on 2016-06-23 for sequencing systems with two slow-step polymerase enzymes.
The applicant listed for this patent is Pacific Biosciences of California, Inc.. Invention is credited to Keith Bjornson, Jonas Korlach, Harold Lee.
Application Number | 20160177384 15/006962 |
Document ID | / |
Family ID | 41316532 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160177384 |
Kind Code |
A1 |
Bjornson; Keith ; et
al. |
June 23, 2016 |
SEQUENCING SYSTEMS WITH TWO SLOW-STEP POLYMERASE ENZYMES
Abstract
Systems for nucleotide sequencing comprising producing
polymerase reactions that exhibit two kinetically observable steps
within an observable phase of the polymerase reaction. Two slow
step systems can be produced, for example, by selecting the
appropriate polymerase enzyme, polymerase reaction conditions
including cofactors, and polymerase reaction substrates including
the primed template and nucleotides.
Inventors: |
Bjornson; Keith; (Fremont,
CA) ; Lee; Harold; (Missouri City, TX) ;
Korlach; Jonas; (Camas, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pacific Biosciences of California, Inc. |
Menlo Park |
CA |
US |
|
|
Family ID: |
41316532 |
Appl. No.: |
15/006962 |
Filed: |
January 26, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14185329 |
Feb 20, 2014 |
9279155 |
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15006962 |
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12537130 |
Aug 6, 2009 |
8658365 |
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14185329 |
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12414191 |
Mar 30, 2009 |
8133672 |
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12537130 |
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61139287 |
Dec 19, 2008 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6874 20130101; C12Q 1/6869 20130101; C12Q 2521/101 20130101;
C12Q 2527/125 20130101; C12Q 2527/137 20130101; C12Q 2527/101
20130101; C12Q 1/6869 20130101; C12Q 2527/119 20130101; C12Q
2565/1015 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A system for single-molecule nucleotide sequencing comprising:
a) a reaction mixture having: (i) a polymerase enzyme, (ii)
polymerase reaction conditions including cofactors, and (iii)
polymerase reaction substrates including a primed template and
nucleotides comprising observable labels, such that a reaction
comprising incorporation of the nucleotides into a growing nucleic
acid occurs, wherein the polymerase enzyme, the polymerase reaction
conditions, and the polymerase reaction substrates are selected
such that the reaction exhibits two kinetically observable steps,
each of which kinetically observable steps proceeds from an
intermediate in which a nucleotide or a polyphosphate product is
bound to the polymerase enzyme or each of which kinetically
observable steps proceeds from an intermediate in which the
nucleotide and the polyphosphate product are not bound to the
polymerase enzyme; and (b) a detector to detect the observable
label to measure the sequential incorporation of nucleotides into
the growing nucleic acid thereby obtaining a sequence of the
template
2. The system of claim 1 wherein the detector comprises a CCD,
ICCD, EMCCD or CMOS detector.
3. The system of claim 1 wherein the observable labels comprise
optical labels.
4. The system of claim 3 wherein the optical labels comprise
fluorescent labels.
5. The system of claim 1 further comprising an illumination
source.
6. The system of claim 1 wherein the two kinetically observable
steps are each steps which proceed from an intermediate in which a
nucleotide or a polyphosphate product is bound to the polymerase
enzyme.
7. The system of claim 1 wherein the two kinetically observable
steps are each steps which proceed from an intermediate in which
the nucleotide and the polyphosphate product are not bound to the
polymerase enzyme.
8. The system of claim 1 wherein the two kinetically observable
steps are selected from a group consisting of enzyme isomerization,
nucleotide incorporation, and product release.
9. The system of claim 1 wherein the two kinetically observable
steps are template translocation and nucleotide binding.
10. The system of claim 1 wherein the ratio of the rate constants
of the kinetically observable steps is from 2:1 to 1:2.
11. The system of claim 1 wherein the rate constant for one of the
kinetically observable steps is less than about 100 per second.
12. The system of claim 1 wherein the rate constant for one of the
kinetically observable steps is between about 0.1 per second and
about 60 per second.
13. The system of claim 1 wherein the rate constant for one of the
kinetically observable steps is between about 1 per second and
about 20 per second.
14. The system of claim 1 wherein the reaction exhibits more than
two kinetically observable steps in an observable phase.
15. The system of claim 1 wherein the polymerase enzyme comprises a
modified recombinant .PHI.29-type polymerase.
16. The system of claim 15 wherein the polymerase enzyme comprises
a modified recombinant .PHI.29, B103, GA-1, PZA, .PHI.15, BS32,
M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or
L-17 polymerase.
17. The system of claim 15 wherein the polymerase enzyme comprises
a modified recombinant .PHI.29 DNA polymerase having at least one
amino acid substitution or combination of substitutions selected
from the group consisting of: an amino acid substitution at
position 484, an amino acid substitution at position 198, and an
amino acid substitution at position 381.
18. The system of claim 15 wherein the polymerase enzyme comprises
a modified recombinant .PHI.29 DNA polymerase having at least one
amino acid substitution or combination of substitutions selected
from the group consisting of E375Y, K512Y, T368F, A484E, A484Y,
N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381 A.
19. The system of claim 1 wherein the polymerase reaction
conditions comprise one or more of metal cofactor concentration,
pH, temperature, an enzyme activity modulator, D.sub.2O, an organic
solvent, and buffer.
20. The system of claim 19 wherein the polymerase reaction
conditions comprise a mixture of divalent metal ions comprising at
least one catalytic metal ion and at least one non-catalytic metal
ion.
21. The system of claim 20 wherein the catalytic metal is selected
from Mg2+, Mn2+ and mixtures thereof, and the non-catalytic metal
is selected from Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, Eu2+ and
mixtures thereof.
22. The system of claim 20 wherein a ratio of catalytic metal to
non-catalytic metal in the reaction mixture is from about 10:1 to
about 1:10.
23. The system of claim 19 wherein the conditions comprise an
organic solvent selected from the group consisting of ethanol,
methanol, THE, dioxane, DMA, DMF, and DMSO.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Patent Application from
Ser. No. 14/185,329, filed Feb. 20, 2014, which is a Continuation
Patent Application from Ser. No. 12/537,130, filed Aug. 6, 2009,
now U.S. Pat. No. 8,658,365 which is a Continuation Patent
Application from U.S. patent application Ser. No. 12/414,191, filed
Mar. 30, 2009, now U.S. Pat. No. 8,133,672, which claims priority
to U.S. Provisional Patent Application No. 61/139,287, filed Dec.
19, 2008, the full disclosures of which are incorporated herein by
reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] The ability to read the genetic code has opened countless
opportunities to benefit humankind. Whether it involves the
improvement of food crops and livestock used for food, the
identification of the causes of disease, the generation of targeted
therapeutic methods and compositions, or simply the better
understanding of what makes us who we are, a fundamental
understanding of the blueprints of life is an integral and
necessary component.
[0003] A variety of techniques and processes have been developed to
obtain genetic information, including broad genetic profiling or
identifying patterns of discrete markers in genetic codes and
nucleotide level sequencing of entire genomes. With respect to
determination of genetic sequences, while techniques have been
developed to read, at the nucleotide level, a genetic sequence,
such methods can be time-consuming and extremely costly.
[0004] Approaches have been developed to sequence genetic material
with improved speed and reduced costs. Many of these methods rely
upon the identification of nucleotides being incorporated by a
polymerization enzyme during a template sequence-dependent nucleic
acid synthesis reaction. In particular, by identifying nucleotides
incorporated against a complementary template nucleic acid strand,
one can identify the sequence of nucleotides in the template
strand. A variety of such methods have been previously described.
These methods include iterative processes where individual
nucleotides are added one at a time, washed to remove free,
unincorporated nucleotides, identified, and washed again to remove
any terminator groups and labeling components before an additional
nucleotide is added. Still other methods employ the "real-time"
detection of incorporation events, where the act of incorporation
gives rise to a signaling event that can be detected. In
particularly elegant methods, labeling components are coupled to
portions of the nucleotides that are removed during the
incorporation event, eliminating any need to remove such labeling
components before the next nucleotide is added (See, e.g., Eid, J.
et al., Science, 323(5910), 133-138 (2009)).
[0005] In any of the enzyme mediated template-dependent processes,
the overall fidelity, processivity and/or accuracy of the
incorporation process can have direct impacts on the sequence
identification process, e.g., lower accuracy may require multiple
fold coverage to identify the sequence with a high level of
confidence.
[0006] The present invention provides methods, systems and
compositions that provide for increased performance of such
polymerization based sequencing methods, among other benefits.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is generally directed to enzyme
reactions, and in particular, nucleic acid synthesis compositions,
systems, and methods that exhibit kinetic mechanisms having two or
more kinetically observable reaction steps within an observable
phase of the polymerase reaction. Such systems can be useful for
observing the activity of a polymerase enzyme in real-time, for
example, for carrying out single-molecule nucleic acid sequencing.
We have discovered that a system in which the reaction kinetics
exhibit two or more rate-limiting, kinetically observable (slow)
steps within an observable phase reduce the relative number of
short, difficult to detect pulses, resulting in more observable
sequencing events, and allowing for a more accurate determination
of a nucleic acid sequence.
[0008] In single-molecule DNA sequencing by synthesis, for example
as described Eid, J. et al., Science, 323(5910), 133-138 (2009),
the incorporation of specific nucleotides can be determined by
observing bright phases and dark phases which correspond, for
example, to reaction steps in which a fluorescent label is
associated with the polymerase enzyme, and steps in which the
fluorescent label is not associated with the enzyme. In some
embodiments of the invention, the polymerase reaction system will
exhibit two slow (kinetically observable) reaction steps wherein
each of the steps is in a bright phase. In some embodiments of the
invention, the system will exhibit two kinetically observable
reaction steps wherein each of the steps is in a dark phase. In
some cases, the system will have four kinetically observable (slow)
reaction steps, two slow steps in a bright phase and two slow steps
in a dark phase.
[0009] Obtaining a system with kinetically observable reaction
steps can involve selection and/or production of 1) the type of
polymerase enzyme, 2) the polymerase reaction conditions, including
the type and levels of cofactors, and 3) the reaction substrates.
We describe herein ways in which each of these aspects can be
controlled in order to obtain a reaction system with two slow steps
within an observable phase of the polymerase reaction.
[0010] In one aspect, the invention provides a method for
nucleotide sequencing comprising: providing a reaction mixture
having: (i) a polymerase enzyme, (ii) polymerase reaction
conditions including cofactors, and (iii) polymerase reaction
substrates including a primed template and nucleotides, such that a
reaction comprising incorporation of the nucleotides into a growing
nucleic acid occurs; and observing the reaction mixture to
determine the incorporation of nucleotides into the growing nucleic
acid; wherein at least one of the polymerase enzyme, the polymerase
reaction conditions, or the polymerase reaction substrates are
selected such that the reaction exhibits two kinetically observable
steps within an observable phase of the polymerase reaction.
[0011] In some embodiments the two kinetically observable steps are
each steps which proceed in a bright phase. In some embodiments the
two kinetically observable steps are each steps which proceed in a
dark phase. In some embodiments the reaction exhibits two
kinetically observable steps which proceed in a bright phase, and
two kinetically observable steps which proceed in a dark phase.
[0012] In some embodiments the two kinetically observable steps are
selected from a group consisting of enzyme isomerization,
nucleotide incorporation, and product release. In some embodiments
two kinetically observable steps are template translocation and
nucleotide binding.
[0013] In some embodiments the ratio of the rate constants of the
kinetically observable steps is from 10:1 to 1:10. In some
embodiments the ratio of the rate constants of the kinetically
observable steps is from 5:1 to 1:5. In some embodiments the ratio
of the rate constants of the kinetically observable steps is from
2:1 to 1:2.
[0014] In some embodiments the rate constant for one of the
kinetically observable steps is less than about 100 per second. In
some embodiments the rate constant for one of the kinetically
observable steps is between about 0.1 per second and about 60 per
second. In some embodiments the rate constant for one of the
kinetically observable steps is between about 1 per second and
about 20 per second. In some embodiments the reaction exhibits more
than two kinetically observable steps.
[0015] In some embodiments the polymerase enzyme comprises a
modified recombinant .PHI.29-type polymerase. In some embodiments
the polymerase enzyme comprises a modified recombinant .PHI.29,
B103, GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5,
Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In some embodiments
the polymerase enzyme comprises a modified recombinant DNA
polymerase having at least one amino acid substitution or
combination of substitutions selected from the group consisting of:
an amino acid substitution at position 484, an amino acid
substitution at position 198, and an amino acid substitution at
position 381. In some embodiments the polymerase enzyme comprises a
modified recombinant DNA polymerase having at least one amino acid
substitution or combination of substitutions selected from the
group consisting of E375Y, K512Y, T368F, A484E, A484Y, N387L,
T372Q, T372L, K478Y, 1370W, F198W, and L381A.
[0016] In some embodiments the polymerase reaction conditions
comprise one or more of metal cofactor concentration, pH,
temperature, an enzyme activity modulator, D2O, an organic solvent,
and buffer. In some embodiments the polymerase reaction conditions
comprise a mixture of divalent metal ions comprising at least one
catalytic metal ion and at least one non-catalytic metal ion. In
some embodiments the catalytic metal is selected from Mg2+, Mn2+
and mixtures thereof, and the non-catalytic metal is selected from
Ca2+, Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, Eu2+ and mixtures
thereof. In some embodiments a ratio of catalytic metal to
non-catalytic metal in the reaction mixture is from about 10:1 to
about 1:10.
[0017] In some embodiments the polymerase reaction conditions
comprise the presence of D2O. In some embodiments the D2O/H2O
volume ratio in the reaction mixture is about 0.1 to about 2. In
some embodiments the D2O/H2O volume ratio in the reaction mixture
is about 0.2 to about 0.5. In some embodiments the D2O/H2O volume
ratio in the reaction mixture is about 0.2 to about 0.3.
[0018] In some embodiments the conditions comprise an organic
solvent selected from the group consisting of ethanol, methanol,
THF, dioxane, DMA, DMF, and DMSO. In some embodiments the solvent
comprises DMA. In some embodiments the solvent comprises DMSO.
[0019] In some embodiments the polymerase conditions comprise an
additive that when added, changes the polymerase enzyme kinetics
relative to a reaction having no additive. In some embodiments the
additive is a thiol containing amino acid. In some embodiments the
additive is L-cysteine.
[0020] In some embodiments one or more of the nucleotides comprise
an optical label. In some embodiments one or more of the
nucleotides comprise tetra, penta, or hexaphosphate groups having
fluorescent labels linked to the terminal phosphate. In some
embodiments the nucleotide comprises one, two, or three
non-bridging thiol groups in its polyphosphate portion. In some
embodiments the nucleotide has one non-bridging thiol. In some
embodiments substantially only one chiral isomer is used.
[0021] In some embodiments the polymerase substrate that is
selected such that the reaction exhibits two kinetically observable
steps comprises a modified primer-template nucleotide complex.
[0022] In some embodiments at least two of the polymerase enzyme,
the polymerase reaction conditions, or the polymerase reaction
substrates are selected such that the reaction exhibits two
kinetically observable steps.
[0023] In some embodiments all of the polymerase enzyme, the
polymerase reaction conditions, or the polymerase reaction
substrates are selected such that the reaction exhibits two
kinetically observable steps.
[0024] In another aspect, the invention provides compositions
useful for nucleotide sequencing comprising: a reaction mixture
having (i) a polymerase enzyme, (ii) polymerase reaction conditions
including cofactors, and (iii) reaction substrates including a
primed template nucleotide and nucleotides, wherein at least one of
the polymerase enzyme, the polymerase reaction conditions, or the
polymerase reaction substrates are selected such that the reaction
resulting in the incorporation of the nucleotides or nucleotide
analogs exhibits two kinetically observable steps.
[0025] In another aspect, the invention provides systems for
single-molecule nucleotide sequencing comprising: a zero-mode
waveguide having, within its core, a reaction mixture comprising
(i) a polymerase enzyme, (ii) polymerase reaction conditions
including cofactors, and (iii) polymerase reaction substrates
including nucleotides or nucleotide analogs and a primed template
nucleotide wherein one or more of the polymerase reaction
substrates is labeled with an optically observable label; and an
optical detection system to detect the optically observable label
to measure the sequential incorporation of nucleotides into a
growing nucleic acid; wherein at least one of the polymerase
enzyme, the polymerase reaction conditions, or the polymerase
reaction substrates are selected such that the incorporation of the
nucleotides or nucleotide analogs exhibits two kinetically
observable steps within an observable phase of the polymerase
reaction.
[0026] In some embodiments the system comprises an array of
zero-mode waveguides.
[0027] In another aspect, the invention provides a method for
identifying a polymerase reaction system having two or more
kinetically observable steps within an observable phase of the
polymerase reaction comprising: selecting a first polymerase
reaction mixture comprising: (i) a polymerase enzyme, (ii)
polymerase reaction conditions including cofactors, and (iii)
polymerase reaction substrates including nucleotides or nucleotide
analogs and a primed template nucleotide such that a polymerase
reaction occurs; observing the polymerase reaction progress over a
time period; and fitting the observed reaction progress over time
in step (b) to a model to determine if the reaction shows two or
more kinetically observable steps within an observable phase of the
polymerase reaction.
[0028] In some embodiments the method is carried out in stop-flow
apparatus.
[0029] In another aspect, the invention provides a method of
sequencing a nucleic acid, comprising:
[0030] providing a complex comprising a polymerase enzyme, a
template nucleic acid, and a primer sequence complementary to at
least a portion of the template nucleic acid; contacting the
complex with a reaction mixture that comprises a mixture of
divalent metal ions comprising at least one catalytic metal ion and
at least one non-catalytic metal ion, and one or more types of
nucleotides or nucleotide analogs; and detecting incorporation of
one or more of the nucleotide into the complex.
[0031] In some embodiments the catalytic metal is selected from
Mg2+, Mn2+ and mixtures thereof, and the non-catalytic metal is
selected from Ca2+, Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, Eu2+
and mixtures thereof. In some embodiments the catalytic metal
comprises Mn2+ and the non-catalytic metal comprises Ca2+. In some
embodiments a ratio of catalytic metal to non-catalytic metal in
the reaction mixture is from about 10:1 to about 1:10. In some
embodiments a ratio of catalytic metal to non-catalytic metal in
the reaction mixture is from about 10:1 to about 1:5. In some
embodiments a ratio of catalytic metal to non-catalytic metal in
the reaction mixture is from about 5:1 to about 1:1. In some
embodiments a ratio of catalytic metal to non-catalytic metal in
the reaction mixture is from about 2.5:1 to about 1.5:1. In some
embodiments the catalytic metal and non-catalytic metal are present
in the reaction mixture at a total concentration of from about 0.1
mM to about 10 mM.
[0032] In some embodiments the detecting step comprises detecting
incorporation of nucleotides in real-time as they are incorporated
into the complex. In some embodiments the complex is immobilized on
a solid support. In some embodiments the complex is immobilized on
a solid support in an individually optically resolvable
configuration.
[0033] In another aspect, the invention provides a method of
sequencing a nucleic acid, comprising: providing a complex
comprising a polymerase enzyme, a template nucleic acid, and a
primer sequence complementary to at least a portion of the template
nucleic acid; contacting the complex with a reaction mixture that
comprises a plurality of types of nucleotides, and a mixture of
divalent metal ions comprising at least one catalytic metal ion and
at least one non-catalytic metal ion at first and second
concentrations, respectively, wherein the mixture of divalent metal
ions is selected to provide improved sequencing accuracy over a
complex exposed to the first concentration of the catalytic metal
in the absence of the non-catalytic metal; and detecting
incorporation of a nucleotide into the complex.
[0034] In another aspect, the invention provides a composition,
comprising: a complex comprising a template nucleic acid, a primer
sequence and a polymerase enzyme; a mixture of divalent metal ions
comprising at least one catalytic metal ion and at least one
non-catalytic metal ion; and at least a first incorporatable
nucleotide or nucleotide analog.
[0035] In another aspect, the invention provides method of
modulating a polymerase activity, comprising: sequestering a bound
nucleotide in a non-exchangeable state with a polymerase enzyme by
contacting the polymerase enzyme with a first non-catalytic
exchangeable co-factor; and contacting the polymerase enzyme with a
catalytic exchangeable co-factor to exchange the non-catalytic
co-factor, rendering the bound nucleotide into an exchangeable
state with the polymerase enzyme.
[0036] In another aspect, the invention provides a kit, comprising:
one or more components of a nucleic acid synthesis complex,
selected from a DNA polymerase enzyme and a primer sequence; a
reaction buffer comprising a mixture of catalytic metal ions and
non-catalytic metal ions; a plurality of types of fluorescently
labeled nucleotide analogs; and instructions for carrying out a
sequence by synthesis reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a schematic illustration of the reaction cycle for
polymerase-mediated nucleic acid primer extension.
[0038] FIG. 2 schematically illustrates an exemplar single-molecule
sequencing-by-incorporation process in which the compositions of
the invention provide particular advantages.
[0039] FIG. 3 shows a theoretical representation of the probability
density for residence time for a polymerase reaction having 1
rate-limiting step or two rate-limiting steps within an observable
phase.
[0040] FIG. 4 schematically illustrates a simplified system for
analysis of sequencing-by-incorporation reactions.
[0041] FIG. 5 shows a plot of the effects of Ca.sup.2+ ion
concentration on nucleotide binding and incorporation by DNA
polymerase in the presence of manganese.
[0042] FIG. 6 shows a plot of the effects of Ca.sup.2+ ion
concentration on the rate of incorporation of nucleotides by a
polymerase, fit to a hyperbolic equation.
[0043] FIG. 7 shows a plot of the effects of Ca.sup.2+ ion
concentration on nucleotide release by DNA polymerase in the
presence of manganese.
[0044] FIG. 8 shows a plot of the effects of Ca.sup.2+ ion
concentration on the rate of nucleotide release by polymerase
enzyme, fit to a hyperbolic equation.
[0045] FIG. 9 shows a plot of Ca.sup.2+ on exonuclease activity of
a DNA polymerase enzyme in the presence of manganese.
[0046] FIG. 10 shows a plot of the effects of Ca.sup.2+
concentration on the exonuclease rate of a DNA polymerase, fit to a
hyperbolic equation.
[0047] FIG. 11 shows data illustrating sequential incorporation of
nucleotides in a polymerase-mediated, template-dependent primer
extension reaction, where the reaction was iteratively initiated
and arrested through the addition of catalytic and non-catalytic
metal co-factors.
[0048] FIG. 12 shows the effect of the addition of Ca.sup.2+ (0.3
mM) to a DNA sequencing reaction on the relative insertion or
deletion errors for such process.
[0049] FIG. 13 shows data for fluorescence versus time for
reactions at varying concentrations of added ZnSO.sub.4.
[0050] FIG. 14 shows polymerase reaction rate as a function of
concentration for various non-catalytic metal cofactors.
[0051] FIG. 15 shows the Ki values determined for various
non-catalytic metal cofactors.
[0052] FIG. 16 shows data for the mean pulse width as a function of
D.sub.2O content in single-molecule sequencing reactions.
[0053] FIGS. 17 (a)-(d) shows data for the interpulse distance as a
function of the amount of dimethylacetamide (DMA) in
single-molecule sequencing reactions for 4 dye channels.
[0054] FIG. 18 shows data for the interpulse distance as a function
of the amount of dimethylsulfoxide (DMSO) in single-molecule
sequencing reactions for 4 dye channels.
[0055] FIG. 19 shows the results of a stopped-flow experiment for a
polymerase reaction system in which the decrease in the fluorescent
signal fits to a single exponential and the increase in signal fits
to a single exponential.
[0056] FIG. 20 shows the results of a stopped-flow experiment for a
polymerase reaction system in which the decrease in the fluorescent
signal fits to a single exponential and the increase in signal is
best described by two exponentials.
[0057] FIG. 21 shows the results of a stopped-flow experiment for a
polymerase reaction system in which the decrease in the fluorescent
signal fits to a single exponential and the increase in signal fits
to a single exponential.
[0058] FIG. 22 shows the results of a stopped-flow experiment for a
polymerase reaction system in which the decrease in the fluorescent
signal fits to a single exponential and the increase in signal is
best described by to two exponentials (22(b)), and is poorly fit by
a single exponential (22(a)).
[0059] FIG. 23 shows a 3-dimensional model of a nucleotide having 6
phosphate units bound to the phi29 polymerase enzyme.
[0060] FIG. 24 shows data for the pulse width as a function of the
amount of added cysteine to single molecule sequencing reactions
for each of four dye channels.
DETAILED DESCRIPTION OF THE INVENTION
[0061] The present invention is generally directed to improved
enzyme reaction compositions, methods, and systems that exhibit
kinetic mechanisms having two or more slow, kinetically observable,
or partially rate-limiting reaction steps within an observable
phase of the polymerase reaction. Such systems can be useful for
example, in single-molecule, real-time observations of such enzyme
activity, which rely, at least in part, on detecting and
identifying the enzyme reaction as it is occurring. By designing
the reaction system to have two or more partially rate-limiting
steps, the relative number of short, difficult to detect, events
can be lowered. Enzymatic reactions often occur at rates that can
far exceed the speed of a variety of detection systems, e.g.,
optical detectors. As such, by providing two or more partially
rate-limiting steps within a phase of an enzyme reaction, one
improves the ability to monitor that reaction using optical
detection systems.
[0062] One particular exemplary system includes compositions for
carrying out single-molecule DNA sequencing. We describe systems
that exhibit two slow steps within an observable phase. An
observable phase will generally have a time period during which the
phase is observable. The time period for a bright phase, for
example, can be represented by the pulse width. The time period for
a dark phase can be represented, for example, by the interpulse
distance. The length of each time period will not be the same for
each nucleotide addition, resulting in a distribution of the length
of the time periods. In some cases, the time periods with the
shortest length will not be detected, leading to errors, for
example in single-molecule sequencing. We have found that by
designing enzyme systems such as polymerase reaction systems in
which there are two slow, or kinetically observable, steps within
an observable phase, the relative number of short, unobservable,
time periods can be reduced, resulting in a higher proportion of
observable sequencing events, and allowing for a more accurate
determination of nucleotide sequence. As used herein, an observable
phase includes phases that are not directly observable, but can be
ascertained by measurements of other, related phases. For example,
the lengths of dark phases can be observed by measuring the times
between optical pulses corresponding to a related bright optical
phase. Also as described herein, a phase which is dark under some
labeling conditions can be bright under other labeling
conditions.
[0063] While primarily described in terms of nucleic acid
polymerases, and particularly DNA polymerases, it will be
appreciated that the approach of providing multiple slow, or
kinetically observable steps, within an enzyme system is applicable
to other enzyme systems where one may wish to directly observe the
enzyme reaction, in real time. Such enzyme systems include, for
example, other synthesizing enzymes, e.g., RNA polymerases, reverse
transcriptases, ribosomal polymerases, as well as other enzyme
systems, such as kinases, phosphatases, proteases, nucleases,
ligases, and the like.
I. POLYMERASE-MEDIATED SYNTHESIS
[0064] In natural polymerase-mediated nucleic acid synthesis, a
complex is formed between a polymerase enzyme, a template nucleic
acid sequence, and a priming sequence that serves as the point of
initiation of the synthetic process. During synthesis, the
polymerase samples nucleotide monomers from the reaction mix to
determine their complementarity to the next base in the template
sequence. When the sampled base is complementary to the next base,
it is incorporated into the growing nascent strand. This process
continues along the length of the template sequence to effectively
duplicate that template. Although described in a simplified
schematic fashion, the actual biochemical process of incorporation
is relatively complex.
[0065] The process can be described as a sequence of steps, wherein
each step can be characterized as having a particular forward and
reverse reaction rate that can be represented by a rate constant.
One representation of the incorporation biochemistry is provided in
FIG. 1. It is to be understood that the scheme shown in FIG. 1 does
not provide a unique representation of the process. In some cases,
the process can be described using fewer steps. For example, the
process is sometimes represented without inclusion of the enzyme
isomerization steps 106 and 110. Alternatively, the process can be
represented by including additional steps such as cofactor binding.
Generally, steps which can be slow, and thus limit the rate of
reaction will tend to be included. The present invention relates to
methods, systems, and compositions in which the polymerization
reaction has two or more slow steps within certain phases of the
polymerase reaction. Various schemes can be used to represent a
reaction having two slow steps that may have more or fewer
identified steps. In some cases the two or more slow steps are
consecutive. In some cases, there can be intervening fast steps
between the two or more slow steps.
[0066] As shown in FIG. 1, the synthesis process begins with the
binding of the primed nucleic acid template (D) to the polymerase
(P) at step 102. Nucleotide (N) binding with the complex occurs at
step 104. Step 106 represents the isomerization of the polymerase
from the open to closed configuration. Step 108 is the chemistry
step where the nucleotide is incorporated into the growing strand
of the nucleic acid being synthesized. At step 110, polymerase
isomerization occurs from the closed to the open position. The
polyphosphate component that is cleaved upon incorporation is
released from the complex at step 112. The polymerase then
translocates on the template at step 114. As shown, the various
steps can include reversible paths and may be characterized by the
reaction constants shown in FIG. 1 where:
[0067] k.sub.on/k.sub.off=DNA binding/release;
[0068] k.sub.1/k..sub.1=nucleotide binding/release;
[0069] k.sub.2/k..sub.2=polymerase isomerization (open/closed);
[0070] k.sub.3/k..sub.3=nucleotide incorporation (chemistry);
[0071] k.sub.4/k..sub.4=polymerase isomerization (closed/open);
[0072] k.sub.5/k..sub.5=polyphosphate release/binding;
[0073] k.sub.6/k..sub.6=polymerase translocation.
[0074] Thus, during steps 104 through 110, the nucleotide is
retained within the overall complex, and during steps 104 and 106,
reversal of the reaction step will yield an unproductive event,
i.e., not resulting in incorporation. For example, a bound
nucleotide at step 104 may be released regardless of whether it is
the correct nucleotide for incorporation.
[0075] By selecting the appropriate polymerase enzyme, polymerase
reaction conditions, and polymerase substrates, the absolute and
relative rates of the various steps can be controlled. We have
found that controlling the reaction such that the reaction exhibits
two or more kinetically observable, or slow steps can produce a
nucleic acid polymerization reaction in which the incorporation of
the nucleotides can be observed more accurately. These
characteristics are particularly useful for sequencing
applications, and in particular single-molecule DNA sequencing.
[0076] In some cases, the invention involves a process having two
or more kinetically observable steps that comprise steps after
nucleotide binding through the step of product release. For the
mechanism shown in FIG. 1, this would be, for example, any of steps
106, 108, 110, and 112. In some cases, steps 108 (nucleotide
incorporation) and 112 (product release) are the two slow, or
kinetically observable steps. As noted previously, where one
desires systems with slow steps in a dark phase, the invention may
involve a process having two or more slow steps that comprise the
steps after product release through nucleotide binding. For the
mechanism shown in FIG. 1, this would include steps 114 and
104.
[0077] In some cases, the invention involves a process in which
there are two or more slow steps in two different observable phases
within the polymerization, for example, two slow steps in a bright
phase and two slow steps in a dark phase. For example, this could
include a system having two slow steps in the steps after
nucleotide binding through product release, and two slow steps for
the steps after product release through nucleotide binding.
[0078] As is described herein, producing a process in which there
are two or more slow steps in these portions of the polymerase
reaction can result in a higher proportion of detectable enzyme
states which can be useful, for example, to observe the sequential
incorporation of nucleotides for nucleotide sequencing.
[0079] By the term slow-step we generally mean a kinetically
observable step or partially rat-limiting step. The slow step need
not be slow in the absolute sense, but will be relatively slow as
compared with other steps in the enzymatic reaction. The slow, or
kinetically observable steps, can be, for example, each partially
rate-limiting, in that the rate of the step has a measurable effect
on the kinetics of the enzymatic reaction. An enzymatic process,
such as nucleic acid polymerization, can have both slower,
kinetically observable steps and faster steps which can be so fast
that they have no measurable effect on the kinetics, or rate, of
the reaction. In some reactions, there can be a single
rate-limiting step. For such reactions, the kinetics can be
characterized by the rate of that single step. Other reactions will
not have a single rate-limiting step, but will have two or more
steps which are close enough in rate such that the characteristics
of each will contribute to the kinetics of the reaction. A
kinetically observable step is generally a step which is slow
enough relative to the other steps in the reaction such that it can
be experimentally ascertained. The experimental identification of a
kinetically observable step can be done by the methods described
herein, or by methods for assessing the kinetics of chemical and
enzymatic reactions known in the art. For the current invention,
the slow, or kinetically observable steps, need not be the slowest
step or the rate-limiting step of the reaction. For example, a
process of the current invention can involve a reaction in which
step 104, nucleotide addition is the slowest (rate-limiting) step,
while two or more of steps 106, 108, 110, or 112 are each
kinetically observable.
[0080] As used herein, the term rate, as applied to the steps of a
reaction can refer to the average rate of reaction. For example,
when observing a single-molecule reaction, there will generally be
variations in the rates as each individual nucleotide is added to a
growing nucleic acid. In such cases the rate of the reaction can be
represented by observing a number of individual events, and
combining the rates, for example, by obtaining an average of the
rates.
[0081] As used herein, the reference to the rate of a step or rate
constant for a step can refer to the forward reaction rate of the
polymerase reaction. As is generally understood in the art,
reaction steps can be characterized as having forward and reverse
rate constants. For example, for step 108, k.sub.3 represents the
forward rate constant, and k..sub.3 represents the reverse rate
constant for the nucleotide incorporation. Some reaction steps,
such as step 108, constitute steps which would be expected to be
first order steps. Other steps, such as the forward reaction of
step 104, with rate constant k.sub.2, would be expected to be
second order rate constants. For the purposes of the invention, for
comparing the rate or the rate constant of a first order to a
second order step, the second order rate constant k.sub.2 can be
treated as a pseudo-first order rate constant with the value
[N]*k.sub.2 where the concentration of nucleotide [N] is known.
[0082] It is generally desirable that the kinetically observable
steps of the invention have rate constants that are lower than
about 1000 per second. In some cases, the rate constants are lower
than about 500 per second, lower than about 200 per second, lower
than about 100 per second, lower than about 60 per second, lower
than about 50 per second, lower than about 30 per second, lower
than about 20 per second, lower than about 10 per second, lower
than about 5 per second, lower than about 2 per second, or lower
than about 1 per second.
[0083] In some embodiments the slowest of the two or more
kinetically observable steps has a rate constant when measured
under single-molecule conditions of between about 500 to about 0.1
per second, about 200 to about 0.1 per second, about 60 to about
0.5 per second, about 30 per second to about 2 per second, or about
10 to about 3 per second.
[0084] The ratio of the rate constants of each the two or more slow
steps is generally greater than 1:10, in some cases the ratio of
the rate constants is about 1:5, in some cases the ratio of the
rate constants is about 1:2, in some cases, the ratio of rate
constants is about 1:1. The ratio of the rate constants can be
between about 1:10 and about 1:1, between about 1:5 and about 1:1,
or between about 1:2 and about 1:1.
[0085] In some cases it is useful to consider the two slow-step
system in terms of rates rather than rate constants. It is
generally desirable that the kinetically observable steps of the
invention have rates that are lower than about 1000 molecules per
second when the reactions are carried out under single-molecule
conditions. In some cases, the rates are lower than about 500
molecules per second, lower than about 200 molecules per second,
lower than about 100 molecules per second, lower than about 60
molecules per second, lower than about 50 molecules per second,
lower than about 30 molecules per second, lower than about 20
molecules per second, lower than about 10 molecules per second,
lower than about 5 molecules per second, lower than about 2
molecules per second, or lower than about 1 molecule per
second.
[0086] In some embodiments the slowest of the two or more
kinetically observable steps has a rate when measured under
single-molecule conditions of between about 500 to about 0.01
molecules per second, between about 200 to about 0.1 molecules per
second, between about 60 to about 0.5 molecules per second, about
30 molecules per second to about 2 molecules per second, or about
10 to about 3 molecules per second.
[0087] The ratio of the rates of each the two or more slow steps is
generally greater than 1:10, in some cases the ratio of the rates
is about 1:5, in some cases the ratio of the rates is about 1:2, in
some cases, the ratio of rates is about 1:1. The ratio can be
between about 1:10 and about 1:1, between about 1:5 and about 1:1,
or between about 1:2 and about 1:1.
[0088] A two or more slow-step system of the present invention can
be obtained by selecting the correct set of polymerase enzyme,
polymerase reaction conditions, and polymerase reaction
substrates.
II. SEQUENCING BY INCORPORATION
[0089] For sequencing processes that rely upon monitoring of the
incorporation of nucleotides into growing nascent strands being
synthesized by the complex, the progress of the reaction through
these steps is of significant importance. In particular, for
certain "real-time" nucleotide incorporation monitoring processes,
the detectability of the incorporation event is improved based upon
the amount of time the nucleotide is incorporated into and retained
within the synthesis complex during its ultimate incorporation into
a primer extension product.
[0090] By way of example, in certain exemplary processes, the
presence of the nucleotide in the synthesis complex is detected
either by virtue of a focused observation of the synthesis complex,
or through the use of interactive labeling techniques that produce
characteristic signals when the nucleotide is within the synthesis
complex. See, e.g., Levene, et al., Science 299:682-686, January
2003, and Eid, J. et al., Science, 323(5910), 133-138 (2009), the
full disclosures of which are incorporated herein by reference in
their entirety for all purposes.
[0091] In the first exemplary technique, as schematically
illustrated in FIG. 2, a nucleic acid synthesis complex, including
a polymerase enzyme 202, a template sequence 204 and a
complementary primer sequence 206, is provided immobilized within
an observation region 200, that permits illumination (as shown by
hv) and observation of a small volume that includes the complex
without excessive illumination of the surrounding volume (as
illustrated by dashed line 208). By illuminating and observing only
the volume immediately surrounding the complex, one can readily
identify fluorescently labeled nucleotides that become incorporated
during that synthesis, as such nucleotides are retained within that
observation volume by the polymerase for longer periods than those
nucleotides that are simply randomly diffusing into and out of that
volume.
[0092] In particular, as shown in panel II of FIG. 2, when a
nucleotide, e.g., A, is incorporated into by the polymerase, it is
retained within the observation volume for a prolonged period of
time, and upon continued illumination yields a prolonged
fluorescent signal (shown by peak 210). By comparison, randomly
diffusing and not incorporated nucleotides remain within the
observation volume for much shorter periods of time, and thus
produce only transient signals (such as peak 212), many of which go
undetected, due to their extremely short duration.
[0093] In particularly preferred exemplary systems, the confined
illumination volume is provided through the use of arrays of
optically confined apertures termed zero-mode waveguides, e.g., as
shown by confined reaction region 100 (ZMWs)(See, e.g., U.S. Pat.
No. 6,917,726, which is incorporated herein by reference in its
entirety for all purposes). For sequencing applications, the DNA
polymerase is provided immobilized upon the bottom of the ZMW (See,
e.g., Korlach et al., PNAS U.S.A. 105(4): 1176-1181. (2008), which
is incorporated herein by reference in its entirety for all
purposes.
[0094] In operation, the fluorescently labeled nucleotides (shown
as A, C, G and T) bear one or more fluorescent dye groups on a
terminal phosphate moiety that is cleaved from the nucleotide upon
incorporation. As a result, synthesized nucleic acids do not bear
the build-up of fluorescent labels, as the labeled polyphosphate
groups diffuses away from the complex following incorporation of
the associated nucleotide, nor do such labels interfere with the
incorporation event. See, e.g., Korlach et al., Nucleosides,
Nucleotides and Nucleic Acids, 27:1072:1083, 2008.
[0095] In the second exemplary technique, the nucleotides to be
incorporated are each provided with interactive labeling components
that are interactive with other labeling components provided
coupled to, or sufficiently near the polymerase (which labels are
interchangeably referred to herein as "complex borne"). Upon
incorporation, the nucleotide borne labeling component is brought
into sufficient proximity to the complex-borne (or complex
proximal) labeling component, such that these components produce a
characteristic signal event. For example, the polymerase may be
provided with a fluorophore that provides fluorescent resonant
energy transfer (FRET) to appropriate acceptor fluorophores. These
acceptor fluorophores are provided upon the nucleotide to be
incorporated, where each type of nucleotide bears a different
acceptor fluorophore, e.g., that provides a different fluorescent
signal. Upon incorporation, the donor and acceptor are brought
close enough together to generate energy transfer signal. By
providing different acceptor labels on the different types of
nucleotides, one obtains a characteristic FRET-based fluorescent
signal for the incorporation of each type of nucleotide, as the
incorporation is occurring.
[0096] In a related aspect, a nucleotide analog may include two
interacting fluorophores that operate as a donor/quencher pair or
FRET pair, where one member is present on the nucleobase or other
retained portion of the nucleotide, while the other member is
present on a phosphate group or other portion of the nucleotide
that is released upon incorporation, e.g., a terminal phosphate
group. Prior to incorporation, the donor and quencher are
sufficiently proximal on the same analog as to provide
characteristic signal, e.g., quenched or otherwise indicative of
energy transfer. Upon incorporation and cleavage of the terminal
phosphate groups, e.g., bearing a donor fluorophore, the quenching
or other energy transfer is removed and the resulting
characteristic fluorescent signal of the donor is observable.
[0097] In exploiting the foregoing processes, where the
incorporation reaction occurs too rapidly, it may result in the
incorporation event not being detected, i.e., the event speed
exceeds the detection speed of the monitoring system. The missed
detection of incorporated nucleotides can lead to an increased rate
of errors in sequence determination, as omissions in the real
sequence. In order to mitigate the potential for missed pulses due
to short reaction times, in one aspect, the current invention can
result in increased reaction time for incorporations. An advantage
of the methods, systems, and compositions that produce a two or
more slow-step process is an increased frequency of longer,
detectable, binding/incorporation events. This advantage may also
be seen as an increased ratio of longer, detectable pulses to
shorter, non-detectable pulses, where the pulses represent
binding/incorporation events.
[0098] Single-molecule sequencing often involves the optical
observation of the polymerase process during the process of
nucleotide incorporation, for example observation of the enzyme-DNA
complex. During this process, there are generally two or more
observable phases. For example, where a terminal-phosphate labeled
nucleotide is used, and the enzyme-DNA complex is observed, there
is a bright phase during the steps where the label is incorporated
with (bound to) the polymerase enzyme, and a dark phase where there
label is not incorporated with the enzyme. For the purposes of this
invention, both the dark phase and the bright phase are generally
referred to as observable phases, because the characteristics of
these phases can be observed.
[0099] Whether a phase of the polymerase reaction is bright or dark
can depend, for example, upon how and where the components of the
reaction are labeled, and also how the reaction is observed. For
example, as described above, the phase of the polymerase reaction
where the nucleotide is bound can be bright where the nucleotide is
labeled on its terminal phosphate. However, where there is a
quenching dye associated with the enzyme or template, the bound
state may be quenched, and therefore be a dark phase. Analogously,
in a ZMW, or other optically confined configuration, the release
and diffusion away of the label-bearing terminal phosphate may
result in a dark phase, whereas in other systems, the release of
the terminal phosphate may be observable, and therefore constitute
a bright phase.
[0100] For example, consider again the reaction scheme of FIG. 1 in
the context of the sequencing by incorporation embodiment described
above which utilizes nucleotides having labels on their terminal
phosphates. For this system, intermediates PDN, P*DN,
P*D.sub.+1PP.sub.i, and PD.sub.+1PP.sub.i would all represent
bright states of a bright phase because for each of these
intermediates, the label is associated with the polymerase enzyme.
In contrast, intermediates PD.sub.+1 and PD correspond to dark
states of a dark phase, because for these intermediates, no dye is
associated with the polymerase enzyme. In one aspect of the
invention, any two of the steps which proceed from a bright
intermediate, e.g. steps 106, 108, 110, and 112 of FIG. 1 are slow.
By having two or more bright steps that are partially
rate-limiting, the relative number of pulses with a longer pulse
width, and/or detectable incorporation events increases.
[0101] Another example of a polymerase reaction with distinct
observable phases is one in which the nucleotide is labeled such
that its label does not dissociated from the enzyme upon product
release, for example where the nucleotide is labeled on the base or
on the sugar moiety. Here, the phase in which the label is
associated with the active site of the enzyme (bright or dark) may
extend past product release until translocation. For this example,
an observable phase may extend from nucleotide binding until
translocation.
[0102] In addition, the systems of the present invention may have
two or more different distinct bright phases, for example, phases
that can be distinguished based on different colors, e.g. different
fluorescent emission wavelengths in the different observable
phases. For all of these cases, we have discovered that it can be
advantageous to have more than one rate-limiting (kinetically
observable) step within a phase. Having more than one rate-limiting
step within a phase can result in a distribution of pulse widths
having relatively fewer undetectable or poorly detectable short
pulses.
[0103] While not being bound by theory, we provide the following
theoretical basis for obtaining improved single-molecule sequencing
results by using a system having two or more slow steps within an
observable phase. While described here for nucleic acid
polymerization, it will be appreciated that the two slow step
systems of the invention can also be used for improved observation
of other enzyme systems. A model for the effect of two slow steps
on the probability density for residence time is described herein.
FIG. 3 shows a plot of calculated probability density for residence
time for cases in which (1) one step is rate-limiting and (2) two
equivalent partially rate-limiting (slow) steps are present for the
observable phase in which the nucleotide is associated with the
enzyme.
[0104] For the case in which one step is rate-limiting, the
probability distribution for the binding time can be represented by
the single exponential equation:
y=A.sub.0e.sup.-kt Eq. 1
[0105] This represents the case in which, for example,
incorporation of nucleotide into the growing nucleic acid (step 108
in FIG. 1) is the single slow step.
[0106] FIG. 3 illustrates that where one slow-step is present in
this phase, there is an exponentially decreasing probability of a
given residence time as the residence time increases, providing a
distribution in which there is a relatively high probability that
the residence time will be short.
[0107] For the case in which there are two slow steps in this
phase, for example where both the incorporation step (step 108 in
FIG. 1) and the release of product (PPi) step (step 112 in FIG. 1)
are slow, the probability density versus residence time can be
represented by a double exponential equation:
y=A.sub.0e.sup.-k.sup.1.sup.t-B.sub.0e.sup.-k.sup.2.sup.t Eq. 2
[0108] FIG. 3 illustrates that for the case in where there are two
slow steps, the probability of very fast residence times is
relatively low as compared to the case having one slow step. In
addition, the probability distribution for two slow steps exhibits
a peak in the plot of probability density versus residence time.
This type of residence time distribution can be advantageous for
single-molecule sequencing where it is desired to measure a high
proportion of binding events and where fast binding events may be
unreliably detected.
[0109] Typically, for a given illumination/detection system there
will be a minimum detection time below which events, such as
binding events, will be unreliably detected or not detected at all.
This minimum detection time can be attributed, for example, to the
frame acquisition time or frame rate of the optical detector, for
example, a CCD camera. A discussion of detection times and
approaches to detection for these types of systems is provided in
U.S. patent application Ser. No. 12/351,173 the full disclosures of
which are incorporated herein by reference in their entirety for
all purposes. FIG. 3 includes a line which indicates a point where
the residence time equals a minimum detection time (Tmin). The area
under the curve in the region below Tmin represents the population
of short pulses which will not be accurately detected for this
system. It can be seen from FIG. 3 that the relative proportion of
binding times that fall below Tmin is significantly lower for the
case in which the reaction exhibits two slow steps as compared to
the case where the reaction exhibits one slow step.
[0110] Thus, as described above, one aspect of the invention
relates to methods, systems, and compositions for performing
nucleic acid sequencing with a nucleic acid synthesis reaction in
which the reaction exhibits two or more slow steps within a bright
phase. In addition, an aspect of the invention relates to nucleic
acid synthesis reactions having two or more slow states wherein
each of the slow steps proceeds from a state in which the labeled
component is associated with the polymerase enzyme.
[0111] In some embodiments of the invention, the two or more slow
steps are within a dark phase. In some cases the two or more slow
steps proceed from states in which the labeled component is not
associated with the enzyme. Having two or more slow states that
proceed from a dark intermediate can be advantageous, for example,
for lowering the frequency of events having a very short dark state
or having a very short interpulse distance. The advantage of this
type of system can be demonstrated by again considering FIG. 1 in
the context of the sequencing by incorporation embodiment described
above which utilizes nucleotides having labels on their terminal
phosphates. In this system, intermediates PD.sub.+1 and PD can
correspond to dark states within a dark phase, for example in a
ZMW, because for these intermediates, no dye is associated with the
polymerase enzyme.
[0112] The steps that comprise the two slow steps can include, for
example, nucleotide addition, enzymatic isomerization such as to or
from a closed state, cofactor binding or release, product release,
incorporation of nucleic acid into the growing nucleic acid, or
translocation.
(i) Determining Whether the Polymerase System Exhibits Two Slow
Steps
[0113] In some cases the presence of two slow steps can be
ascertained by the characteristics of the polymerase reaction run
under single-molecule sequencing conditions, for example by
measuring the distribution of pulse widths. For example, a
distribution of pulse widths can be determined using systems
described herein where the components of the system are labeled
such that a bright state is observed during nucleotide binding, and
a dark state is observed from after product release until the next
nucleotide binding event. Under these conditions a bright pulse
will be observed that corresponds to bound nucleotide. The width of
the pulse corresponds to the amount of time that the nucleotide is
bound. By measuring the width of a number of pulses, corresponding
to a number of nucleotide incorporation events, a distribution of
pulse widths can be obtained. From this distribution of pulse
widths, in some cases, it can be determined that a polymerase
reaction having two slow steps is occurring, and in particular, a
polymerase reaction having two slow steps during the bright state
during which the nucleotide is associated with the polymerase
enzyme. The use of a distribution of pulses to determine a kinetic
mechanism having two slow (kinetically observable) steps is
described, for example, in Miyake et al. Analytical Chemistry 2008
80 (15), 6018-6022. The determination of the steps in a multistep
reaction such as a polymerase reaction is described, for example,
in Zhou, et al. J. Phys. Chem. B, 2007, 111, 13600-13610.
[0114] Analogously, the presence of two slow steps in the dark
phase of a polymerase reaction can in some cases be detected by
determining the distribution of the time between pulses (interpulse
time). Where the system exhibits two slow steps, a distribution
described by a double exponential can be seen.
[0115] In some cases, it is not possible or not practical to
determine under single-molecule conditions whether a system is
exhibiting two slow-step kinetics. For example, in some cases, the
frame time of the detection optics will be slow enough that a
significant number of pulses or interpulse times are not detected,
precluding a reliable determination of pulse width or interpulse
time distribution. In some cases, the short pulses are not detected
because the short pulses generally have a smaller number of
photons, making the pulses difficult to detect even were a short
camera frame time is available. In such cases, the presence of two
slow-step kinetics under such polymerase reaction conditions can be
determined by running a reaction under substantially the same
polymerase reaction conditions, but not under single-molecule
conditions. For example, a reaction can be run under substantially
the same polymerase reaction conditions as the single-molecule
sequencing system, but with a higher concentration of polymerase
enzyme and in some cases, a higher concentration of primer and/or
template nucleotide. The reaction run under substantially the same
polymerase reaction conditions, but with higher concentrations of
polymerase enzyme, primer, and/or template can be used to determine
whether the system shows two slow steps as described herein. The
reaction to determine two slow-step kinetics may have labels on
different components of the reaction than that for single-molecule
sequencing, such as having labels on the template nucleic acid.
[0116] For example, a stopped-flow reaction such as described in
the examples below can be used to determine whether the polymerase
reaction conditions exhibit two slow steps. As described in the
examples, stopped-flow experiments can be used to establish that
the polymerase reaction is exhibiting two slow step kinetics either
in a bright phase or in a dark phase for single-molecule
sequencing.
[0117] A higher enzyme/primer/template concentration reaction such
as a stopped-flow reaction can be used to identify systems having
two slow steps for single-molecule sequencing. Alternatively, the
reaction run under substantially the same conditions but higher
concentration of enzyme/primer/template can be used to verify that
a single-molecule sequencing system is being carried out under
polymerase reaction conditions that exhibit two slow steps.
A. Polymerase Enzyme
[0118] One important aspect of obtaining a two slow-step system of
the invention is selection of the enzyme that is used. The
polymerase enzyme can be modified in a manner in which the relative
rates of the steps of the polymerase reactions are changed such
that the enzyme will be capable of showing two slow-step
characteristics. Recombinant enzymes useful in the present
invention are described, for example, in copending U.S. Patent
Application [unassigned] entitled "Generation of Modified
Polymerases for Improved Accuracy in Single-molecule Sequencing",
docket number 105-004901US, filed Mar. 30, 2009.
[0119] A modified polymerase (e.g., a modified recombinant
.PHI.29-type DNA polymerase for example, a modified recombinant
.PHI.29, B103, GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1,
PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase) that
exhibits one or more slow steps optionally includes a mutation
(e.g., an amino acid substitution or insertion) at one or more of
positions 484, 249, 179, 198, 211, 255, 259, 360, 363, 365, 370,
372, 378, 381, 383, 387, 389, 393, 433, 478, 480, 514, 251, 371,
379, 380, 383, 458, 486, 101, 188, 189, 303, 313, 395, 414, 497,
500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392, 402, 422,
496, 529, 538, 555, 575, 254, 390, 372-397, and 507-514, where
numbering of positions is relative to wild-type .PHI.29 polymerase.
For example, relative to wild-type .PHI.29 a modified recombinant
polymerase can include at least one amino acid substitution or
combination of substitutions selected from the group consisting of:
an amino acid substitution at position 484; an amino acid
substitution at position 198; an amino acid substitution at
position 381; an amino acid substitution at position 387 and an
amino acid substitution at position 484; an amino acid substitution
at position 372, an amino acid substitution at position 480, and an
amino acid substitution at position 484; an amino acid substitution
at position 372, an amino acid substitution at position 387, and an
amino acid substitution at position 480; an amino acid substitution
at position 372, an amino acid substitution at position 387, and an
amino acid substitution at position 484; an amino acid substitution
at position 372, an amino acid substitution at position 387, an
amino acid substitution at position 478, and an amino acid
substitution at position 484; A484E; A484Y; N387L; T372Q; T372Y;
T372Y and K478Y; K478Y; 1370W; F198W; L381A; T368F; A484E, E375Y,
K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L, E375Y,
K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y,
K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; 1370W,
E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A,
E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F. A K512F
substitution (or K512W, K512L, K512I, K512V, K512H, etc.) is
optionally employed, e.g., where a K512Y substitution is listed
herein. As another example, the modified polymerase can include an
insertion of at least one amino acid (e.g., 1-7 amino acids, e.g.,
glycine) within residues 372-397 and/or 507-514. For example, a
glycine residue can be introduced after residue 374, 375, 511,
and/or 512 (designated as 374.1G, 375.1G, etc.). In some
embodiments the enzyme has one or more of the amino acid
substitutions E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q,
T372L, K478Y, 1370W, F198W, and L381A.
[0120] A list of exemplary mutations and combinations thereof is
provided in Table 1, and additional exemplary mutations are
described herein. Essentially any of these mutations, or any
combination thereof, can be introduced into a polymerase to produce
a modified recombinant polymerase (e.g., into wild-type .PHI.29, an
exonuclease deficient .PHI.29-type polymerase, and/or
E375Y/K512Y/T368F .PHI.29, as just a few examples).
TABLE-US-00001 TABLE 1 Mutation Rationale D249E metal coordination
A484E metal coordination D249E/A484E metal coordination A484D metal
coordination A484H metal coordination A484Y metal coordination
D249E/A484D metal coordination D249E/A484H metal coordination
D249E/A484Y metal coordination 374.1G/375.1A dye interaction
374.1Gins/375.1Gins dye interaction V514Y dye interaction V514F dye
interaction 511.1G/K512Y/512.1G dye interaction T372H closed
conformation of fingers T372V closed conformation of fingers T372I
closed conformation of fingers T372F closed conformation of fingers
T372Y closed conformation of fingers T372N closed conformation of
fingers T372Q closed conformation of fingers T372L closed
conformation of fingers T372L/K478Y closed conformation of fingers
T372Y/K478Y closed conformation of fingers T372Y/K478L closed
conformation of fingers K478Y closed conformation of fingers D365N
closed conformation of fingers D365Q closed conformation of fingers
L480H closed conformation of fingers L480F closed conformation of
fingers L381A closed conformation of finger and exo I179A closed
conformation of finger and exo I378A closed conformation of finger
and exo I179A/L381A closed conformation of finger and exo
I179A/I378A/L381A closed conformation of finger and exo I370A/I378A
closed conformation of finger and exo I179A/I370A/I378A/L381A
closed conformation of finger and exo I179W closed conformation of
finger and exo I179H closed conformation of finger and exo F211A
closed conformation of finger and exo F211W closed conformation of
finger and exo F211 H closed conformation of finger and exo F198A
closed conformation of finger and exo F198W closed conformation of
finger and exo F198H closed conformation of finger and exo P255A
closed conformation of finger and exo P255W closed conformation of
finger and exo P255H closed conformation of finger and exo Y259A
closed conformation of finger and exo Y259W closed conformation of
finger and exo Y259H closed conformation of finger and exo F360A
closed conformation of finger and exo F360W closed conformation of
finger and exo F360H closed conformation of finger and exo F363A
closed conformation of finger and exo F363H closed conformation of
finger and exo F363W closed conformation of finger and exo I370W
closed conformation of finger and exo I370H closed conformation of
finger and exo K371A closed conformation of finger and exo K371W
closed conformation of finger and exo I378H closed conformation of
finger and exo I378W closed conformation of finger and exo L381W
closed conformation of finger and exo L381H closed conformation of
finger and exo K383N closed conformation of finger and exo K383A
closed conformation of finger and exo L389A closed conformation of
finger and exo L389W closed conformation of finger and exo L389H
closed conformation of finger and exo F393A closed conformation of
finger and exo F393W closed conformation of finger and exo F393H
closed conformation of finger and exo I433A closed conformation of
finger and exo I433W closed conformation of finger and exo I433H
closed conformation of finger and exo K383L phosphate backbone
interaction K383H phosphate backbone interaction K383R phosphate
backbone interaction Q380R phosphate backbone interaction Q380H
phosphate backbone interaction Q380K phosphate backbone interaction
K371L phosphate backbone interaction K371H phosphate backbone
interaction K371R phosphate backbone interaction K379L phosphate
backbone interaction K379H phosphate backbone interaction K379R
phosphate backbone interaction E486A phosphate backbone interaction
E486D phosphate backbone interaction N387L incoming nucleotide base
and translocation N387F incoming nucleotide base and translocation
N387V incoming nucleotide base and translocation N251H phosphate
interaction N251Q phosphate interaction N251D phosphate interaction
N251E phosphate interaction N251K phosphate interaction N251R
phosphate interaction A484K phosphate interaction A484R phosphate
interaction K383A phosphate interaction K383N phosphate interaction
K383T phosphate interaction K383S phosphate interaction K383A
phosphate interaction I179H/I378H closed conformation I179W/I378W
closed conformation I179Y/I378Y closed conformation K478L I378Y
I370A I179Y N387L/A484E N387L/A484Y T372Q/N387L/A484E
T372Q/N387L/A484Y T372L/N387L/A484E T372L/N387L/K478Y/A484Y
T372Y/N387L/K478Y/A484E T372Y/N387L/K478Y/A484Y
[0121] Table 2 presents exemplary .PHI.29 mutants that can exhibit
two slow step behavior under appropriate reaction conditions. The
first three modified polymerases exhibit the most pronounced two
slow step behavior, followed by the next six. As noted, the
polymerases are optionally exonuclease-deficient; for example, they
can also include an N62D substitution.
TABLE-US-00002 TABLE 2 A484E/E375Y/K512Y/T368F
A484Y/E375Y/K512Y/T368F N387L/E375Y/K512Y/T368F
T372Q/E375Y/K512Y/T368F T372L/E375Y/K512Y/T368F
T372Y/K478Y/E375Y/K512Y/T368F I370W/E375Y/K512Y/T368F
F198W/E375Y/K512Y/T368F L381A/E375Y/K512Y/T368F
E375Y/K512Y/T368F
[0122] Compositions, kits, and systems (e.g., sequencing systems)
including the modified recombinant polymerases with decreased rate
constants are features of the invention, as are methods employing
the modified polymerases (e.g., methods of sequencing or making
DNA). Methods for generating recombinant polymerases are also
featured, as described in greater detail below, as are the
resulting polymerases. Thus, one aspect provides a modified
recombinant .PHI.29-type DNA polymerase comprising one or more
mutations (e.g., amino acid substitutions or insertions) relative
to a parental polymerase at one or more positions selected from the
group consisting of: a) positions that form a binding site for a
metal ion that interacts with an epsilon and/or digamma phosphate
of a bound nucleotide analog having five or more phosphate groups;
b) positions 372-397 and 507-514; c) positions that form a binding
site for a terminal fluorophore on a phosphate-labeled nucleotide
analog, particularly hexaphosphate analogs; d) positions at an
intramolecular interface in a closed conformation of a ternary
complex comprising the polymerase, a DNA, and a nucleotide or
nucleotide analog; e) positions that form a binding site for a
polyphosphate group of a bound nucleotide or nucleotide analog; f)
positions that interact with the base of a bound nucleotide or
nucleotide analog; and g) positions that interact with a bound DNA;
wherein numbering of positions is relative to wild-type .PHI.29
polymerase. Preferably, the one or more mutations comprise at least
one mutation other than a 514Y, 514W, 514F, 5141, 514K, 259S, 370V,
370K, 372D, 372E, 372R, 372K, 372N, 372L, 387A, 387D, 478D, 478E,
478R, 480K, 480M, 480R, 371Q, 379E, 379T, 486D, 486A, 188A, 188S,
254F, 254V, 254A, 390F, or 390A substitution. The modified
polymerase optionally exhibits a decreased first rate constant,
balanced first and second rate constants, and the like as for the
embodiments described above.
[0123] A number of relevant positions and mutations are described
herein. For example, the modified polymerase can comprise at least
one amino acid substitution at at least one residue selected from
the group consisting of positions 484, 249, 179, 198, 211, 255,
259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433,
478, 480, 514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189,
303, 313, 395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574,
64, 305, 392, 402, 422, 496, 529, 538, 555, 575, 254, and 390.
Exemplary modified polymerases include those with at least one
amino acid substitution or combination of substitutions selected
from the group consisting of: an amino acid substitution at
position 484; an amino acid substitution at position 198; an amino
acid substitution at position 381; A484E; A484Y; N387L; T372Q;
T372Y; T372Y and K478Y; K478Y; 1370W; F198W; L381A; T368F; A484E,
E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L,
E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L,
E375Y, K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F;
1370W, E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F;
L381A, E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F, as
well as others described herein. As another example, the modified
polymerase can include an insertion of at least one amino acid
(e.g., 1-7 amino acids, e.g., glycine) within residues 372-397
and/or 507-514 (e.g., after residue 374, 375, 511, and/or 512).
[0124] The polymerase mutations and mutational strategies noted
herein can be combined with each other and with essentially any
other available mutations and mutational strategies to confer
additional improvements in, e.g., nucleotide analog specificity,
enzyme processivity, improved retention time of labeled nucleotides
in polymerase-DNA-nucleotide complexes, and the like. For example,
the mutations and mutational strategies herein can be combined with
those taught in, e.g., WO 2007/076057 POLYMERASES FOR NUCLEOTIDE
ANALOGUE INCORPORATION by Hanzel et al. and WO 2008/051530
POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACID
SEQUENCING by Rank et al. This combination of mutations/mutational
strategies can be used to impart several simultaneous improvements
to a polymerase (e.g., decreased branch fraction formation,
improved specificity, improved processivity, altered rates,
improved retention time, improved stability of the closed complex,
etc.). In addition, polymerases can be further modified for
application-specific reasons, such as to improve activity of the
enzyme when bound to a surface, as taught, e.g., in WO 2007/075987
ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al. and WO
2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF
SURFACE ATTACHED PROTEINS by Hanzel et al., or to include
purification or handling tags as is taught in the cited references
and as is common in the art.
[0125] Specific mutations noted herein can be used alone or in
combination with each other and/or with available mutations as
described in the references noted above, or can be used in
polymerases that lack such previously described mutations. As just
one example, essentially any mutation or combination thereof noted
herein can be introduced into an E375Y/K512Y/T368F .PHI.29
polymerase, optionally, an exonuclease-deficient E375Y/K512Y/T368F
.PHI.29 polymerase.
[0126] For example, enzymological approaches have been reported for
enhancing the reaction kinetics of the polymerization reaction
(See, e.g., published U.S. Patent Application Nos. 2007-0196846 and
2008-0108082, and Provisional Patent Application 61/094,843, the
full disclosures of which are incorporated herein by reference in
their entirety for all purposes), to increase the residence time of
an incorporating nucleotide in the active site of a polymerase.
While such reactions yield improvements in detectability of a bound
nucleotide, and thus, an incorporation event, for a number of
circumstances, it has been shown that increasing the retention time
of a nucleotide complexed with a polymerase, also results in an
increased likelihood that the nucleotide will be released
unproductively.
B. Polymerase Reaction Conditions
[0127] The polymerase reaction conditions can also be important for
obtaining a two slow-step enzyme system. In particular, polymerase
reaction conditions include components selected to produce two
slow-step kinetics. The polymerase reaction conditions include the
type and concentration of buffer, the pH of the reaction, the
temperature, the type and concentration of salts, the presence of
particular additives which influence the kinetics of the enzyme,
and the type, concentration, and relative amounts of various
cofactors, including metal cofactors. The term "polymerase reaction
conditions" as used herein generally excludes the concentration of
the polymerase enzyme or the concentration of the primer-template
complex. Thus, two reactions are run under substantially the same
polymerase reaction conditions where the first reaction has a small
amount of polymerase enzyme, such as a single polymerase enzyme,
and a small amount of primer template complex, such as a single
primer-template complex associated with a single polymerase enzyme,
and the second reaction has a higher concentration of polymerase
enzyme, for example a concentration of polymerase enzyme of about
0.05 .mu.M to 0.5 .mu.M and about 0.01 .mu.M to about 0.1
.mu.M.
[0128] It some embodiments the type and concentration of buffer are
chosen in order to produce a reaction having two slow steps.
Enzymatic reactions are often run in the presence of a buffer,
which is used, in part, to control the pH of the reaction mixture.
We have found that in some cases the type of buffer can influence
the kinetics of the polymerase reaction in a way that can lead to
two slow-step kinetics. For example, in some cases, we have found
that the use of TRIS as buffer is useful for obtaining a two
slow-step reaction. Buffers suitable for the invention include, for
example, TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic
acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), TRIS
(tris(hydroxymethyl)methylamine), ACES
(N-(2-Acetamido)-2-aminoethanesulfonic acid), Tricine
(N-tris(hydroxymethyl)methylglycine), HEPES
4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES
(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), PIPES
(piperazine-N,N'-bis(2-ethanesulfonic acid)), and MES
(2-(N-morpholino)ethanesulfonic acid).
[0129] The pH of the reaction can influence the kinetics of the
polymerase reaction, and can be used as one of the polymerase
reaction conditions to obtain a reaction exhibiting two slow-step
kinetics. The pH can be adjusted to a value that produces a two
slow-step reaction mechanism. The pH is generally between about 6
and about 9. In some cases, the pH is between about 6.5 and about
8.0. In some cases, the pH is between about 6.5 and 7.5. In some
cases, the pH is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,
7.4, or 7.5.
[0130] The temperature of the reaction can be adjusted in order to
obtain a reaction exhibiting two slow-step kinetics. The reaction
temperature may depend upon the type of polymerase which is
employed. Temperatures between 15.degree. C. and 90.degree. C.,
between 20.degree. C. and 50.degree. C., between 20.degree. C. and
40.degree. C., or between 20.degree. C. and 30.degree. C. can be
used.
[0131] In some cases, additives can be added to the reaction
mixture that will change the kinetics of the polymerase reaction in
a manner that can lead to two slow-step kinetics. In some cases,
the additives can interact with the active site of the enzyme,
acting for example as competitive inhibitors. In some cases,
additives can interact with portions of the enzyme away from the
active site in a manner that will change the kinetics of the
reaction so as to produce a reaction exhibiting two slow steps.
Additives that can influence the kinetics include, for example,
competitive, but otherwise unreactive substrates or inhibitors in
analytical reactions to modulate the rate of reaction as described
in copending U.S. Utility patent application Ser. No. 12/370,472
the full disclosures of which is incorporated herein by reference
in its entirety for all purposes.
[0132] One aspect of the invention is the use of a kinetic isotope
effect, such as the addition of deuterium to the system in order to
control the kinetics of the polymerase reaction in single-molecule
sequencing. In some cases, the isotope, such as deuterium can be
added to influence the rate of one or more step in the polymerase
reaction for improving single-molecule sequencing. In some cases,
the deuterium can be used to slow one or more steps in the
polymerase reaction due to the deuterium isotope effect. By
altering the kinetics of steps of the polymerase reaction, in some
instances, two slow-step kinetics, as described herein, can be
achieved. As described in the examples below, in some cases, the
addition of deuterium can be used to increase the mean pulse width
in a single-molecule sequencing system.
[0133] The substitution of deuterium for hydrogen in a chemical
reaction such as the polymerase reaction can result in a change in
the kinetics of the reaction. An isotopic substitution can
significantly modify the reaction rate when the isotopic
replacement is in a chemical bond that is broken or formed in the
rate-limiting step. In such a case, the change is generally termed
a primary isotope effect. When the substitution is not involved in
the bond that is breaking or forming, a smaller rate change,
generally termed a secondary isotope effect can be observed. The
magnitude of the kinetic isotope effect has been used to elucidate
reaction mechanisms. If other steps are partially rate-determining,
the effect of isotopic substitution can be masked. The presence of
a deuterium isotope effect for polymerase enzymes has been
described in Castro et al., PNAS, 104(11), 4267-4272 (2007), the
full disclosure of which is incorporated here by reference in its
entirety for all purposes. We describe here the use of a kinetic
isotope effect to control the kinetics of a polymerase reaction for
single-molecule sequencing, for example to improve the accuracy of
sequencing by influencing the characteristics of the light pulses
which are measured. The deuterium isotope effect could be used, for
example, to control the rate of incorporation of nucleotide, for
example by slowing the incorporation rate.
[0134] The amount of deuterium isotope that is substituted for
hydrogen can be used to control the characteristics of the
reaction. For example, in some cases, the more deuterium that is
added, the more of a rate effect on a given polymerase step can be
obtained. In some cases, the deuterium is added to a readily
exchangeable proton/deuterium position, such as to water, a
hydroxyl or a carboxylic acid proton/deuterium. In these positions,
the proton/deuterium in the system would be expected to rapidly
exchange. In other cases, the deuterium could be added to a
position that experiences less exchange, such as, for example, a
carbon-hydrogen bond alpha to a hydroxyl group. In some cases, the
use of a statistical mixture of D.sub.2O/H.sub.2O is advantageous.
For example, it allows one to change the incorporation rate and
therefore the nucleotide residence time while keeping the other
conditions of the polymerase reaction relatively unchanged. The
volume percent of deuterium substituted for hydrogen can be, for
example about 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70,
75, 80, 90, 95, 99 percent or higher. In some cases, the amount of
deuterium can be expressed as the percent of D.sub.2O out of the
total D.sub.2O plus H.sub.2O. In some cases, a range of D2O can be
between about 1% and about 80%, between about 10% and about 60%,
between about 20% and about 40%, or between about 20% and 30%. In
some cases, adding a high level of deuterium can slow the reaction
so as to diminish the yield of the polymerase reaction. The amount
of deuterium can be controlled in order to improve the accuracy
while retaining acceptable yield.
[0135] Other isotopes than deuterium can be used to control
single-molecule sequencing. For example, isotopes of carbon (e.g.
.sup.13C), nitrogen, oxygen, sulfur, or phosphorous could also be
used.
[0136] Additives that can be used to control the kinetics of the
polymerase reaction include the addition of organic solvents. The
solvent additives are generally water soluble organic solvents. The
solvents need not be soluble at all concentrations, but are
generally soluble at the amounts used to control the kinetics of
the polymerase reaction. While not being bound by theory, it is
believed that the solvents can influence the three dimensional
conformation of the polymerase enzyme which can affect the rates of
the various steps in the polymerase reaction. For example, the
solvents can provide affect steps involving conformational changes
such as the isomerization steps shown in FIG. 1. Added solvents can
also affect, and in some cases slow, the translocation step. The
slowing of the translocation step can increase interpulse
distances, and can be used in conjunction with slowing the
nucleotide binding step, for example, to obtain two slow steps in
the steps in which the nucleotide is not associated with the
enzyme, for instance resulting in two slow steps in the dark phase
of a polymerase reaction. In some cases, the solvent additives can
increase the interpulse distance without substantially affecting
the pulse widths in single-molecule sequencing. In some cases, the
solvents act by influencing hydrogen bonding interactions. In some
case, the addition of solvent can be used to change the rate of one
or more steps in the polymerase reaction. For example, the solvent
may slow one or more steps in the polymerase reaction. By
influencing the rates of various steps of the polymerization, the
solvent additives can be used, in some cases, to obtain two
slow-step kinetics. The addition of organic solvents can be used,
for example to increase the mean time between pulses (interpulse
distance).
[0137] The water miscible organic solvents that can be used to
control the rates of one or more steps of the polymerase reaction
in single-molecule sequencing include alcohols, amines, amides,
nitriles, sulfoxides, ethers, and esters and small molecules having
more than one of these functional groups. Exemplary solvents
include alcohols such as methanol, ethanol, propanol, isopropanol,
glycerol, and small alcohols. The alcohols can have one, two,
three, or more alcohol groups. Exemplary solvents include small
molecule ethers such as tetrahydrofuran (THF), and dioxane. In some
embodiments the solvent is dimethylacetamide (DMA). In some
embodiments the solvent is dimethylsulfoxide (DMSO). In some
embodiments, the solvent is dimethylformamide (DMF). In some
embodiments the solvent is acetonitrile.
[0138] The water miscible organic solvent can be present in any
amount sufficient to control the kinetics of the polymerase
reaction. The solvents are generally added in an amount less than
40% of the solvent weight by weight or volume by volume. In some
embodiments the solvents are added between about 0.1% and 30%,
between about 1% and about 20%, between about 2% and about 15%, and
between about 5% and 12%. The effective amount for controlling the
kinetics can be determined by the methods described herein and
those known in the art.
[0139] A suitable additive for obtaining a two slow-step system is
the amino acid, cysteine, having the chemical formula
HO.sub.2CCH(NH.sub.2)CH.sub.2SH. Cysteine can be added to the
reaction mixture as a salt, for example, as the hydrochloride salt.
Generally, the naturally occurring L-cysteine (Cys) is used. Other
additives with chemical structures related to cysteine can also be
used. For example, homocysteine or any other suitable natural or
artificial amino acid having an S atom, and in particular, a thiol
group. We have found that the addition of cysteine can lead to an
increase in both overall yield and in accuracy of single molecule
sequencing. While not being bound by theory, Cys, because of its
thiol side chain and AA polar moiety may have beneficial effects on
both polymerase and nucleotides during sequencing. An increase in
the pulse width with the addition of Cys has also been observed.
The effect could be different from or cumulative to that of
dithiothreitol (DTT), which can also be added to the sequencing
reaction, owing to only a single --SH functionality in Cys and,
therefore, larger tendency to participate in intermolecular
interactions. In addition, Cys may influence the analog binding to
polymerase via linking the two with hydrogen and S--S bonds.
Cysteine can be added at any level suitable for improving the
properties of the enzymatic reaction. For example, cysteine can be
added at amounts greater than about 0.1 mM, greater than about 0.5
mM, greater than about 1 mM, greater than about 5 mM, greater than
about 10 mM. In some cases, the cysteine can be added in amounts
less than about 200 mM, less than about 100 mM, less than about 50
mM, less than about 20 mM, or less than about 10 mM. In some cases,
the cysteine is present in amounts between about 1 mM and about 100
mM, between about 5 mM and about 50 mM, or between about 10 mM and
about 30 mm.
[0140] Additives such as dithiothreitol (DTT), can also be present
in the reaction. In some cases, such additives, which are often
used in enzymatic systems, do not directly lead to two slow-step
systems, but are useful for the functioning of the enzyme during,
for example, nucleic acid synthesis.
[0141] One aspect of controlling the polymerase reaction conditions
relates to the selection of the type, level, and relative amounts
of cofactors. For example, during the course of the polymerase
reaction, divalent metal co-factors, such as magnesium or
manganese, will interact with the enzyme-substrate complex, playing
a structural role in the definition of the active site. For a
discussion of metal co-factor interaction in polymerase reactions,
see, e.g., Arndt, et al., Biochemistry (2001) 40:5368-5375.
[0142] For example, and without being bound to any particular
theory of operation, it is understood that metal cofactor binding
in and around the active site serves to stabilize binding of
incoming nucleotides and is required for subsequent catalysis,
e.g., as shown in steps 106 and 108. Other metal cofactor binding
sites in polymerases, e.g., in the exonuclease domains, are
understood to contribute to different functionality of the overall
proteins, such as exonuclease activity.
[0143] In the context of the present invention, however, it has
been discovered that modulation, and particularly competitive
modulation of divalent metal cofactors to the synthesis reaction
can provide substantial benefits in terms of reaction kinetics
without a consequent increase in negative reaction events.
[0144] In the synthesis reaction, certain divalent or trivalent
metal cofactors, such as magnesium and manganese are known to
interact with the polymerase to modulate the progress of the
reaction (See, e.g., U.S. Pat. No. 5,409,811). Other divalent metal
ions, such as Ca.sup.2+, have been shown to interact with the
polymerase, such as phi29 derived polymerases, to negative effect,
e.g., to halt polymerization. As will be appreciated, depending
upon the nature of the polymerization reaction, environmental
conditions, the polymerase used, the nucleotides employed, etc.,
different metal co-factors will have widely varying catalytic
effects upon the polymerization reaction. In the context of the
present invention, different metal co-factors will be referred to
herein based upon their relative catalytic impact on the
polymerization reaction, as compared to a different metal included
under the same reaction conditions. For purposes of discussion, a
first metal co-factor that interacts with the polymerase complex to
support the polymerization reaction to a higher level than a second
metal co-factor under the same conditions is termed a "catalytic
metal ion" or "catalytic metal". In preferred aspects, such
catalytic metals support the continued, iterative or processive
polymerization of nucleic acids under the particular polymerase
reaction conditions, e.g., through the addition on multiple bases,
while in some cases, a given type of metal cofactor may only
support addition of a single base. Such metals may be sufficiently
catalytic, depending upon the specific application.
[0145] In certain cases, particularly preferred divalent metal ions
or catalytic metals, include, e.g., Mn.sup.2+, and in some cases
will include Mg.sup.2+. Less preferred multivalent metal ions that
may provide a sufficient level of catalytic activity depending upon
the desired application include, e.g., zinc.
[0146] For purposes of the invention, metal ions that interact with
the polymerase, but that do not promote the polymerization
reaction, and in many cases act to arrest or prevent
polymerization, are termed "non-catalytic metals". Included among
the non-catalytic metals for various polymerase systems are
calcium, barium, strontium, iron, cobalt, nickel, tin, zinc, and
europium. For example, these metals can be added to the
polymerization reaction in salt form such as Sr(OAc).sub.2,
Sr(OAc).sub.2, CoCl.sub.2, SnCl.sub.2, CaCl.sub.2, or ZnSO.sub.4.
As will be appreciated, a first metal co-factor that might be
deemed to be catalytic under a first set of reaction conditions or
relative to second metal co-factor, may be deemed to be a
non-catalytic metal under another different set of reaction
conditions, or with respect to a third metal co-factor. By way of
example, as noted previously, magnesium is generally known to
support DNA polymerization. However, under certain conditions,
and/or relative to manganese, magnesium can operate as a
non-catalytic co-factor. For purposes of the present invention, a
catalytic co-factor will support polymerization to a greater degree
than the non-catalytic metal under the same reaction conditions.
The relative catalytic impact will typically be a function of the
reactant turnover rate of the polymerization complex, with
catalytic metal co-factors promoting a turnover that is at least
2.times., more preferably at least 5.times., still more preferably,
at least 10.times., and in some cases 20.times., 50.times. or more
than that of the non-catalytic metal co-factor under the same
reaction conditions. Accordingly, in the context of various aspects
of the invention, the polymerization complex is exposed to two
different co-factors that have substantially different impacts on
the polymerization reaction under the given set of reaction
conditions, where the first metal co-factor promotes polymerization
to a substantially greater degree than the second metal co-factor,
or restated in the negative context, the second metal co-factor
arrests or halts polymerization to a substantially greater degree
than the first.
[0147] In particular, and without being bound to any particular
theory of operation, it is believed that the presence of a
non-catalytic metal in the polymerase complex, through binding in
or around the active site, results in the inability for the
synthesis reaction to proceed out of the complexed state. In
particular, the presence of calcium ions has been shown to modulate
both the forward progress of the polymerase reaction at step 106
and/or 108 (also shown as k2 and k3, respectively), as well as the
reverse progress of the reaction at step 106 and/or 104 (also shown
as k-2 and k-1, respectively). As a result, in the presence of
calcium or other non-catalytic metals, the complexed nucleotide is
effectively sequestered in the complex; unable to proceed forward
to incorporation, or in reverse to the release of the
unincorporated nucleotide, in an unproductive nucleotide binding
event, to yield a free polymerase.
##STR00001##
[0148] Such unproductive binding, and subsequent release of an
otherwise correct nucleotide by a polymerization complex is
referred to herein as "branching". For real-time sequence by
incorporation processes, such branching can lead to incorrect
repeat calls or insertion errors for a single base.
[0149] Because these non-catalytic metal ions interact with
polymerase enzymes to promote the tight, non-exchangeable binding
of nucleotides to polymerases, the use of such metals in polymerase
based sequencing processes is counterintuitive. In particular, it
would not be expected that the use of such non-catalytically
competent metal ions would provide benefits in polymerization based
sequencing processes, because they specifically interfere with the
desired interaction.
[0150] Surprisingly however, it has been discovered that mixtures
of both catalytic and non-catalytic metal ions in the
polymerization reaction mixture yields surprisingly beneficial
results in this process. In particular, it has been observed that
the competitive exchange rate for catalytic and non-catalytic metal
ions in nucleic acid polymerases is sufficiently fast, that one can
exchange catalytic for non-catalytic ions in the reaction complex.
Restated, upon exchange of the calcium ion with a catalytically
more competent metal ion, e.g., manganese or magnesium, the
polymerization reaction is again capable of proceeding forward to
incorporation, or in reverse to release a bound nucleotide to
return to the free polymerase state. Thus, these exchangeable
catalytic and non-catalytic cofactors can be contacted with the
polymerase complex to first sequester the nucleotide in a
non-exchangeable state within the polymerase complex, from which it
is substantially less likely to be released. Upon exchange of a
non-catalytic cofactor with a catalytic co-factor, the nucleotide
will be transitioned into an exchangeable state within the complex,
from which it can proceed through an incorporation reaction.
Further, the rate of the exchange is such that one can effectively
modulate the speed of the polymerase reaction by modulating the
relative proportion of catalytic/non-catalytic metal ions in the
reaction mixture. In particular, modulating the relative
concentrations of these ions effectively modulates the reaction
kinetics of individual enzymes, rather than just in bulk.
Furthermore, because the nature of the interaction of the complex
with calcium ions interferes with both the forward progress of
incorporation and the reverse progress of release or branching, one
can effectively slow the reaction, or more specifically, increase
the time the "to be incorporated" nucleotide is bound, without a
consequent increase in the amount of nucleotide released or
branching. In contrast, other approaches that have been exploited
to increase the retention time of a nucleotide by a polymerase
complex generally do so by slowing the kinetics of the forward
reaction out of a given state, without concurrently slowing the
reverse of the reaction into that state. Such methods include both
enzymological approaches, as well as adjustment of the polymerase
reaction conditions, e.g., temperature and pH, to slow the
reaction. As such, the slowed forward progress of the reaction can
result in a concurrent increase of the unproductive release of
correct nucleotides for incorporation of similar magnitude.
[0151] Although generally described in terms of mixtures of a first
and second metal co-factors, where the first has higher catalytic
impact than the second, it will be appreciated that the reaction
mixtures may include more than two metal co-factors of differing
catalytic impact upon the polymerization complex. For example, the
reaction mixtures may include three, four, five or more different
metal co-factors that have differing catalytic impacts, i.e.,
promotion or inhibition of polymerization reaction under the given
reaction conditions. Thus, in its broadest sense, the invention
includes polymerization reaction mixtures that include mixtures of
different metal co-factors that interact with the polymerization
complex, where the different metal co-factors have different
catalytic impacts upon the polymerization reaction, e.g., different
effects on enzyme turnover rates, relative to each other. Such
reaction mixtures can include two, three, four, five or more
different metal co-factors that are capable of interacting with the
polymerization complex, and particularly the polymerase itself, to
promote or inhibit the polymerization reaction, relative to one or
more other metal co-factors that are present.
[0152] In addition to the benefits of enhanced retention time
without a substantial concurrent increase in branching, the
presence of non-catalytic ions also provides additional advantages,
such as increased ternary stability and reduced Km values. Further,
the presence of such metals can provide an inhibitory effect on any
exonuclease activity present in the reaction mixture, either as an
activity of the polymerase enzyme, or otherwise. See FIGS. 9 and
10, and Soengas, et al., EMBO (1992) 11(11):4227-4237.
[0153] In an alternative aspect, the reaction rate of the
polymerase may be modulated through the iterative modulation of
catalytic and non-catalytic metals in the reaction mixture, rather
than through the real-time modulation of metal ions in the complex.
As a result, one can proceed, step wise, along the template
sequence, monitoring the incorporation of nucleotides into the
nascent strand.
[0154] In an exemplary operation, one introduces the four types of
nucleotides, e.g., each labeled with a detectably different
fluorophore on its terminal phosphate group or other portion of the
nucleotide released upon incorporation, to the
polymerase/template/primer complex that is immobilized upon a
substrate, e.g., either in a spotted array format where all
template/primers in a single spot represent the same sequence, or
in a single-molecule observable configuration. The nucleotides are
introduced along with a sufficient concentration of non-catalytic
metal ions, e.g., Ca.sup.2+, and without catalytic metal ions,
e.g., Mn.sup.2+. In the context of this reaction mixture, a cognate
nucleotide (the correct nucleotide for incorporation into the
nascent strand based upon the template), is bound by the polymerase
which proceeds through the first portion of the incorporation
reaction, e.g., through step 104 and/or 106 of FIG. 1. However, due
to the presence of Ca.sup.2+ and the lack of catalytic metals, the
nucleotide is sequestered in the active site of the polymerase,
unable to proceed to incorporation or be released from the complex.
Excess labeled nucleotides are then washed from the complex,
typically still in the presence of Ca.sup.2+ ions. The remaining
complex bound cognate nucleotides are then observed and identified
based upon their fluorescent label, e.g., using a fluorescent
microscope, array scanner, or the like.
[0155] The complex is then allowed to proceed with incorporation of
the nucleotide and consequent release of the label group by washing
the complex with catalytic metal ions, e.g., Mn.sup.2+ to allow
incorporation to proceed, e.g., through step 108 of FIG. 1,
resulting in a single base extended primer. The complex is then
washed to remove catalytic metal ions from the complex and reaction
mixture, and the process is repeated with a new wash of labeled
nucleotides in a Ca.sup.2+ containing buffer. See, for example,
FIG. 11.
[0156] Accordingly, in one aspect, the present invention is
directed to the use of a mixture of catalytic and non-catalytic
metal ions in a nucleic acid synthesis reaction, to modulate the
reaction kinetics of the complex. Thus, in at least one aspect, the
invention is directed to nucleic acid synthesis reaction mixtures
that include both catalytic and non-catalytic metals. The molar
ratio of catalytic to non-catalytic metals in the reaction mixture
will generally vary depending upon the type of kinetic modulation
desired for a given synthesis reaction, where slower incorporation
would suggest higher levels of non-catalytic metal ions. Typically,
such ratios of catalytic to non-catalytic metals in the reaction
mixture will vary from about 10:1 to about 1:10, and preferably,
from about 10:1 to about 1:5, depending upon the desired level of
modulation, the particular enzyme system employed, the catalytic
and non-catalytic metal cofactors that are used, and the reaction
conditions. In particularly preferred aspects, the ratios of
catalytic to non-catalytic metals will be in the range of from
about 5:1 to about 1:1, with ratios of from about 2.5:1 to about
1.5:1 being particularly preferred.
[0157] In addition to the presence of such metals at the ratios
described herein, the absolute concentration of such metals in the
reaction mixtures will typically range from about 0.05 mM to about
50 mM, in some cases from about 0.1 mM to about 10 mM, in some
cases from about 0.1 mM to about 5 mM. The composition can include,
for example, from about 0.1 mM MnCl.sub.2 to about 1 mM MnCl.sub.2
and from about 0.1 mM CaCl.sub.2 to about 2 mM CaCl.sub.2; or from
about 0.2 mM MnCl.sub.2 to about 1 mM MnCl.sub.2 and from about 0.4
mM CaCl.sub.2 to about 1.5 mM CaCl.sub.2.
[0158] In addition to the catalytic and/or non-catalytic metal
components, the compositions of the invention will typically
include one or more of the other components of a nucleic acid
synthesis reaction. In particular, such complexes typically will
include one, and preferably more than one of the other various
components for a nucleic acid synthesis reaction. Such components
include, for example, a nucleic acid polymerizing enzyme. In
preferred aspects, the nucleic acid polymerizing enzymes are
selected from DNA polymerases, although RNA polymerases, reverse
transcriptases, or the like are also envisioned. In the case of DNA
polymerases, a variety of polymerases may be employed in the
compositions of the invention, including for example, strand
displacing polymerases, such as Phi29 derived polymerases (e.g.,
those described in U.S. Pat. No. 5,001,050, and published U.S.
Patent Application No. 2007-0196846, the full disclosures of which
are incorporated herein by reference in their entirety for all
purposes), Taq polymerases, KOD polymerases, Klenow, 9.degree.N
polymerase, T7 DNA polymerase, E. coli pol I, Bacillus
stearothermophilus pol I, DNA polymerases .alpha., .delta.,
.epsilon., and .gamma., RB 69 polymerase, polIV (DINB), polV
(UmuD'2C), and others.
C. Polymerase Reaction Substrates
[0159] The polymerase reactions of the invention include polymerase
reaction substrates. The substrates that are selected can be
selected to influence the kinetics of the polymerase reaction, and
can be utilized to prepare a polymerase reaction system that
exhibits two slow-step kinetics. The polymerase reaction substrates
include the template nucleic acid, a primer, and one or more
nucleotides. The template nucleic acid is the molecule for which
the complimentary sequence is synthesized in the polymerase
reaction. In some cases, the template nucleic acid is linear, in
some cases, the template nucleic acid is circular. The template
nucleic acid can be DNA, RNA, or can be a non-natural RNA analog or
DNA analog. Any template nucleic acid that is suitable for
replication by a polymerase enzyme can be used herein.
[0160] By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, nucleic acid
analogs are included that may have alternate backbones, comprising,
for example, phosphoramide, phosphorothioate, phosphorodithioate,
and peptide nucleic acid backbones and linkages. Other analog
nucleic acids include those with positive backbones; non-ionic
backbones, and non-ribose backbones, including those described in
U.S. Pat. Nos. 5,235,033 and 5,034,506. The template nucleic acid
may also have other modifications, such as the inclusion of
heteroatoms, the attachment of labels, such as dyes, or
substitution with functional groups which will still allow for base
pairing and for recognition by the enzyme.
[0161] The synthesis reaction will typically include a template or
target nucleic acid sequence that is sought to be replicated, as
well as a primer sequence that specifically hybridizes to a portion
of the template or target sequence. The nucleic acid template and
primer can be selected to influence the kinetics of the polymerase
reaction, and can be utilized to prepare a system in which two
slow-step kinetics is observed.
[0162] The template sequence may be provided in any of a number of
different format types depending upon the desired application. For
example, in some cases, the template sequence may be a linear
single or double stranded nucleic acid sequence. In still other
embodiments, the template may be provided as a circular or
functionally circular construct that allows redundant processing of
the same nucleic acid sequence by the synthesis complex. Use of
such circular constructs has been described in, e.g., U.S. Pat. No.
7,315,019 and U.S. patent application Ser. No. 12/220,674, filed
Jul. 25, 2008. Alternate functional circular constructs are also
described in U.S. Patent Application [unassigned], Attorney docket
number 105-005902US, entitled "Method and Compositions for Nucleic
Acid Sample Preparation" filed Mar. 27, 2009, and U.S. patent
application Ser. No. 12/413,258, the full disclosures of each of
which are incorporated herein by reference in their entirety for
all purposes.
[0163] Briefly, such alternate constructs include template
sequences that possess a central double stranded portion that is
linked at each end by an appropriate linking oligonucleotide, such
as a hairpin loop segment. Such structures not only provide the
ability to repeatedly replicate a single molecule (and thus
sequence that molecule), but also provide for additional redundancy
by replicating both the sense and antisense portions of the double
stranded portion. In the context of sequencing applications, such
redundant sequencing provides great advantages in terms of sequence
accuracy.
[0164] The polymerase enzymes of the invention generally require a
primer, which is usually a short oligonucleotide that is
complementary to a portion of the template nucleic acid. The
primers of the invention can comprise naturally occurring RNA or
DNA oligonucleotides. The primers of the invention may also be
synthetic analogs. The primers may have alternative backbones as
described above for the nucleic acids of the invention. The primer
may also have other modifications, such as the inclusion of
heteroatoms, the attachment of labels, such as dyes, or
substitution with functional groups which will still allow for base
pairing and for recognition by the enzyme. Primers can select
tighter binding primer sequences, e.g., GC rich sequences, as well
as employ primers that include within their structure non-natural
nucleotides or nucleotide analogs, e.g., peptide nucleic acids
(PNAs) or locked nucleic acids (LNAs), that can demonstrate higher
affinity pairing with the template.
[0165] The primer can be selected to influence the kinetics of the
polymerase reaction, and to prepare a system in which two slow-step
kinetics is observed.
[0166] As used in the art, the term nucleotide refers both to the
nucleoside triphosphates that are added to a growing nucleic acid
chain in the polymerase reaction, and also to refer to the
individual units of a nucleic acid molecule, for example the units
of DNA and RNA. Herein, the term nucleotide used in this manner.
Whether the term nucleotide refers to the substrate molecule to be
added to the growing nucleic acid or to the units in the nucleic
acid chain can be derived from the context in which the term
used.
[0167] The nucleotides or set of nucleotides of the invention can
be naturally occurring nucleotides or modified nucleotides
(nucleotide analogs). The nucleotides used in the invention,
whether natural, unnatural, modified or analog are suitable for
participation in the polymerase reaction. For example, the term
nucleotide is used to refer to nucleotides that are labeled with
fluorescent dye group. The term nucleotide may also be used to
refer to nucleotides having other than three phosphate groups, for
example 4, 5, 6, 7 or more phosphate groups. Such nucleotides have
been described, for example in U.S. Pat. Nos. 6,936,702 and
7,041,812. Labels such as fluorescent dye group may be located in
various positions on the nucleotide. In some cases, a fluorescent
dye is located on the terminal phosphate of the nucleotide. The
term nucleotide as used herein also comprises nucleotide
analogs.
[0168] The type of nucleotide or set of nucleotides in the
polymerase reaction can be selected to obtain a system that
exhibits two slow-step kinetics.
[0169] The nucleotide compositions may include nucleoside
triphosphates, or analogs of such compounds. For example, in some
cases, the reaction mixtures will include nucleotide analogs having
longer phosphate chains, such as nucleoside tetra, penta-, hexa- or
even heptaphosphates. In addition, the nucleotide analogs of the
compositions of the invention may additionally include other
components, such as detectable labeling groups. Such detectable
labeling groups will typically impart an optically or
electrochemically detectable property to the nucleotide analogs
being incorporated into the synthesis reaction. In particularly
preferred aspects, fluorescent labeling groups, i.e., labeling
groups that emit light of one wavelength when excited with light of
another wavelength, are used as the labeling groups. For purposes
of the present disclosure, the foregoing or later discussed
nucleotide or nucleotide analog compositions whether labeled or
unlabeled, possessing of three or more phosphate groups, or
otherwise modified, are generally referred to herein as
nucleotides.
[0170] Typically, each of the different types of nucleotide analogs
will be labeled with a detectably different fluorescent labeling
group, e.g., that possesses a detectably distinct fluorescent
emission and/or excitation spectrum, such that it may be identified
and distinguished from different nucleotides upon incorporation.
For example, each of the different types of nucleotides, e.g., A,
T, G and C, will be labeled with a fluorophore having a different
emission spectrum. For certain embodiments, the nucleotide may
include a fluorescent labeling group coupled to a portion of the
nucleotide that is incorporated into the nascent nucleic acid
strand being produced during synthesis, e.g., the nucleobase or
sugar moiety. Nucleotide compositions having fluorophores coupled
to these portions have been previously described (See, e.g., U.S.
Pat. Nos. 5,476,928 and 4,711,955 to Ward et al.). As a result of
the label group being coupled to the base or sugar portion of the
nucleotide, upon incorporation, the nascent strand will include the
labeling group. This labeling group may then remain or be removed,
e.g., through the use of cleavable linkages joining the label to
the nucleotide (See, e.g., U.S. Pat. No. 7,057,026). A variety of
different fluorophore types, including both organic and inorganic
fluorescent materials, have been described for biological
applications and are likewise applicable in the instant
invention.
[0171] Alternatively and preferably, the labeling group is coupled
to a portion of the polyphosphate chain that is removed by the
polymerase action during the incorporation event, e.g., the beta,
gamma or further distal phosphate group. Examples of such phosphate
labeled nucleotide analogs and their use in sequencing applications
are described in, e.g., U.S. Pat. Nos. 6,399,335, 6,762,048,
7,041,812 and published U.S. Patent Application No. 2006-0063173.
Because the label is included on a portion of the nucleotide that
is cleaved during incorporation, the labeling group is not actually
incorporated into the nascent strand, but instead, diffuses away
from the synthesis complex. As described previously, where the
complex is provided within an optical confinement, e.g., a
zero-mode waveguide, the act of incorporation provides a
characteristic retention of the label prior to its cleavage and
diffusion away, so as to permit the recognition of an incorporation
event. Further, by identifying the spectral characteristics of the
label associated with the base being incorporated, one can identify
the specific type of base.
[0172] In certain embodiments, the nucleotides or the complex as a
whole may be provided with cooperative fluorescent labeling groups,
e.g., that act cooperatively as a donor-quencher or fluorescent
resonant energy transfer pair, to provide labeling. As noted above,
in this context, the necessity for optical confinement to eliminate
background signal from unincorporated labels or nucleotides is
reduced, as substantially only interacting labels brought into
sufficient proximity by the incorporation event (in the case of
complex and nucleotide bound interactive labels), or only labels
separated by cleavage of the polyphosphate chain upon
incorporation, will produce a characteristic signal indicative of
incorporation.
[0173] Other fluorescent labeling groups may likewise be employed
in the nucleotide compositions, including inorganic fluorescent
materials, such as semiconductor nanocrystals, like II-VI or III-V
semiconductor nanocrystals, including CdSe, CdTe, InS, ZnS or other
nanocrystal compositions, available from, e.g., e-Biosciences, Inc.
(San Diego, Calif.), and Life Technologies, Inc.
[0174] The nucleotides of the present invention include nucleotides
having the structure:
B--S--P-L,
wherein B is a natural or non-natural nucleobase, S is selected
from a sugar moiety, an acyclic moiety or a carbocyclic moiety, P
is a modified or unmodified polyphosphate, and L is a detectable
label optionally including a linker.
[0175] The base moiety, B, incorporated into the compounds of the
invention is generally selected from any of the natural or
non-natural nucleobases or nucleobase analogs, including, e.g.,
purine or pyrimidine bases that are routinely found in nucleic
acids and nucleic acid analogs, including adenine, thymine,
guanine, cytidine, uracil, and in some cases, inosine. For purposes
of the present description, nucleotides and nucleotide analogs are
generally referred to based upon their relative analogy to
naturally occurring nucleotides. As such, an analog that operates,
functionally, like adenosine triphosphate, may be generally
referred to herein by the shorthand letter A. Likewise, the
standard abbreviations of T, G, C, U and I, may be used in
referring to analogs of naturally occurring nucleosides and
nucleotides typically abbreviated in the same fashion. In some
cases, a base may function in a more universal fashion, e.g.,
functioning like any of the purine bases in being able to hybridize
with any pyrimidine base, or vice versa. The base moieties used in
the present invention may include the conventional bases described
herein or they may include such bases substituted at one or more
side groups, or other fluorescent bases or base analogs, such as
1,N6 ethenoadenosine or pyrrolo C, in which an additional ring
structure renders the B group neither a purine nor a pyrimidine.
For example, in certain cases, it may be desirable to substitute
one or more side groups of the base moiety with a labeling group or
a component of a labeling group, such as one of a donor or acceptor
fluorophore, or other labeling group. Examples of labeled
nucleobases and processes for labeling such groups are described
in, e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0176] In the nucleotides of the invention, the S group is
generally a sugar moiety that provides a suitable backbone for a
synthesizing nucleic acid strand. In it most preferred aspect, the
sugar moiety is selected from a D-ribosyl, 2' or 3' D-deoxyribosyl,
2',3'-D-dideoxyribosyl, 2',3'-D-didehydrodideoxyribosyl, 2' or 3'
alkoxyribosyl, 2' or 3' aminoribosyl, 2' or 3' mercaptoribosyl, 2'
or 3' alkothioribosyl, acyclic, carbocyclic or other modified sugar
moieties. A variety of carbocyclic or acyclic moieties may be
incorporated as the "S" group in place of a sugar moiety,
including, e.g., those described in published U.S. Patent
Application No. 2003/0124576, previously incorporated herein by
reference in its entirety for all purposes.
[0177] The P groups in the nucleotides of the invention are
modified or unmodified polyphosphate groups. The number of
phosphates in the polyphosphate can be 1, 2, 3, 4, 5, 5, 7, 8 or
more modified or unmodified phosphates. The unmodified phosphates
have linearly linked --O--P(O).sub.2-units, for example a
monophosphate, diphosphate, triphosphate, tetraphosphate,
pentaphosphate, hexaphosphate, heptaphosphate, or octaphosphate.
The P groups also include modified polyphosphates, for example by
virtue of the inclusion of one or more phosphonate groups,
effectively substituting a non-ester linkage in the phosphorous
containing chain of the analog, with a more stable linkage.
Examples of preferred linkages include, e.g., CH.sub.2, methylene
derivatives (e.g., substituted independently at one or more
hydrogens with F, Cl, OH, NH2, alkyl, alkenyl, alkynyl, etc.),
CCl.sub.2, CF.sub.2, NH, S, CH.sub.2CH.sub.2, C(OH)(CH.sub.3),
C(NH.sub.2)[(CH.sub.2).sub.6CH.sub.3], CH(NHR) (R is H or alkyl,
alkenyl, alkynyl, aryl, C(OH)[(CH.sub.2).sub.nNH2] (n is 2 or 3),
and CNH.sub.2. In particularly preferred aspects, methylene, amide
or their derivatives are used as the linkages.
[0178] Other P groups of the invention have phosphate or modified
phosphates in which one or more non-bridging oxygen is substituted,
for example with S, or BH3. In one aspect of the invention, one or
more, two or more, three or more, or four or more non-bridging
oxygen atoms in the P group has an S substituted for an O. The
substitution of, sulfur atoms for oxygen can change the polymerase
reaction kinetics such that a system having two slow steps can be
selected. While not being bound by theory, it is believed that the
properties of the nucleotide, such as the metal chelation
properties, electronegativity, or steric properties are the
nucleotide can be altered by the substitution of non-bridging
oxygen for sulfur in P. In some cases, it is believed that the
substitution of two or more non-bridging oxygen atoms with sulfur
can affect the metal chelation properties so as to lead to a two
slow-step system.
[0179] Suitable nucleotides include nucleotides having 4, 5, 6, or
7 phosphates in which a sulfur is substituted for one of the
non-bridging oxygens. In some embodiments, the single sulfur
substitution is made such that substantially only one stereoisomer
is present. The nucleotide can have 7 phosphates in which phosphate
2, 3, 4, 5, 6, or 7 has a non-bridging sulfur in place of oxygen.
The nucleotide can have 6 phosphates in which phosphate 2, 3, 4, 5,
or 6 has a non-bridging sulfur in place of oxygen. The nucleotide
can have 5 phosphates in which phosphate 2, 3, 4, or 5 has a
non-bridging sulfur in place of oxygen. The substituted phosphate
in the nucleotide can be the R or the S stereoisomer.
[0180] The nucleotide can have 6 phosphates in which phosphate 2
has sulfur substituted for oxygen. The nucleotide can have 6
phosphates in which phosphate 2 has sulfur substituted for oxygen
and phosphate 2 is the R stereoisomer. The nucleotide can have 6
phosphates in which phosphate 2 has sulfur substituted for oxygen
and phosphate 2 is the S stereoisomer. The nucleotide can have 6
phosphates in which phosphate 6 has sulfur substituted for oxygen.
The nucleotide can have 6 phosphates in which phosphate 6 has
sulfur substituted for oxygen and phosphate 6 is the R
stereoisomer. The nucleotide can have 6 phosphates in which
phosphate 6 has sulfur substituted for oxygen and phosphate 6 is
the S stereoisomer. The nucleotide can have 7 phosphates in which
phosphate 2 has sulfur substituted for oxygen and phosphate 2 is
the S stereoisomer. The nucleotide can have 7 phosphates in which
phosphate 6 has sulfur substituted for oxygen. The nucleotide can
have 7 phosphates in which phosphate 6 has sulfur substituted for
oxygen and phosphate 6 is the R stereoisomer. The nucleotide can
have 7 phosphates in which phosphate 6 has sulfur substituted for
oxygen and phosphate 6 is the S stereoisomer.
[0181] While not being bound by theory, it is believed that
two-slow-step kinetics can be obtained from the stabilized metal
ion coordination between the non-bridging sulfur on the nucleotide
and the manganese or other metal cofactor atoms in the enzyme
complex. Based on the structural analysis of a crystal structure of
phi29 DNA polymerases, specific non-bridging oxygen atoms on the
phosphate are coordinated with manganese atom. FIG. 23 shows a
model of a nucleotide having 6 phosphate units bound to the enzyme.
The phosphates are labeled 1 through 6. The non-bridging oxygens
(or substituted sulfurs) can be seen as extending from the
phosphorous atoms. Hydrogen bonding interactions and metal ion
coordination are represented as black dashed lines. The manganese
ions are shown as spheres. FIG. 23 shows that some non-bridging
oxygen atoms on the phosphate are hydrogen bound to the positively
charged residues on the polymerase L-helix. Other non-bridging
oxygen atoms are in coordination with manganese atoms in the
complex. Thus, in some cases, a specific stereoisomer can be useful
for obtaining two slow-step kinetics, while the other isomer will
not be effective. Note, for example, that the oxygens on the 2nd
and 6th phosphates have contacts to manganese ions.
[0182] L generally refers to a detectable labeling group that is
coupled to the terminal phosphorus atom via the R.sub.4 (or
R.sub.10 or R.sub.12) group. The labeling groups employed in the
analogs of the invention may comprise any of a variety of
detectable labels. As used herein, labels or detectable labels
generally denote a chemical moiety that provides a basis for
detection of the analog compound separate and apart from the same
compound lacking such a labeling group. Examples of labels include,
e.g., optical labels, e.g., labels that impart a detectable optical
property to the analog, electrochemical labels, e.g., labels that
impart a detectable electrical or electrochemical property to the
analog, physical labels, e.g., labels that impart a different
physical or spatial property to the analog, e.g., a mass tag or
molecular volume tag. In some cases individual labels or
combinations may be used that impart more than one of the
aforementioned properties to the nucleotide analogs of the
invention.
[0183] In preferred aspects, the labeling groups incorporated into
the analogs of the invention comprise optically detectable
moieties, including luminescent, chemiluminescent, fluorescent,
fluorogenic, chromophoric and/or chromogenic moieties, with
fluorescent and/or fluorogenic labels being particularly preferred.
A variety of different label moieties are readily employed in
nucleotide analogs, and particularly, the compound of the
invention. Such groups include fluorescein labels, rhodamine
labels, cyanine labels (i.e., Cy3, Cy5, and the like, generally
available from the Amersham Biosciences division of GE Healthcare),
the Alexa family of fluorescent dyes and other fluorescent and
fluorogenic dyes available from Molecular Probes/Invitrogen, Inc.,
and described in `The Handbook--A Guide to Fluorescent Probes and
Labeling Technologies, Tenth Edition` (2005) (available from
Invitrogen, Inc./Molecular Probes). A variety of other fluorescent
and fluorogenic labels for use with nucleoside polyphosphates, and
which would be applicable to the compounds of the present invention
are described in, e.g., Published U.S. Patent Application No.
2003/0124576, the full disclosure of which is incorporated herein
in its entirety for all purposes.
[0184] The label group may be directly coupled to the terminal
phosphorus atom of the analog structure, in alternative aspects, it
may additionally include a linker molecule to provide the coupling
through, e.g., an alkylphosphonate linkage. A wide variety of
linkers and linker chemistries are known in the art of synthetic
chemistry may be employed in coupling the labeling group to the
analogs of the invention. For example, such linkers may include
organic linkers such as alkane or alkene linkers of from about C2
to about C20, or longer, polyethyleneglycol (PEG) linkers, aryl,
heterocyclic, saturated or unsaturated aliphatic structures
comprised of single or connected rings, amino acid linkers, peptide
linkers, nucleic acid linkers, PNA, LNAs, or the like or phosphate
or phosphonate group containing linkers. In preferred aspects,
alkyl, e.g., alkane, alkene, alkyne alkoxy or alkenyl, or ethylene
glycol linkers are used. Some examples of linkers are described in
Published U.S. Patent Application No. 2004/0241716, which is
incorporated herein by reference in its entirety for all purposes.
Additionally, such linkers may be selectively cleavable linkers,
e.g., photo- or chemically cleavable linkers or the like. The
linkers can be alkyl, aryl, or ester linkers. The linkers can be,
amino-alkyl linkers, e.g., amino-hexyl linkers. In some cases, the
linkers can be rigid linkers such as disclosed in U.S. patent
application Ser. No. 12/403,090.
[0185] The B, S, P, and L groups can be connected directly, or can
be connected using an linking unit such as an --O--, --S--, --NH--,
or --CH.sub.2-- unit.
III. SINGLE-MOLECULE SEQUENCING PROCESSES AND SYSTEMS
[0186] As noted, the mixtures of catalytic and non-catalytic metals
in the reaction mixture provide for the modulation of the reaction
kinetics of individual complexes. Accordingly, in particularly
preferred aspects, the synthesis complexes in such reaction
mixtures are arrayed so as to permit observation of the individual
complexes that are being so modulated. In arraying individual
complexes to be individually optically resolvable, the systems of
the invention will position the complexes on solid supports such
that there is sufficient distance between adjacent individual
complexes as to allow optical signals from such adjacent complexes
to be optically distinguishable from each other.
[0187] Typically, such complexes will be provided with at least 50
nm and more preferably at least 100 nm of distance between adjacent
complexes, in order to permit optical signals, and particularly
fluorescent signals, to be individually resolvable. Examples of
arrays of individually resolvable molecules are described in, e.g.,
U.S. Pat. No. 6,787,308.
[0188] In some cases, individual complexes may be provided within
separate discrete regions of a support. For example, in some cases,
individual complexes may be provided within individual optical
confinement structures, such as zero-mode waveguide cores. Examples
of such waveguides and processes for immobilizing individual
complexes therein are described in, e.g., Published International
Patent Application No. WO 2007/123763, the full disclosure of which
is incorporated herein by reference in its entirety for all
purposes.
[0189] As noted previously, in preferred aspects, the synthesis
complexes are provided immobilized upon solid supports, and
preferably, upon supporting substrates. The complexes may be
coupled to the solid supports through one or more of the different
groups that make up the complex. For example, in the case of
nucleic acid polymerization complexes, attachment to the solid
support may be through an attachment with one or more of the
polymerase enzyme, the primer sequence and/or the template sequence
in the complex. Further, the attachment may comprise a covalent
attachment to the solid support or it may comprise a non-covalent
association. For example, in particularly preferred aspects,
affinity based associations between the support and the complex are
envisioned. Such affinity associations include, for example,
avidin/streptavidin/neutravidin associations with biotin or
biotinylated groups, antibody/antigen associations, GST/glutathione
interactions, nucleic acid hybridization interactions, and the
like. In particularly preferred aspects, the complex is attached to
the solid support through the provision of an avidin group, e.g.,
streptavidin, on the support, which specifically interacts with a
biotin group that is coupled to the polymerase enzyme.
[0190] Methods of providing binding groups on the substrate surface
that result in the immobilization of optically resolvable complexes
are described in, e.g., published U.S. Patent Application No.
2007-0077564, incorporated herein by reference in its entirety for
all purposes, and WO 2007123763, previously incorporated herein by
reference.
[0191] The sequencing processes, e.g., using the substrates
described above and the synthesis compositions of the invention,
are generally exploited in the context of a fluorescence microscope
system that is capable of illuminating the various complexes on the
substrate, and obtaining detecting and separately recording
fluorescent signals from these complexes. Such systems typically
employ one or more illumination sources that provide excitation
light of appropriate wavelength(s) for the labels being used. An
optical train directs the excitation light at the reaction
region(s) and collects emitted fluorescent signals and directs them
to an appropriate detector or detectors. Additional components of
the optical train can provide for separation of spectrally
different signals, e.g., from different fluorescent labels, and
direction of these separated signals to different portions of a
single detector or to different detectors. Other components may
provide for spatial filtering of optical signals, focusing and
direction of the excitation and or emission light to and from the
substrate.
[0192] One such exemplary system is shown in FIG. 4. As shown, the
overall system 300 generally includes an excitation illumination
source 302. Typically, such illumination sources will comprise high
intensity light sources such as lasers or other high intensity
sources such as LEDs, high intensity lamps (mercury, sodium or
xenon lamps), laser diodes, and the like. In preferred aspects, the
sources will have a relatively narrow spectral range and will
include a focused and/or collimated or coherent beam. For the
foregoing reasons, particularly preferred light sources include
lasers, solid state laser diodes, and the like. An exemplar system
is also described in Lundquist et al., Optics Letters, Vol. 33,
Issue 9, pp. 1026-1028, the full disclosure of which is
incorporated herein by reference in its entirety for all
purposes.
[0193] The excitation illumination source 302 is positioned to
direct light of an appropriate excitation wavelength or wavelength
range, at a desired fluorescent signal source, e.g., substrate 304,
through an optical train. As shown, the optical train includes a
number of elements to appropriately direct excitation illumination
at the substrate 304, and receive and transmit emitted signals from
the substrate to an appropriate detection system such as detector
328. The excitation illumination from illumination source 302 is
directed first through an optical multiplex element 306, or
elements, to multiply the number of illumination beams or spots
from an individual beam or spot from the illumination source 302.
The multiplexed beam(s) is then directed via focusing lens 308
through optional first spatial filter 310, and focusing lens 312.
As discussed in greater detail below, spatial filter 310 optionally
provides control over the extent of multiplex beams continuing
through the optical train reduces the amount of any scattered
excitation light from reaching the substrate. The spatially
filtered excitation light is then passed through dichroic 314 into
objective lens 316, whereupon the excitation light is focused upon
the substrate 304. Dichroic 314 is configured to pass light of the
spectrum of the excitation illumination while reflecting light
having the spectrum of the emitted signals from the substrate 304.
Because the excitation illumination is multiplexed into multiple
beams, multiple discrete regions of the substrate are separately
illuminated.
[0194] Fluorescent signals that are emitted from those portions of
the substrate that are illuminated, are then collected through the
objective lens 316, and, because of their differing spectral
characteristics, they are reflected by dichroic 314, through
focusing lens 318, and second spatial filter, such as confocal mask
320, and focusing lens 322. Confocal mask 320 is typically
positioned in the focal plane of lens 318, so that only in-focus
light is passed through the confocal mask, and out-of focus light
components are blocked. This results in a substantial reduction in
noise levels from the system, e.g., that derive from out of focus
contributors, such as autofluorescence of the substrate and other
system components.
[0195] As with the excitation illumination, the signals from the
multiple discrete illuminated regions on the substrate are
separately passed through the optical train. The fluorescent
signals that have been subjected to spatial filtering are then
passed through a dispersive optical element, such as prism assembly
324, to separately direct spectrally different fluorescent signal
components, e.g., color separation, which separately directed
signals are then passed through focusing lens 326 and focused upon
detector 328, e.g., an imaging detector such as a CCD, ICCD, EMCCD
or CMOS based detection element. Again, the spectrally separated
components of each individual signal are separately imaged upon the
detector, so that each signal from the substrate will be imaged as
separate spectral components corresponding to that signal from the
substrate. For a discussion of the spectral separation of discrete
optical signals, see, e.g., Published U.S. Patent Application No.
2007-0036511, incorporated herein by reference in its entirety for
all purposes.
[0196] As will be appreciated, a more conventional configuration
that employs reflected excitation light and transmitted
fluorescence may also be employed by altering the configuration of
and around dichroic 314. In particular, dichroic 314 could be
selected to be reflective of the excitation light from illumination
source 302, and transmissive to fluorescence from the substrate
304. The various portions of the optical train are then arranged
accordingly around dichroic 314. Notwithstanding the foregoing,
fluorescence reflective optical trains are particularly preferred
in the applications of the systems of the invention. For a
discussion on the advantages of such systems, see, e.g., U.S.
patent application Ser. No. 11/704,689, filed Feb. 9, 2007, Ser.
No. 11/483,413, filed Jul. 7, 2006, and Ser. No. 11/704,733, filed
Feb. 9, 2007, the full disclosures of which are incorporated herein
by reference in their entirety for all purpose.
[0197] In addition to the foregoing composition components,
additional components may also be included within the compositions
of the invention. For example, such compositions will typically
include buffering agents, salts, and other agents that facilitate
the desired reactions.
[0198] In certain embodiments, the sequencing compositions
described herein will be provided in whole, or in part, in kit form
enabling one to carry out the processes described herein. Such kits
will typically comprise one or more components of the reaction
complex, such as the polymerase enzyme and primer sequences. Such
kits will also typically include buffers and reagents that provide
the catalytic and non-catalytic metal co-factors employed in the
processes described herein. The kits will also optionally include
other components for carrying out sequencing applications in
accordance with those methods described herein. In particular, such
kits may include ZMW array substrates for use in observing
individual reaction complexes as described herein.
[0199] In addition to the various components set forth above, the
kits will typically include instructions for combining the various
components in the amounts and/or ratios set forth herein, to carry
out the desired processes, as also described or referenced herein,
e.g., for performing sequence by incorporation reactions.
VI. EXAMPLES
Example 1
Single-Molecule Sequencing in Zero-Mode Waveguides
[0200] Sequencing reactions are carried out in a zero-mode
waveguide array having 3000 discrete cores. The reaction is
observed using a highly multiplexed confocal fluorescent microscope
providing a targeted illumination profile, e.g., a separate spot
for each core (See, e.g., U.S. patent application Ser. No.
12/151,979, filed May 9, 2008, and incorporated herein by reference
in its entirety for all purposes). Fluorescent signals from the
various ZMWs are detected on an EMCCD camera for 5-7 minutes, and
are subjected to pulse recognition and base calling processes (See,
e.g., Published U.S. Patent Application No. 2009-0024331, and
incorporated herein by reference in its entirety for all
purposes).
Example 2
Catalytic and Non-Catalytic Metals
[0201] The effects of catalytic and non-catalytic metal ions and
mixtures thereof on nucleotide incorporation in polymerase mediated
template dependent primer extension reactions.
[0202] A. Stopped Flow Incorporation Assays.
[0203] The oligonucleotides that constitute the template/primer
complex were purchased from Integrated DNA Technologies
(Coralville, Iowa). The position iAmMC6T has an Int amino modified
C6 dT substituted for dT at this position. The "template"
oligonucleotide was labeled at position "iAmMC6T" with alexa fluor
488 fluorescent dye.
[0204] Sequence of oligonucleotides used for the assays.
TABLE-US-00003 5'-GGT GAT GTA GAT AGG TGG TAG GTG GTG TCA________
GAT C 3'-CCA CTA CAT CTA TCC ACC ATC CAC CAC AG/iAmMC6T/ CTA GGC
ATA ATA ACA GTT GCA GCA
[0205] This stopped flow assay relies on the quenching, for example
by fluorescent resonance energy transfer (FRET) of the fluorescence
of the Alexa fluor 488 attached to the template by a dye labeled
nucleotide. A nucleotide having an Alexa fluor 555 as a terminal
phosphate label, such as Alexa fluor 555-O-aminohexyl-dT6P
(A555-O-dC6P), having six phosphates, is used in the polymerase
reaction, which will quench the fluorescence of the Alexa fluor 488
dye attached to the template only when the nucleotide is associated
with (bound to) the polymerase enzyme.
[0206] The drop in the fluorescent signal, measured at 535 nm, is
attributed to binding of the Alexa-555-dC6P nucleotide to the
enzyme-DNA complex. Because quenching only occurs when the two dyes
are in close proximity, a significant drop in the fluorescence of
alexa fluor 488 due to the presence of alexa fluor 555 in solution
would not be expected to occur. Alexa-555-dC6P bound in the active
site of the enzyme, however, will cause a drop in the fluorescence
of alexa fluor 488 labeled oligonucleotide. The rate of drop of the
measured fluorescence signal is a function of the rate of binding
of the nucleotide to the active site of the enzyme.
[0207] Once bound, the nucleotide analog can undergo nucleotidyl
transfer catalyzed by the polymerase enzyme, extending the
oligonucleotide. Subsequent to extension of the oligonucleotide,
the product, the alexa fluor 555-pentaphosphate is released from
the enzyme. Once released from the enzyme DNA complex, the alexa
fluor 555-pentaphosphate no longer quenches the alexa fluor 488
attached to the template in the enzyme-DNA complex, and the
measured fluorescence signal increases at a rate that is a function
of the release of product.
[0208] The DNA polymerase (recombinant DNA polymerase (see
published U.S. Patent Application No. 2007-0196846, which is
incorporated herein by reference in its entirety for all purposes),
at 150 nM) was incubated with an oligonucleotide primer-template
complex (20 nM) in a buffer solution containing 50 mM ACES, pH 7.1,
75 mM potassium acetate, and 5 mM dithiothreitol (Buffer A). This
solution was rapidly mixed with a solution containing Buffer A and
6 .mu.M Alexa Fluor 555-dC6P, 1.4 mM manganese chloride, and
varying concentrations of calcium chloride from 0 to 5 mM using a
SF-2004 stopped flow instrument (Kintek Corporation, Austin, Tex.).
The observed fluorescent trace was fit to a double exponential
equation
(y=A.sub.1e.sup.-k.sup.1.sup.t+A.sub.2e.sup.-k.sup.2.sup.t+c) to
extract the observed rate constant for nucleotide binding and the
observed rate constant for incorporation. This was performed over a
series of CaCl.sub.2 concentrations (0, 0.25, 0.5, 1.25, 2.5, and 5
mM) in order to map the effects of CaCl.sub.2 on the rate constants
for nucleotide binding and incorporation. The fluorescence traces
are shown in FIG. 5. The rate constant for incorporation decreased
from 8.5.+-.0.1 s.sup.-1 (at 0 mM CaCl.sub.2) to 0.110.+-.0.001
s.sup.-1 (at 5 mM CaCl.sub.2). The single-exponential nature of the
fluorescence increase and the equivalence of the magnitude of the
fluorescence increase over all concentrations of CaCl.sub.2 assayed
implies rapid exchange of the divalent metal ions in this assay.
The observed rate constant for incorporation was then plotted as a
function of the CaCl.sub.2 concentration and then fitted to a
hyperbolic equation
( k obs = k max * ( 1 - [ CaCl 2 ] K i + [ CaCl 2 ] ) + c ) .
##EQU00001##
The hyperbolic fit generated a maximum rate of incorporation of
8.6.+-.0.5 s.sup.-1 and an apparent K.sub.i for CaCl.sub.2 of
0.29.+-.0.6 mM. The hyperbolic fit of the observed incorporation
rate constants vs. [CaCl.sub.2] is shown in FIG. 6.
[0209] B. Alexa-555-dC6P Release Assay
[0210] This experiment was carried out using the stopped flow
instrument in "double-mixing mode" which allows the mixing of two
samples prior to the addition of a third solution. The DNA
polymerase (250 nM) was incubated with an oligonucleotide
primer-template complex (50 nM) in Buffer A). The sequences of the
primer and the template for this assay are identical to those in
the incorporation assay, except that the primer for this assay has
a 3' terminal dideoxy-CMP. This solution was mixed with a solution
containing 6 .mu.M Alexa Fluor-555-dC6P in Buffer A with 1.4 mM
manganese chloride, and varying concentrations of CaCl.sub.2 (0,
0.5, 1, 2.5, 5, and 10 mM). This mixture was allowed to incubate
for 0.4 seconds prior to mixing with a solution containing 750
.mu.M dCTP in Buffer A with 0.7 mM manganese chloride, and varying
concentrations of CaCl.sub.2 (0, 0.25, 0.5, 1.25, 2.5, 5 mM). The
Alexa Fluor-488 dye on the DNA template was excited at 488 nm and
emission was monitored at 515 nm. The FRET quenching of the
fluorescence signal, observed in the stopped flow incorporation
assay, occurs during the unobservable first mixing event. Because
the primer for this experiment is 3'-dideoxyCMP terminated, no
incorporation of the Alexa Fluor-555-dC6P can occur. The observed
increase in the fluorescent signal is attributed to the release of
the Alexa Fluor-555-dC6P from the enzyme-DNA-nucleotide complex.
The fluorescence change was plotted versus time (FIG. 7) and fit to
a single exponential equation (y=Ae.sup.-kt+c). The rate of the
change was plotted versus CaCl.sub.2 concentration and fit to a
hyperbolic equation (FIG. 8). This fit generated a maximum rate of
release of 0.065.+-.0.002 s.sup.-1 and an apparent K.sub.i for
CaCl.sub.2 of 0.39.+-.0.04 mM.
[0211] C. Exonuclease Assay
[0212] The DNA polymerase was preincubated with an oligonucleotide
primer-template complex in Buffer A and varying concentrations of
CaCl.sub.2 (0, 0.1, 0.25, 0.5, 0.75, 1, 2.5, and 5 mM). This
solution was rapidly mixed with Buffer A with 1.4 mM manganese
chloride, and varying concentrations of CaCl.sub.2 (0, 0.1, 0.25,
0.5, 0.75, 1, 2.5, and 5 mM). This reaction mixture was allowed to
incubate for 30 minutes, with time points taken periodically from
zero to 30 minutes. The time points were quenched in 0.5 M EDTA to
stop the reaction, the products of the reactions were separated
using 16% polyacrylamide gel electrophoresis, and visualized using
a Typhoon 9400 variable mode scanner (Molecular Dynamics). The
intensities of the product bands were quantified in order to
determine the amount of substrate primer remaining at each time
point. The substrate remaining was plotted against time and fit to
a single exponential equation (FIG. 9). The observed rate constant
for exonuclease activity was plotted against [CaCl.sub.2] and fit
to a hyperbolic equation (FIG. 10). The hyperbolic fit generated a
maximum rate of exonuclease activity of 0.0019.+-.0.0001 s.sup.-1
and an apparent K.sub.i for CaCl.sub.2 of 0.5.+-.0.1 mM.
[0213] D. Cycle Sequencing
[0214] Nucleotide incorporation was monitored using an iterative
process of cycling catalytic and noncatalytic metals through the
reaction mixture as provided below.
[0215] A recombinant DNA polymerase covalently modified with biotin
was incubated for 30 minutes with a primed DNA template (1 .mu.M
each) in buffer B (50 mM Aces pH 7.1, 130 mM KOAc, 5 mM DTT, 0.03%
Tween20). Four wells of a Streptavidin Coated High Binding Capacity
Clear 96-well plate (Prod#15500 from ThermoScientific) were briefly
hydrated and rinsed with 50 mM Tris pH 7.5. The buffer was
completely removed from the four wells and 30 .mu.l of the DNA
polymerase-DNA complexes were added at room temperature and allowed
to incubate for 30 minutes to adhere the complexes to the
streptavadin-coated plate. The solution was removed and the wells
were rinsed with 50 .mu.l Buffer of Buffer B. A different
sequencing mix (30 .mu.l) was added to each of four wells in the
first column of the plate. Well A1 contained 1 .mu.M Alexa555-dA6P
(See, e.g., Eid et al.,) plus 1 .mu.M each of dCTP, dGTP, and dTTP
in Buffer B with 1 mM CaCl.sub.2 Well B1 contained 1 .mu.M
Alexa555-dC6P plus 1 .mu.M each of dATP, dGTP, and dTTP in Buffer B
with 1 mM CaCl.sub.2. Well Cl contained 1 .mu.M Alexa555-dT6P plus
1 .mu.M each of dATP, dGTP, and dCTP in Buffer B with 1 mM
CaCl.sub.2. Well D1 contained 1 .mu.M Alexa555-dG6P plus 1 .mu.M
each of dATP, dCTP, and dTCP in Buffer B with 1 mM CaCl.sub.2. The
sequencing mix was removed from each well and replaced with 50
.mu.l Buffer B with 1 mM CaCl.sub.2. The plate was read in a
fluorescent plate reader (Beckman Paradigm with excitation
wavelength 535 nm and emission wavelength 595 nm). The raw
fluorescence intensity is plotted for each well (designated by the
inclusion of fluorescently labeled base) in FIG. 11. The calcium
buffer was removed and replaced with 40 .mu.l Buffer B with 0.7 mM
MnCl.sub.2 to allow the bound base to be incorporated. The
manganese buffer was removed and the wells were then rinsed with 50
.mu.l Buffer B with 1 mM CaCl.sub.2. A next cycle of sequencing was
then performed in an identical manner by replacing the calcium
buffer in each well with the appropriate sequencing mix detailed
above. Three consecutive rounds of cycle sequencing with calcium
are demonstrated in FIG. 11. The first three incorporations should
be "C" then "T" and then "G". The first and second round clearly
distinguishes the correct bases demonstrating the principle of the
technique. Rising background fluorescence confounds the third base
read which presumable could be mitigated by more stringent washes
between cycles or shorter cycle time by an automated procedure.
[0216] Error analysis was performed as a function of addition of
0.3 mM CaCl.sub.2 to a single-molecule, real-time DNA sequencing
reaction. The control sequencing reactions and error analysis were
carried out as described in Eid, J. et al., Science, 323(5910),
133-138 (2009). For the 0.3 mM CaCl.sub.2 condition, 0.3 mM
CaCl.sub.2 was included in the immobilization, wash and reaction
buffers. Because the addition of CaCl.sub.2 increases the
nucleotide incorporation residence times, the errors caused by
missed pulses is reduced, while extra pulses due to premature
release events are unchanged within the error of the measurement.
The results are plotted in FIG. 12 for each of the four types of
bases. As can be seen, the error rates for insertion and deletions
are reduced upon the inclusion of non-catalytic metals.
Example 3
Non-Catalytic Metal Cofactors--Inhibition of DNA Polymerase
[0217] The degree by which different metal cofactors can inhibit
DNA polymerization by phi29 DNA pol was surveyed in the presence of
a constant concentration of catalytic manganese metal cofactor (0.7
mM MnCl.sub.2). DNA synthesis rate was measured using a real-time,
steady-state DNA polymerization assay utilizing 4-MU derivitized
nucleotides (4 methylumbelliferyl coumarin, M. Kozlov, V.
Bergendahl, R. Burgess, A. Goldfarb, A. Mustaev, Anal. Biochem.
342, 206 (2005)). The assay utilizes rolling circle DNA
polymerization on a primed 72 base circular single-stranded DNA
template. Three of the deoxyribonucleotides (A, T, and G) are
phospholinked with 4-MU. Incorporation of the derivatized
nucleotides releases the non-fluorescent pentaphosphate 4-MU.
[0218] In a fast coupled reaction, Shrimp Alkaline Phosphatase
(SAP) hydrolyzes the pendant phosphates creating the nascent
fluorescent 7-hydroxyl methylumbelliferyl coumarin (Eid et al.
Science. 2009 Jan. 2; 323(5910):133-8). The increase in the
fluorescent signal with time is proportional to the rate of DNA
polymerization. Steady state polymerization reactions were carried
out using 25 nM phi29 DNA polymerase mutant, 5 nM primed circular
DNA template, 10 .mu.M 4MU-dA6P, 4MU-dG6P, 4MU-dT6P, and 5 .mu.M
Alexa 555-dC6P in 50 mM ACES pH 7.1, 130 mM KOAc, 5 mM DTT, 0.7 mM
MnCl.sub.2, and 0.04 U/ul SAP. The fluorescence was monitored in
plate format using a Beckman Paradigm fluorescence plate reader
(excitation 360 nm, emission 465 nm).
[0219] FIG. 13 shows the fluorescence plotted as a function of time
at varying concentrations of added ZnSO.sub.4. The slope (rate) of
each time course was determined by fitting the data using linear
regression. The rate is plotted as a function of the metal ion
concentration in FIG. 14 along with a similar analysis performed
for Sr, Ba, Co, Sn, and Ca. The degree of inhibition of polymerase
activity can be compared by fitting the inhibition profiles using
nonlinear regression to the equation:
rate = rate 0 ( 1 - [ metal ] [ metal ] + K i ) ##EQU00002##
where rate.sub.0 is the rate of the reaction without additional
metal added and Ki is the inhibition constant for a given metal
ion. The inhibition constants are plotted in FIG. 15 where lower
values of Ki indicate a greater degree of inhibition. Assays of
this type can be used to identify potential non- or lower catalytic
metal cofactors for phi29 DNA polymerase. Experiments performed
using single metals can likewise be performed to identify metal
that can support DNA polymerization (catalytic).
Example 4
Deuterium Addition
[0220] This example demonstrates the increase in mean pulse width
for single molecule sequencing observed with the addition of
deuterium in the form of D.sub.2O. Experiments were conducted using
a Single Molecule Real Time (SMRT.TM.) 4 color sequencing
technology instrument as described herein. A modified phi29 DNA
polymerase having the mutations N62D/T368F/E375Y/K512Y and modified
for streptavidin binding (polymerase R, 5 nM) was mixed with a
circular template/primer complex (30 nM) as described in U.S.
Patent Application [unassigned] Attorney docket number
105-005902US, entitled "Method and Compositions for Nucleic Acid
Sample Preparation" filed Mar. 27, 2009, and U.S. patent
application Ser. No. 12/413,258, and other reagents (e.g. Ca2+
salt, 1 mM and A555-T nucleotide analog, 500 nM) in MOPS pH 7.4
buffer and kept above room temperature for at least 1 hr to form a
polymerase R/template/primer complex. Then solution of the
Polymerase R/template/primer complex was diluted by MOPS pH 7.4
buffer, an aliquot was added to the chip and kept at room
temperature in high humidity chamber for least 15 min. The chip was
then washed at least 5 times with ACES pH 7.1 buffer and a solution
containing 4 fluorescently labeled analogs
(A555-O-aminohexyl-dT6P(A555-T)--channel 1, A568-O-aminohexyl-dG6P
(A568-G)--channel 2, A647-O-aminohexyl-dA6P (A647-A)--channel 3,
and
Cy5.5-NH(CH.sub.2).sub.5C(O)NH(CH.sub.2).sub.6O-dC6P--(Cy5.5-C)--channel
4)--all 500 nM) in ACES pH 7.1 buffer was added to the chip. The
chip was then placed inside the prototype sequencing instrument and
sequencing reaction was started by adding another solution
containing 4 fluorescently labeled analogs and Mn2+ (0.7 mM). Seven
minute data movies were recorded for each condition and data was
processed and analyzed. D2O (99.95+% isotopic purity) was purchased
from Alfa Aesar and used as received. To obtain the final
concentration on the chip, D2O was introduced into the concentrated
ACES buffer and/or used as diluting agent instead of H2O.
[0221] FIG. 16 shows the mean pulse widths for each of the four
dyes corresponding to the four nucleotides. It can be seen that for
each of the four dyes, the mean pulse width increases with the
addition of higher percentages of D.sub.2O. It was determined that
the yield at 25% D.sub.2O was comparable to the yield at 100% H2O,
while the mean pulse width increased by a factor of about 1.5 for
all of the nucleotide analogs tested.
Example 5
Solvent Additives
[0222] Experiments were conducted using a Single Molecule Real Time
(SMRT.TM.) 4-color sequencing instrument as described herein to
collect the data. Polymerase R (5 nM) was mixed with a circular
template/primer complex (30 nM) as described in U.S. Patent
Application [unassigned], Attorney docket number 105-005902US,
entitled "Method and Compositions for Nucleic Acid Sample
Preparation" filed Mar. 27, 2009, and U.S. patent application Ser.
No. 12/413,258 and other reagents (e.g. Ca2+ salt, 1 mM and A555-T
analog, 500 nM) in MOPS pH 7.4 buffer and kept above room
temperature for at least 1 hr to form a Polymerase
R/template/primer complex. Then solution of the Polymerase
R/template/primer complex was diluted 10 times by MOPS pH 7.4
buffer, an aliquot was added to the chip and kept at room
temperature in high humidity chamber for least 15 min. The chip was
then washed at least 5 times with 8 ACES pH 7.1 buffer and a
solution containing 4 fluorescently labeled analogs (A555-T,
A568-G, A647-A, Cy5.5-C--all 500 nM) in ACES pH 7.1 buffer was
added to the chip. The chip was then placed inside the prototype
sequencing instrument and sequencing reaction was started by adding
another solution containing 4 fluorescently labeled analogs and
Mn2+ (0.7 mM). Seven minute data movies were recorded for each
condition and data was processed and analyzed.
[0223] Solvents (dimethylacetamide (DMA--anhydrous, 99.8%),
dimethylsulfoxide (DMSO--99.5%), ethanol (absolute), dioxane
(anhydrous, 99.8%), tetrahydrofuran (THF--99.9%, Chromasolv
grade)--all from Aldrich and methanol (HPLC grade, 99.8%),
acetonitrile (HPLC grade, 99.8%), dimethylformamide (DMF--Drysolv
grade, 99.8%)--all from EMD) were used as received. To obtain the
final concentration, organic solvent additives were introduced into
the concentrated buffers and/or used as diluting agents in place of
water.
[0224] FIG. 17 shows the mean interpulse distance in milliseconds
for each of the nucleotides for a single molecule sequencing
reaction run with various concentrations of dimethylacetamide
(DMA). The data for 5 separate experiments and the average for the
5 experiments is shown in the figure. It can be seen that as the
concentration of DMA is increased, the interpulse distance also
increases for all four of the nucleotides. The measured pulse
widths showed very little change with the addition of DMA (no
measurable change for channels 1-3 and a slight increase for
channel 4).
[0225] FIG. 18 shows the mean interpulse distance in milliseconds
for the each of the 4 dye channels for a single molecule sequencing
reaction run with various concentrations of dimethysulfoxide
(DMSO). The data show that as the DMSO concentration is increased,
the interpulse distance also increases. The pulse widths increased
on the addition of DMSO in channels 1, 2, and 4, and were unchanged
in channel 3.
Example 6
Polymerase Systems Having Two Kinetically Observable Steps--Stopped
Flow Measurements
[0226] This experiment describes the observation of a polymerase
system having two kinetically observable steps (two slow steps)
where the two kinetically observable steps occur while the
nucleotide is associated with the enzyme (after nucleotide binding
and through product release. In the experiment described here, the
two kinetically observable steps would correspond to steps
occurring in the bright state of a single-molecule sequencing
system using nucleotides having dyes attached to the terminal
phosphate of the nucleotides.
[0227] For this assay we use a SF-2004 stopped flow instrument
(Kintek Corp, Austin, Tex.) to monitor the fluorescence at 535 nm
(using a band pass filter), to measure Alexa fluor 488 emission.
The experimental design is the same as for example 2. The enzyme,
DNA, buffer, potassium acetate, and dithiothreitol (DTT) are mixed
in one sample and allowed to equilibrate. Alexa-555-dC6P (a
terminally labeled hexaphosphate nucleotide substrate), buffer,
potassium acetate, DTT, MnCl.sub.2, and CaCl.sub.2 are mixed in a
second sample. The stopped flow instrument rapidly mixes these
samples and reads the fluorescent signal at 535 nm as a function of
time.
[0228] The binding of the nucleotide to the enzyme-DNA complex is
often observed to occur as a single exponential decrease in the
fluorescence signal, indicating a process with a single kinetically
observable step. Where the steps of the polymerase reaction from
after binding through release of the pentaphosphate-dye molecule
are governed by a single rate-limiting step a single exponential
increase in the fluorescent signal is expected. Thus, in the
scenario where nucleotide binding and the subsequent steps through
product release are each governed by single rate-limiting steps, we
observe a fluorescent signal that is adequately described by a sum
of two exponentials.
[0229] FIG. 19 shows the data from a polymerase reaction system in
which the decrease in the fluorescent signal fits to a single
exponential having an observed rate constant of 156.+-.3 s.sup.-1,
and the increase in signal fits to a single exponential having an
observed rate constant of 8.5.+-.0.1 s.sup.-1. FIG. 19 includes
both the experimental data and the curve fits for single
exponential decay and rise in fluorescence. The polymerase reaction
shown in FIG. 19 involved the polymerase enzyme Polymerase R in 50
mM ACES buffer at a pH of 7.1. The assay was performed with the
following components and amounts: 0.125 .mu.M polymerase R enzyme,
0.025 .mu.M DNA, 50 mM ACES, pH 7.1, 0.7 mM MnCl.sub.2, 75 mM
potassium acetate, 5 mM dithiothreitol, 3 .mu.M alexa 555-dC6P. The
observed fluorescent signal was fit to a sum of two exponentials,
where the rate of the drop is 156.+-.3 s.sup.-1, and the rate of
the increase in signal is 8.5.+-.0.1 s.sup.-1.
[0230] FIG. 20 shows the data for a polymerase reaction system
which exhibits two kinetically observable steps for the steps after
nucleotide binding through product release. The polymerase reaction
used the enzyme polymerase R in 50 mM Tris buffer, at pH 7.1, with
0.25 mM CaCl.sub.2. The assay used 0.125 .mu.M polymerase R enzyme,
0.025 .mu.M DNA, 50 mM Tris, pH 7.1, 0.7 mM MnCl.sub.2, 0.25 mM
CaCl.sub.2, 75 mM potassium acetate, 5 mM dithiothreitol, 3 .mu.M
alexa 555-dC6P. A good fit to the data could not be obtained with
two exponentials. However, a good quality fit was obtained using
the sum of three exponentials. The drop in fluorescence occurs with
a single exponential having an observed rate constant of 172.+-.12
s.sup.-1. The increase in fluorescence is best described as the sum
of two exponentials, where the faster of the two steps occurs with
an observed rate constant of 60.+-.10 s.sup.-1, and the slower of
the two steps occurs with an observed rate constant of 12.0.+-.0.1
s.sup.-1. The behavior of this system is best described by two
kinetically observable steps during the part of the polymerase
reaction in which the nucleotide is associated with the enzyme.
Each of the steps is partially rate-limiting. The observed
fluorescent signal is fit to a sum of three exponentials, where the
observed rate constant for the drop in fluorescence is 172.+-.12
s.sup.-1, and the increase in fluorescence exhibits two kinetically
observable rate constants, one at 60.+-.10 s.sup.-1 and the other
at 12.0.+-.0.1 s.sup.-1.
[0231] FIG. 21 shows stopped flow experimental data for a
polymerase having a drop in fluorescence and a rise in fluorescence
which each can be fit to a single exponential. FIG. 21 shows the
incorporation of Alexa 555-dC6P by a phi29 DNA polymerase enzyme
having the mutations N62D/T368F/E375Y/A484E/K512Y and modified for
streptavidin binding (polymerase T) in 50 mM Tris buffer, pH 7.1.
The assay used 0.125 .mu.M polymerase T enzyme, 0.025 .mu.M DNA, 50
mM Tris, pH 7.1, 0.7 mM MnCl2, 75 mM potassium acetate, 5 mM
dithiothreitol, 3 .mu.M alexa 555-dC6P. The observed fluorescent
signal is fit to a sum of two exponentials, where the rate of the
drop has an observed rate constant of 118.+-.4 s.sup.-1, and the
increase in the signal rate-limiting step occurs with an observed
rate constant of 46.+-.1 s.sup.-1.
[0232] FIG. 22 illustrates how changing the polymerase reaction
conditions can produce a polymerase reaction system which exhibits
two kinetically observable rate-limiting steps for the steps after
nucleotide binding through product release. In this case, we
believe that specific enzyme mutations in the polymerase T enzyme,
coupled with the presence of Ca.sup.++ under the conditions of the
polymerase reaction described results in additional mutations of
the enzyme has changed the kinetic performance of the system to
obtain a system in which there are two kinetically observable rate
constants between nucleotide binding through product release with
almost equal rate constants. FIG. 22 shows stopped flow data for
the incorporation of Alexa 555-dC6P by polymerase enzyme polymerase
Tin 50 mM Tris buffer, pH 7.1, with 1.25 mM CaCl.sub.2. The assay
used 0.125 .mu.M polymerase T enzyme, 0.025 .mu.M DNA, 50 mM Tris,
pH 7.1, 0.7 mM MnCl.sub.2, 1.25 mM CaCl.sub.2, 75 mM potassium
acetate, 5 mM dithiothreitol, 3 .mu.M alexa 555-dC6P. FIG. 22(a)
shows an attempt to fit the data with two exponentials, one for the
decay, and the other for the rise in fluorescence. It can be seen
from FIG. 22(a) that the data is not well described in this manner.
22(b) shows the observed fluorescent signal fit to a sum of three
exponentials where the rate constant for the drop in fluorescence
is 157.+-.5 s.sup.-1, and the increase in the signal exhibits two
kinetically observable steps, where one step exhibits an observed
rate constant of 9.+-.2 s.sup.-1 and the other step exhibits a rate
constant of 7.+-.1 s.sup.-1. We note that the conditions that
resulted in the two kinetically observable steps of FIG. 22(b) are
the same as those for the experiment shown in FIG. 21, except for
the presences of CaCl.sub.2 at a concentration of 1.25 mM in this
experiment, illustrating that a polymerase reaction exhibiting two
slow steps can be produced by controlling the polymerase reaction
conditions.
Example 7
Rapid Chemical Quench Experiment to Observe Two Kinetically
Observable Steps for the Steps after Product Release Through
Nucleotide Binding
[0233] The presence of two kinetically observable steps after
product release through nucleic acid binding can be observed by
measuring the difference in the kinetics of single incorporation
and multiple incorporations. First, a transient incorporation
nucleotide incorporation assay (rapid chemical quench flow or
stopped flow fluorescence) is run in order to determine the
apparent rate constant for binding of a first nucleotide. Next, the
experiment is run such that two nucleotides are incorporated. By
comparing the kinetic parameters for the incorporation of two
nucleotides as compared to those for incorporating one nucleotide,
it can be determined whether there is an intervening step, such as
translocation or isomerization which significantly limits the rate.
Where such a step is identified, the pseudo first order rate
constant of the nucleotide binding step can be lowered by lowering
the concentration of nucleotide. In this manner, a system having
two slow steps in the phase after product release and through
nucleotide binding can be produced by matching the apparent rate
constant of nucleotide binding with that the preceding
isomerization or translocation event.
Example 8
Effect of L-Cysteine on Single Molecule Sequencing
[0234] Experiments were conducted using a Single Molecule Real Time
(SMRT.TM.) 4-color sequencing instrument as described above for
solvent additives. L-Cysteine, Hydrochloride (99.6%) was purchased
from Calbiochem and used as received. Cysteine solution was
introduced in a solution of ACES buffer. FIG. 24 shows that the
pulse width increases with increasing amounts of added cysteine to
the sequencing reaction mixture. The effect is seen in all four
nucleotide/dye channels. In addition to the increase in pulse
width, the addition of cysteine led to increases in accuracy and in
yield. When 0.25 mM cysteine was added, the overall yield increased
2.5 times, and accuracy was increased by 4.7%. over a control
reaction having no added cysteine.
Example 9
High Throughput Screen for Polymerase Mutants with Slow Product
Release
[0235] As described above, polymerases exhibiting slow release of
polyphosphate product are of particular interest, e.g., in
producing polymerases exhibiting two slow steps for use in single
molecule sequencing. Screening polymerase mutants using a
stopped-flow assay to determine kinetic parameters, however, can be
time-consuming. A higher throughput format for identifying
polymerase variants exhibiting slow product release has thus been
developed.
[0236] In the screen, each candidate polymerase mutant is employed
in a primer extension reaction using a DNA template (e.g., a
circular DNA template) and four dNTPs or analogs, in the presence
or absence of a competitive inhibitor. Nucleotide incorporation is
measured based upon elongation rate of the polymerization reaction,
as determined from the change in synthesis product size (e.g., as
determined by agarose gel electrophoresis).
[0237] Suitable competitive inhibitors include, but are not limited
to, Z-6-aminohexylpentaphosphate (Cbz-X-5P). Synthesis of Cbz-X-5P
has been described in U.S. patent application Ser. No. 12/370,472,
which also describes additional exemplary inhibitors. Without
limitation to any particular mechanism, Cbz-X-5P mimics the
polyphosphate reaction product and competes with dNTP binding,
slowing primer extension. The assay is predicated on product
affinity as an indication of slow product release; that is, mutants
with slower product release are expected to have greater affinity
for the competitive inhibitor and thus show a slower extension
rate. Candidate mutants identified by the primer extension screen
as potentially having decreased product release rates can be
verified if desired, e.g., by stopped-flow measurements. The screen
is optionally automated or partially automated.
[0238] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations. All publications, patents, patent applications,
and/or other documents cited in this application are incorporated
by reference in their entirety for all purposes to the same extent
as if each individual publication, patent, patent application,
and/or other document were individually and separately indicated to
be incorporated by reference for all purposes.
* * * * *