U.S. patent application number 13/169991 was filed with the patent office on 2012-06-28 for apparatus and method for performing nucleic acid analysis.
This patent application is currently assigned to Pacific Biosciences of California, Inc.. Invention is credited to Jonas Korlach, Stephen Turner.
Application Number | 20120164631 13/169991 |
Document ID | / |
Family ID | 36072938 |
Filed Date | 2012-06-28 |
United States Patent
Application |
20120164631 |
Kind Code |
A1 |
Turner; Stephen ; et
al. |
June 28, 2012 |
APPARATUS AND METHOD FOR PERFORMING NUCLEIC ACID ANALYSIS
Abstract
The present invention relates to optical confinements, methods
of preparing and methods of using them for analyzing molecules
and/or monitoring chemical reactions. The apparatus and methods
embodied in the present invention are particularly useful for
high-throughput and low-cost single-molecular analysis.
Inventors: |
Turner; Stephen; (Menlo
Park, CA) ; Korlach; Jonas; (Menlo Park, CA) |
Assignee: |
Pacific Biosciences of California,
Inc.
Menlo Park
CA
|
Family ID: |
36072938 |
Appl. No.: |
13/169991 |
Filed: |
June 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11925675 |
Oct 26, 2007 |
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13169991 |
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11228759 |
Sep 16, 2005 |
7315019 |
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11925675 |
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10944106 |
Sep 17, 2004 |
7170050 |
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11228759 |
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60649009 |
Jan 31, 2005 |
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60651846 |
Feb 9, 2005 |
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Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
G01N 21/77 20130101;
G01N 21/648 20130101; C12Q 1/6869 20130101; G01N 21/6452 20130101;
G01N 21/6428 20130101; G01N 2201/06113 20130101; C12Q 1/6806
20130101; G01N 2021/6439 20130101; G01N 2021/7786 20130101; G01N
2021/6463 20130101; G01N 21/00 20130101; G01N 21/645 20130101 |
Class at
Publication: |
435/6.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 21/64 20060101 G01N021/64; G01N 21/75 20060101
G01N021/75 |
Claims
1. A method of increasing accuracy of nucleic acid sequencing, the
method comprising the steps of: a) performing a first single
molecule sequencing reaction on at least a first region of a
template, thereby obtaining a nucleotide sequence of at least the
first region of the template; and b) performing a second single
molecule sequencing reaction on at least the first region of the
template to resequence the first region of the template, thereby
increasing the accuracy of nucleic acid sequencing.
2. The method of claim 1, wherein the sequence obtained in a) is
compared with the sequence obtained in b).
3. The method of claim 1, further comprising the step of performing
at least a third single molecule sequencing reaction on at least
the first region of the template.
4. The method of claim 1, wherein: the first single molecule
sequencing reaction comprises exposing a template/first primer
duplex to a polymerase and one or more nucleotides comprising a
detectable label under conditions sufficient for template-dependent
nucleotide addition to the first primer, the first primer being
hybridized to the first region of the template, wherein the
template/first primer duplex is individually optically
resolvable.
5. The method of claim 4, wherein the second single molecule
sequencing reaction comprises continuing the template-dependent
nucleotide addition to the first primer.
6. The method of claim 4, wherein the label is an
optically-detectable label.
7. The method of claim 6, wherein the optically-detectable label is
a fluorescent label.
8. The method of claim 7, wherein the fluorescent label is selected
from the group consisting of fluorescein, rhodamine, cyanine, Cy5,
Cy3, BODIPY, alexa, and derivatives thereof.
9. The method of claim 4, wherein the template/first primer duplex
is attached to a surface.
10. The method of claim 1, wherein a plurality of regions are
sequenced.
11. The method of claim 10, wherein a plurality of regions are
resequenced.
12. A method of increasing accuracy of nucleic acid sequencing, the
method comprising the steps of: a) performing a first set of single
molecule sequencing reactions on a plurality of regions of a
template, thereby obtaining a nucleotide sequence of at least one
of the plurality of regions of the template; and b) performing a
second set of single molecule sequencing reactions on the plurality
of regions of the template to resequence the at least one of the
plurality of regions of the template, thereby increasing the
accuracy of nucleic acid sequencing.
13. The method of claim 12, wherein the sequence obtained in a) is
compared with the sequence obtained in b).
14. The method of claim 12, further comprising the step of
performing at least a third set of single molecule sequencing
reactions on the at least one of the plurality of regions of the
template.
15. The method of claim 12, wherein the sequencing reactions
comprise exposing a duplex comprising the template and at least one
primer to a polymerase and to one or more nucleotides comprising a
detectable label under conditions for template-dependent nucleotide
addition to the primer, which is hybridized to the template,
wherein the detectable label is an optically-detectable label, and
further wherein the duplex is individually optically
resolvable.
16. The method of claim 15, wherein the optically-detectable label
is a fluorescent label.
17. The method of claim 16, wherein the fluorescent label is
selected from the group consisting of fluorescein, rhodamine,
cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof.
18. The method of claim 17, wherein the duplex is attached to a
surface.
19. The method of claim 12, wherein the template is a circular
template.
20. The method of claim 12, wherein the template is a concatemeric
template.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/925,675, filed Oct. 26, 2007, which is a
continuation of U.S. patent application Ser. No. 11/228,759, filed
Sep. 16, 2005, now U.S. Pat. No. 7,315,019, which (1) is a
continuation-in-part of U.S. patent application Ser. No.
10/944,106, filed Sep. 17, 2004, now U.S. Pat. No. 7,170,050; and
(ii) claims the benefit of U.S. Provisional Application Nos.
60/649,009 and 60/651,846 filed on Jan. 31, 2005 and Feb. 9, 2005,
respectively, the disclosures of all of which are incorporated
herein by reference in their entireties for all purposes.
BACKGROUND OF THE INVENTION
[0002] Confinement of illumination and signal detection has long
been recognized as an important tool in molecular diagnostics since
the application of Fluorescence Correlation Spectroscopy (FCS). FCS
involves illumination of a sample volume containing
fluorophore-labeled molecules, and detection of fluctuations in
fluorescence signal produced by the molecules as they diffuse into
and out of an effective observation volume. The fluorescence
intensity fluctuations can best be analyzed if the volume under
observation contains only a small number of fluorescent molecules,
and if the background signal is low. This can be accomplished by
the combination of a drastically limited detection volume and a low
sample concentration. The detection volume of traditional FCS is
approximately 0.5 femtoliters (or 0.5.times.10.sup.-15 liters), and
is achieved through the use of a high numerical aperture microscope
objective lens to tightly focus a laser beam. In this detection
volume, single molecules can be observed in solutions at
concentrations of up to approximately one nanomolar. This
concentration range is unacceptably low for most biochemical
reactions, which have reaction constants that are typically in or
above the micromolar range. At lower concentrations, these
reactions either do not proceed acceptably fast, or behave in a
qualitatively different fashion than is useful in most analyses. To
observe single molecules at higher, more relevant concentrations,
the observation volume would typically need to be reduced to far
smaller dimensions.
[0003] In recent years, the advancement in nanofabrication
technology enabled the production of nanoscale devices that are
integrated with electrical, optical, chemical and/or mechanical
elements.
[0004] However, there still remains a considerable need for
chemical and biological analyses that are faster, cheaper and of
greater accuracy, to provide for the ability to observe single
molecule reactions under conditions that are more biologically or
diagnostically relevant. There also exists a need for small, mass
produced, and disposable devices that can aid in these goals by
providing optical confinements that are amenable to single-molecule
analysis at a higher concentration. The present invention satisfies
these needs and provides related advantages as well.
SUMMARY OF THE INVENTION
[0005] A principal aspect of the present invention is the design of
optical devices and methods for characterizing molecules and/or
monitoring chemical reactions. The devices and methods of the
present invention are particularly suited for single-molecule
analysis.
[0006] Accordingly, the present invention provides an array of
optical confinements having a surface density exceeding
4.times.10.sup.4 confinements per mm.sup.2, preferably exceeding
10.sup.5 confinements per mm.sup.2. In one aspect, the individual
confinement in the array provides an effective observation volume
that is less than one nanoliter (10.sup.-9 liter), less than one
picoliter, or less than one femtoliter, preferably on the order of
zeptoliters. In other aspects, each of the individual confinements
provides an effective observation volume that is less than 1000
zeptoliters, 100 zeptoliters, 80 zeptoliters, or less than 50
zeptoliters, or even less than 10 zeptoliters.
[0007] In other aspects, each of the individual confinements yields
an effective observation volume that permits resolution of
individual molecules present at a concentration that is higher than
one nanomolar, or higher than 100 nanomolar, or on the order of
micromolar range. In certain preferred aspects, each of the
individual confinements yields an effective observation volume that
permits resolution of individual molecules present at a
physiologically relevant concentration, e.g., at a concentration
higher than about 1 micromolar, or higher than 50 micromolar range
or even higher than 100 micromolar. The array may comprise a
zero-mode waveguide or other nanoscale optical structure. The array
of optical confinements may further comprise another array of
confinements that does not yield the above-described effective
observation volume or does not permit resolution of individual
molecules. For example, the array of optical confinements may be
coupled with or integrated into a microtiter plate, where a
separate array of optical confinements may be disposed within each
of several different wells on a multiwell reaction plate. The array
of optical confinements may comprise at least about
2.times.10.sup.5 optical confinements, or at least about 10.sup.6,
or at least about 10.sup.7 optical confinements.
[0008] In another embodiment, the present invention provides a
method of creating a plurality of optical confinements having the
aforementioned characteristics. The method involves the steps of
(a) providing a substrate; and (b) forming an array of optical
confinements having a surface density exceeding 4.times.10.sup.4
confinements per mm.sup.2, wherein the individual confinement
comprises a zero-mode waveguide comprising: a cladding surrounding
a core, wherein said cladding is configured to preclude propagation
of electromagnetic energy of a wavelength longer than a cutoff
wavelength longitudinally through the core of the zero-mode
waveguide; and (c) illuminating the array with an electromagnetic
radiation of a frequency less than the cutoff frequency, thereby
creating the plurality of optical confinements.
[0009] In another embodiment, the present invention provides a
method of creating an optical observation volume that permits
resolution of individual molecules. The method involves providing a
zero-mode waveguide that comprises a cladding surrounding a core,
wherein said cladding is configured to preclude propagation of
electromagnetic energy of a frequency less than a cutoff frequency
longitudinally through the core of the zero-mode waveguide, wherein
upon illuminating the zero-mode waveguide with an electromagnetic
radiation of a frequency less than the cutoff frequency, the
zero-mode waveguide yields an effective observation volume that
permits resolution of individual molecules. In certain aspects, the
effective observation volume is less than one nanoliter (10.sup.-9
liter), less than one picoliter, or less than one femtoliter,
preferably on the order of zeptoliters. Using the zero-mode
waveguide of the present invention, one typically can obtain an
effective observation volume that is less than 100 zeptoliters
(100.times.10.sup.-21 liters) or less than 50 zeptoliters, or even
less than 10 zeptoliters. In other aspects, the method yields an
effective observation volume that permits resolution of individual
molecules present at a concentration that is higher than one
nanomolar, more often higher than 100 nanomolar, and preferably on
the order of micromolar range. In preferred embodiments, individual
molecules present at a concentration higher than about 5
micromolar, or higher than 7.5 micromolar, or even higher than 50
micromolar range, can be resolved by the method of the present
invention.
[0010] The present invention also provides a method of detecting
interactions among a plurality of molecules. The method comprises
the steps of (a) placing the plurality of molecules in close
proximity to an array of zero-mode waveguides, wherein individual
waveguides in the array are separated by a distance sufficient to
yield detectable intensities of diffractive scattering at multiple
diffracted orders upon illuminating the array with an incident
wavelength; (b) illuminating the array of zero-mode waveguides with
an incident wavelength; and (c) detecting a change in the
intensities of diffractive scattering of the incident wavelength at
the multiple diffracted orders, thereby detecting the interactions
among a plurality of molecules.
[0011] The present invention also provides a method of reducing
diffractive scattering upon illuminating an array of optical
confinements with an incident wavelength, wherein the array
comprises at least a first optical confinement and a second optical
confinement, said method comprising: forming the array of optical
confinements wherein the optical confinement is separated from the
second optical confinement by a distance such that upon
illumination with the incident wavelength, intensity of diffractive
scattering resulting from the first optical confinement at a given
angle is less than that if the first optical confinement were
illuminated with the same incident wavelength in the absence of the
optical confinement. In preferred aspects, the aforementioned
optical confinements are zero mode waveguides.
[0012] The present invention also includes a method of detecting a
biological analyte using an array of optical confinements having a
density on a substrate exceeding 4.times.10.sup.4 confinements per
mm.sup.2 or any other density described herein or equivalents
thereof. The method typically involves illuminating at least one
optical confinement within the array that is suspected to contain
the analyte with an incident light beam. The invention also
provides a method of using of an array of optical confinements
having a density on a substrate exceeding 4.times.10.sup.4
confinements per mm.sup.2 any other density described herein or
equivalents thereof for performing multiple chemical reactions. The
method comprises the steps of placing the plurality of reaction
samples comprising labeled reactants into the optical confinements
in the array, wherein a separate reaction sample is placed into a
different confinement in the array; subjecting the array to
conditions suitable for formation of products of the chemical
reactions; and detecting the formation of the products with said
optical system.
[0013] In addition, the invention provides a method of sequencing a
plurality of target nucleic acid molecules. The method typically
involves (a) providing an array of optical confinements having a
density on a substrate exceeding 4.times.10.sup.4 confinements per
mm.sup.2, or any other density described herein or equivalents
thereof, wherein said optical confinements provide an effective
observation volume that permits observation of individual
molecules; and an optical system operatively coupled to the optical
confinements that detects signals from the effective observation
volume of said confinement; (b) mixing in the optical confinements
the plurality of target nucleic acid molecules, primers
complementary to the target nucleic acid molecules, polymerization
enzymes, and more than one type of nucleotides or nucleotide
analogs to be incorporated into a plurality of nascent nucleotide
strands, each strand being complementary to a respective target
nucleic acid molecule; (c) subjecting the mixture of step (b) to a
polymerization reaction under conditions suitable for formation of
the nascent nucleotide strands by template-directed polymerization
of the nucleotides or nucleotide analogs; (d) illuminating the
optical confinements with an incident light beam; and (e)
identifying the nucleotides or the nucleotide analogs incorporated
into the each nascent nucleotide strand.
[0014] The present invention also provides an apparatus comprising
an array of waveguides on a solid support having a fill fraction
greater than about 0.0001, wherein said waveguides are suitable for
holding a biological reagent, and wherein waveguides provide an
effective observation volume that permits observation of individual
molecules present in said biological reagent; and an optical system
that detects said individual molecules in said waveguides, by e.g.,
detecting signals from the effective observation volume. In one
aspect, the array has a fill fraction greater than about 0.001. In
another aspect, the array has a fill fraction greater than about
0.01, in some instances greater than 0.1, or within the range about
0.001 to about 0.1.
[0015] The present invention also provides various methods of using
such high fill fraction array. In one embodiment, the present
invention provides a method of detecting a biological analyte. The
method comprises optically capturing the analyte within an optical
confinement that is created by (a) providing an array of waveguides
having a fill fraction greater than about 0.0001; and (b)
illuminating at least one waveguide within the array that is
suspected to contain the analyte with an incident light beam
thereby detecting the analyte.
[0016] In another embodiment, the present invention provides a
method of performing multiple chemical reactions involving a
plurality of reaction samples using the subject high fill fraction
array. The method involves (a) providing a subject high fill
fraction array; (b) placing the plurality of reaction samples
comprising labeled reactants into the waveguides in the array,
wherein a separate reaction sample is placed into a different
waveguide in the array; (c) subjecting the array to conditions
suitable for formation of products of the chemical reactions; and
(d) detecting the formation of the products with an optical system.
The step of detecting may comprise illuminating the different
waveguides with an incident light beam and detecting an optical
signal emitted from the reaction samples. Applicable chemical
reactions may involve protein-protein interactions, nucleic
acid-protein interactions, and nucleic acid-nucleic acid
interactions. Specifically, the present invention provides a method
of sequencing a plurality of target nucleic acid molecule using a
fill fraction greater than about 0.0001.
[0017] The present invention further provides a method of
sequencing nucleic acid using an array having a high fill faction.
The method typically involves a) providing an array of waveguides
having a fill fraction greater than about 0.0001, or 0.001, or 0.01
or even 0.1; (b) mixing in the waveguides the plurality of target
nucleic acid molecules, primers complementary to the target nucleic
acid molecules, polymerization enzymes, and more than one type of
nucleotides or nucleotide analogs to be incorporated into a
plurality of nascent nucleotide strands, each strand being
complementary to a respective target nucleic acid molecule; (c)
subjecting the mixture of step (b) to a polymerization reaction
under conditions suitable for formation of the nascent nucleotide
strands by template-directed polymerization of the nucleotides or
nucleotide analogs; (d) illuminating the waveguides with an
incident light beam; and (e) identifying the nucleotides or the
nucleotide analogs incorporated into each nascent nucleotide
strand.
[0018] Also included in the present invention is a redundant
sequencing method. The method comprises (a) subjecting a target
nucleic acid molecule to a template-directed polymerization
reaction to yield a nascent nucleic acid strand that is
complementary to the target nucleic acid molecule in the presence
of a plurality of types of nucleotides or nucleotide analogs, and a
polymerization enzyme exhibiting strand-displacement activity; and
(b) registering a time sequence of incorporation of nucleotides or
nucleotide analogs into the nascent nucleotide strand. In one
aspect of this embodiment, the target nucleic acid molecule is a
circular nucleic acid, or is a linear or circular template strand
synthesized from a circular nucleic acid sequence such that the
synthesized strand includes multiple repeated copies of the
original circular strand, and is thus is subject to the sequencing
operations of the invention. In another aspect of this embodiment,
the target nucleic acid molecule is sequenced multiple times, e.g.,
more than once, or more than twice by the polymerization enzyme. In
yet another aspect of this embodiment, the polymerization enzyme is
a DNA polymerase, such as a modified or unmodified .PHI.29
polymerase.
[0019] Further included in the present invention is a solid support
having a surface wherein the surface has a polymerization enzyme
array attached to it, wherein members of the array comprise
individually and optically resolved polymerization enzymes
possessing strand-displacement activities.
[0020] Also provided is a zero mode waveguide, comprising a first
molecular complex immobilized therein, said molecular complex
comprising a polymerization enzyme complexed with a target nucleic
acid, wherein the polymerization enzyme processes a sequence of
nucleotides in said target nucleic acid multiple times via
template-dependent replication of the target nucleic acid.
[0021] Further provided by the present invention is a method of
fabricating an array of optical confinements that exhibits a
minimal intensity of diffractive scattering of an incident
wavelength. The method comprises providing a substrate; and forming
the array of optical confinements on the substrate such that
individual confinements in the array are separated from each other
at a distance less than one half of the wavelength.
[0022] Finally, the present invention includes a method of
fabricating an optical confinement the method comprises a cladding
surrounding a core, comprising: (a) providing a substrate coated
with a layer of photoresist; (b) patterning said layer of
photoresist to define boundaries of said core; (c) removing said
layer of photoresist surrounding said defined boundaries so that a
sufficient amount of photoresist remains to occupy said core; (d)
depositing a layer of cladding material over said remaining
photoresist and said substrate; (e) removing at least a portion of
said cladding material deposited over said remaining photoresist;
and (f) removing said photoresist of step (e) to form said core
surrounded by said cladding of said optical confinement. In one
aspect, the photoresist is negative and said patterning step
employs a positive pattern. In another aspect, the photoresist is
positive and said patterning step employs a negative pattern. The
removing step can be effected by a technique selected from the
group consisting of etching, mechanical polishing, ion milling, and
solvent dissolution. The layer of cladding material can be
deposited by a thermal evaporation method or vapor deposition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts a top view of an array of illustrative
optical confinements, here zero-mode waveguides arranged in a
square format.
[0024] FIG. 2 depicts a top view of an array of illustrative
optical confinements, here zero-mode waveguides arranged in a
non-square format.
[0025] FIG. 3 depicts a top view of an illustrative 2-dimentional
array with an illustrative angle and two different unit vector
lengths.
[0026] FIG. 4 depicts a top view of an illustrative regular
disposition of ZMWs.
[0027] FIG. 5 depicts an array of arrays, in which a subarray 71 is
part of a super array 72.
[0028] FIG. 6 illustrates a process of negative tone
fabrication.
[0029] FIG. 7 illustrates an array of ZMWs optically linked to an
optical system.
[0030] FIGS. 8A and 8B depict scanning electron micrographs of ZMW
structures fabricated by positive tone resist (left panels) or
negative tone resist (right panels). The grain structure of the
polycrystalline film is visible in the image as flecks, and the
ZMWs as dark round structures.
[0031] FIGS. 9A, 9B and 9C depict a single-molecule DNA sequence
pattern recognition in ZMWs using artificial pre-formed replication
forks.
[0032] FIG. 10, depicts a coated ZMW 101 that is bound to a
substrate 105. The ZMW comprises a sidewall 102, a coating 103 on
the upper surface, and a metal film 104.
[0033] FIG. 11 depicts one alignment strategy and optical
setup.
[0034] FIG. 12 depicts an alternative optical confinement made of
porous film 91 on a substrate 93. 92 represents the pores in the
film.
[0035] FIGS. 13A-B depict an alignment detection system and the
associated components.
[0036] FIGS. 14A, 14B, 14C and 14D depict several exemplary
photocleavable blockers and the applicable wavelength applied to
cleave the blocking groups.
[0037] FIG. 15 depicts an exemplary reversible extension terminator
in which the photocleavable blacker is conjugated to a detectable
label (e.g., fluorescent label).
[0038] FIG. 16 depicts an exemplary profile of fluorescent bursts
corresponding to the time sequence of incorporation of two types of
labeled nucleotides or nucleotide analogs in single-molecule
sequencing reaction using the subject optical confinement.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of Integrated Circuit
(IC) processing biochemistry, chemistry, molecular biology,
genomics and recombinant DNA, which are within the skill of the
art. See, e.g., Stanley Wolf et al., SILICON PROCESSING FOR THE
VLSI ERA, Vols 1-4 (Lattice Press); Michael Quirk et al.,
SEMICONDUCTOR MANUFACTURING TECHNOLOGY; Sambrook, Fritsch and
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd edition
(1989); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995), all of which are incorporated herein by
reference.
DEFINITIONS
[0040] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise.
[0041] "Luminescence" refers to the emission of light from a
substance for any reason other than a rise in its temperature. In
general, atoms or molecules emit photons of electromagnetic energy
(e.g., light) when then move from an "excited state" to a lower
energy state (usually the ground state); this process is often
referred to as "decay". There are many causes of excitation. If
exciting cause is a photon, the luminescence process is referred to
as "photoluminescence". If the exciting cause is an electron, the
luminescence process is referred to as "electroluminescence". More
specifically, electroluminescence results from the direct injection
and removal of electrons to form an electron-hole pair, and
subsequent recombination of the electron-hole pair to emit a
photon. Luminescence which results from a chemical reaction is
usually referred to as "chemiluminescence". Luminescence produced
by a living organism is usually referred to as "bioluminescence".
If photoluminescence is the result of a spin allowed transition
(e.g., a single-singlet transition, triplet-triplet transition),
the photoluminescence process is usually referred to as
"fluorescence". Typically, fluorescence emissions do not persist
after the exciting cause is removed as a result of short-lived
excited states which may rapidly relax through such spin allowed
transitions. If photoluminescence is the result of a spin forbidden
transition (e.g., a triplet-singlet transition), the
photoluminescence process is usually referred to as
"phosphorescence". Typically, phosphorescence emissions persist
long after the exciting cause is removed as a result of long-lived
excited states which may relax only through such spin-forbidden
transitions. A "luminescent label" or "luminescent signal" may have
any one of the above-described properties.
[0042] The term "electromagnetic radiation" refers to
electromagnetic waves of energy including, for example, in an
ascending order of frequency (or alternatively, in a descending
order of wavelength), infrared radiation, visible light,
ultraviolet (UV) light, X-rays, and gamma rays.
[0043] As used herein, an "effective observation volume" typically
refers to that volume that is observable by the detection means
employed for a given application. For example, in the case of
fluorescence based detection, it is that volume which is exposed to
excitation radiation and/or from which emission radiation is
gathered by an adjacent optical train/detector. By way of example,
in the case of a zero mode waveguide used for certain applications,
an effective observation volume is dictated by the propagation of
excitation radiation into the waveguide core, and particularly that
volume that is exposed to light that is at least 1%, and preferably
at least 10% of the original intensity of excitation radiation
entering the waveguide core. Such intensities and volumes are
readily calculable from the particular conditions of the
application in question, including the wavelength of the excitation
radiation and the dimensions of the waveguide core (See, e.g., U.S.
Pat. No. 6,917,726, incorporated herein by reference in its
entirety for all purposes).
[0044] A "primer" is a short polynucleotide, generally with a free
3' OH group, that binds to a target nucleic acid (or template)
potentially present in a sample of interest by hybridizing with the
target nucleic acid, and thereafter promoting polymerization of a
polynucleotide complementary to the target.
[0045] The terms "operatively linked to" or "operatively coupled
to" are used interchangeably herein. They refer to a juxtaposition
wherein the components so described are in a relationship
permitting them to function in their intended manner.
[0046] The term "nucleotide" generally refers to a molecule
comprising a base, sugar and one or more anionic groups, preferably
phosphates. The molecule may comprise one, two, three, four, five
or more phosphates groups and/or other groups such as sulfate. The
term also encompasses nucleotide analogs that are structurally
analogous to naturally occurring nucleotides and are capable of
acting substantially like nucleotides, for example exhibiting base
complementarity with one or more of the bases that occur in DNA or
RNA, and/or being capable of base-complementary incorporation in
synthesizing nucleotide strand by a polymerization enzyme.
[0047] The term "polynucleotide" refers to a polymeric form of
"nucleotides" of any length.
[0048] A "type of nucleotide" refers to a set of nucleotides that
share a common characteristic that is to be detected. For instance,
the types of nucleotides can be classified into four categories: A,
T, C, and G for DNA, or A, U, C and G for RNA. In some embodiments,
each type of nucleotides used in the a reaction will be labeled
with a unique label that is distinguishable from the rest.
[0049] The term "optical confinement" refers to an area in which
the reactants for an intended reaction within the confinement are
confined and resolved by optical means.
[0050] A "polynucleotide probe" refers to a polynucleotide used for
detecting or identifying its corresponding target polynucleotide in
a hybridization reaction.
[0051] The term "hybridize" as applied to a polynucleotide refers
to the ability of the polynucleotide to form a complex that is
stabilized via hydrogen bonding between the bases of the nucleotide
residues in a hybridization reaction. The hydrogen bonding may
occur by Watson-Crick base pairing, Hoogstein binding, or in any
other sequence specific manner. The complex may comprise two
strands forming a duplex structure, three or more strands forming a
multi-stranded complex, a single self-hybridizing strand, or any
combination of these. The hybridization reaction may constitute a
step in a more extensive process, such as the initiation of a PCR
reaction, or the enzymatic cleavage of a polynucleotide by a
ribozyme.
[0052] Hybridization can be performed under conditions of different
"stringency". Relevant conditions include temperature, ionic
strength, time of incubation, the presence of additional solutes in
the reaction mixture such as for a ide, and the washing procedure.
Higher stringency conditions are those conditions, such as higher
temperature and lower sodium ion concentration, which require
higher minimum complementarity between hybridizing elements for a
stable hybridization complex to form. In general, a low stringency
hybridization reaction is carried out at about 40.degree. C. in
10.times.SSC or a solution of equivalent ionic
strength/temperature. A moderate stringency hybridization is
typically performed at about 50.degree. C. in 6.times.SSC, and a
high stringency hybridization reaction is generally performed at
about 60.degree. C. in 1.times.SSC.
[0053] When hybridization occurs in an antiparallel configuration
between two single stranded polynucleotides, the reaction is called
"annealing" and those polynucleotides are described as
"complementary". A double-stranded polynucleotide can be
"complementary" or "homologous" to another polynucleotide, if
hybridization can occur between one of the strands of the first
polynucleotide and the second. "Complementarity" or "homology" (the
degree that one polynucleotide is complementary with another) is
quantifiable in terms of the proportion of bases in opposing
strands that are expected to form hydrogen bonding with each other,
according to generally accepted base pairing rules.
[0054] Structure of the Optical Confinements of the Present
Invention
[0055] One aspect of the present invention is the design of optical
devices and methods for characterizing molecules and/or monitoring
chemical reactions. The optical devices of the present invention
allow multiplexing of large numbers of single-molecule analyses
under physiologically relevant conditions.
[0056] In one embodiment, the present invention provides a high
density array of optical confinements having a surface density
exceeding 4.times.10.sup.4 confinements per mm.sup.2, preferably
exceeding 10.sup.5, wherein the individual confinement in the array
provides an effective observation volume on the order of
zeptoliters. The array may contain at least about 2.times.10.sup.5,
at least about 10.sup.6, or at least about 10.sup.7 optical
confinements. Preferably, the individual confinement in the array
provides an effective observation volume less than about 1000
zeptoliters, more preferably less than about 900, more preferably
less than about 80, even more preferably less than about 10
zeptoliters. Where desired, an effective observation volume less
than 1 zeptoliter can be provided. In a preferred aspect, the
individual confinement yields an effective observation volume that
permits resolution of individual molecules present at a
physiologically relevant concentration. The physiologically
relevant concentrations for most biochemical reactions range from
micro-molar to millimolar because most of the enzymes have their
Michaelis constants in these ranges. Accordingly, preferred array
of optical confinements has an effective observation volume for
detecting individual molecules present at a concentration higher
than about 1 micromolar (.mu.M), or more preferably higher than 50
.mu.M, or even higher than 100 .mu.M.
[0057] To achieve the required observation volume for
single-molecule analysis under physiologically relevant conditions,
the array may comprise zero-mode waveguides or alternative
nanoscale optical structures. Such alternative structures include
but are not limited to porous films with reflective index media,
and confinements using index matching solids.
[0058] As used herein, "zero-mode waveguide" refers to an optical
guide in which the majority of incident radiation is attenuated,
preferably more than 80%, more preferably more than 90%, even more
preferably more than 99% of the incident radiation is attenuated.
As such high level of attenuation, no significant propagating modes
of electromagnetic radiation exist in the guide. Consequently, the
rapid decay of incident electromagnetic radiation at the entrance
of such guide provides an extremely small observation volume
effective to detect single molecules, even when they are present at
a concentration as high as in the micromolar range.
[0059] The zero-mode waveguide of the present invention typically
comprises a cladding surrounding a core (i.e., partially or fully),
wherein the cladding is configured to preclude propagation of
electromagnetic energy of a wavelength higher than the cutoff
wavelength longitudinally through the core of the zero-mode
waveguide. The cladding is typically made of materials that prevent
any significant penetration of the electric and the magnetic fields
of an electromagnetic radiation. Suitable materials for fabricating
the cladding include but are not limited to alloys, metals, and
semi-conducting materials, and any combination thereof. Alloys
include any of the numerous substances having metallic properties
but comprising two or more elements of which at lest one is a
metal. Alloys may vary in the content or the amount of the
respective elements-whether metallic or non metallic. Preferred
alloys generally improve some desirable characteristics of the
material over a pure elemental material. Characteristics that can
be improved through the use of mixtures of materials include,
chemical resistance, thermal conductivity, electrical conductivity,
reflectivity, grain size, coefficient of thermal expansion,
brittleness, temperature tolerance, conductivity, and/or reduce
grain size of the cladding.
[0060] In general, alloys suitable for the present invention may
involve mixtures where one component is present at fractions as low
as 0.0001%. In other instances, alloys with large fractions of more
than one compound will be desirable. One embodiment of the ZMW uses
aluminum as the cladding of the ZMW structure. As an example of how
alloys can be beneficial to a ZMW structure, it is useful to
consider different alloys of aluminum in how they would affect a
ZMW. In the art of metalurgy, numerous materials are alloyed with
aluminum. Non-limiting examples of materials suitable to alloy with
aluminum are antimony, arsenic, beryllium, bismuth, boron, cadmium,
calcium, carbon, cerium, chromium, cobalt, copper, gallium,
hydrogen, indium, iron, lead, lithium, magnesium, manganese,
mercury, molybdenum, nickel, niobium, phosphorous, silicon,
vanadium, zinc and others. By way of example of how the
introduction of another element could beneficially impact the ZMW
performance, the introduction of boron to aluminum is known to
increase the conductivity of aluminum. An increase in conductivity
of the metal film may improve the performance by decreasing the
penetration depth thereby decreasing the observation volume. A
preferred embodiment includes an alloy of aluminum that is more
than 0.0001% of a dopant. A more preferred embodiment includes an
alloy of aluminum that is more than 0.005% of a dopant. A still
more preferred embodiment includes an allow of aluminum that is
more than 0.1% of a dopant.
[0061] In contrast, some materials are expected to decrease the
performance of the ZMW structure, and in these instances it will be
desirable to take measures to eliminate certain impurities. For
example, in certain applications it may be desirable to decrease
the amount of lead or arsenic if toxicity of the device is a
concern. A preferred embodiment of the device includes a metal film
that is less than 1% arsenic. A more preferred embodiment of the
device includes a metal films that is less than 0.1% arsenic. A
still more preferred embodiment includes a metal film that is less
than 0.001% arsenic. A still more preferred embodiment includes a
metal film that is less than 0.00001% arsenic. An additional
preferred embodiment includes a metal film that is less than 1%
lead. A still more preferred embodiment includes a metal film that
is less than 0.1% lead. A still more preferred embodiment includes
a metal film that is less than 0.01% lead. A still more preferred
embodiment includes a metal film that is less than 0.001% lead. A
still more preferred embodiment includes a film that is less than
0.00001% lead. In other applications where optical confinement
performance is especially important, impurities that tend to reduce
the conductivity, thereby worsening the confinement, will be
undesirable. For example, vanadium is known in the art of
metallurgy to reduce the conductivity of aluminum. A preferred
embodiment includes a metal film that is less than 0.1% vanadium. A
still more preferred embodiment includes a metal film that is less
than 0.01% vanadium. A still more preferred embodiment includes a
film that is less than 0.001% vanadium.
[0062] Semi-conducting materials suitable for fabricating the
cladding are generally opaque, and they include silicon, silicates,
silicon nitride, gallium phosphide, gallium arsenide, or any
combinations thereof.
[0063] The cladding of the subject zero-mode waveguide may be
coated with materials to improve the surface quality. For instance,
coating may enhance the durability of the cladding material. In
addition, coating is particularly desirable if the reactants
contained in the core are prone to interact or adhere to the
cladding material. A variety of appropriate coating materials are
available in the art. Some of the materials may covalently adhere
to the surface, others may attach to the surface via non-covalent
interactions. Non-limiting examples of coating materials include
aluminum oxide film, silanization reagent such as
dimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane or
trimethylchlorosilane, polymaleimide, and siliconizing reagents
such as silicon oxide, Aquasil.TM., and Surfasil.TM.. An
illustrative coated ZMW (101) is shown in FIG. 10. The ZMW (101) is
bound to a substrate 105. The ZMW comprises a sidewall 102, a
coating 103 on the upper surface, and a metal film 104.
[0064] In certain embodiments, it may be advantageous to construct
the confinement from metal compositions that are inhomogeneous
combinations of more than one material. For example, for certain
applications, it may be beneficial to provide a composition that
comprises more than one layer, each layer having a different
composition, or composition that varies within a layer. This can
have beneficial effects on several aspects of the performance of
the confinement, including but not limited to the nature of the
optical confinement, the structural strength and behavior of the
device, the characteristics of the surface chemistry of the device
or the like. In one embodiment the confinement comprises two layers
in which one of the layers serves to enhance the adhesion of the
second layer to a substrate. In another embodiment, the composition
of the cladding film varies as a function of the axial position
relative to the confinement, so as to provide different optical
performance than would be obtained from a layer of uniform
composition. In a particular version of this embodiment, the film
comprises a composition that has a larger value of skin depth close
to the surface of the substrate, and comprises a composition that
has a smaller value of skin depth farther from the surface of the
substrate, so that the nature of the confinement is to be more
uniform in shape near the surface and then tapering off more
quickly a larger distances away from the substrate. In another
embodiment, the thicknesses of two different layers comprising the
cladding of the confinement are chosen so that a specific optical
condition is achieved at the substrate of the device, such as
constructive or destructive interference.
[0065] The internal cavity (i.e., the core) surrounded by the
cladding may adopt a convenient size, shape or volume so long as
propagating modes of electromagnetic radiation in the guide is
effectively prevented. The core typically has a lateral dimension
less than the cutoff wavelength (.lamda..sub.c). For a circular
guide of diameter d and having a clad of perfect conductor,
.lamda..sub.c is approximately 1.7.times.d. The cross sectional
area of the core may be circular, elliptical, oval, conical,
rectangular, triangular, polyhedral, or in any other shape. The
various shapes can have particular suitability for certain
applications. For instance, elongated cross-sections can be useful
to provide enhanced access to molecules with mechanical persistence
or stiffness, such as DNA. Cross sections ranging from extended
slots to ovals of various aspect ratio will significant increase
the accessibility of the persistent molecule to the detection zone
of the structure, without excessive compromise in the axial
attenuation of radiation. Although uniform cross sectional area is
preferred, the cross sectional area may vary at any given depth of
the guide if desired. Preferred average cross sectional areas range
from 100 nm.sup.2 to 10,000 nm.sup.2.
[0066] In a preferred embodiment, the core is non-cylindrical. In
one aspect of this embodiment, a non-cylindrical core comprises an
opening on the upper surface and a base at the bottom surface that
is entirely surrounded by the cladding, wherein the opening is
narrower in lateral dimension than the base. This configuration
significantly restricts the diffusion of reactants, and hence
increases the average residence time in the observation volume.
Such configuration is particularly useful for measuring the
association rate constant (on-rate) of a chemical reaction. In
another aspect, the core comprises an opening that is wider in
lateral dimension than the base. Such configuration allows easier
access to large molecules that impose a steric or entropic
hindrance to entering the structure if the open end of the zero
mode waveguide was as small as the base needed to be for optical
performance reasons. Examples include the accessibility for long
strand polyelectrolytes such as DNA molecules that are subject to
entropic forces opposing entry into small openings.
[0067] The zero-mode waveguides embodied in the present invention
have a relatively high fill fraction ratio, typically above 0.0001,
preferably above 0.001, more preferably above 0.01, and even more
preferably above 0.1. As used herein, "fill fraction" of a pattern
refers to the ratio of the area occupied by the foreground of the
pattern to the total area occupied by the pattern (foreground and
background, together). The terms "fill fraction ratio" and "fill
faction" are used interchangeably. In the context of zero-mode
waveguide, the foreground is considered to be the area occupied by
the core of the zero-mode waveguide, and the background is the area
between the zero-mode waveguide (e.g., the aluminum film that forms
the cladding in certain designs). The zero-mode waveguides with
high fill fraction ratios are particularly useful for performing
homogenous assays. The fill fraction can be calculated by summing
the total areas of all of the zero-mode waveguides in the array and
dividing by the total available area including both the zero-mode
waveguides and the spaces between them. For example, if a zero-mode
waveguide has a diameter of 50 nm, then the area of this zero-mode
waveguide is one fourth of 7,850 square nanometers or 1962.5
nm.sup.2. If these zero-mode waveguides are in a square array
separated by 100 nm, the total available area is 10,000 square
nanometers for each zero-mode waveguide. Therefore, the array has a
fill fraction of one fourth of 78% or 19.6%, which would provide
nearly four orders of magnitude higher signal strength in a surface
binding assay than a zero-mode waveguide having a fill fraction on
the order of 0.01%.
[0068] In a bioassay such as an ELISA or other molecular binding
bioassay, one limitation is the inability to operate
"homogeneously", or in a mode where solutions may be added to a
mixture but nothing removed. This complicates highly multiplexed
assays, as provisions for both adding and removing material from a
large number of wells is significantly more complex than the
provisions for simply adding materials. In the case of the ELISA
assay, the removal of materials is necessary, because the
fluorescent (or other) markers that remain free in solution at the
end of the assay would interfere with the ability to detect markers
bound to the reaction surface. Techniques to overcome this have
been devised to exploit the short range of radioactive emissions
from certain radioisotopes, but these techniques have inherent
difficulties associated with personnel safety and waste disposal.
Other methods for confining the sensitivity of the assay to the
surface have been devised, such as total internal reflection
confinement (TIR), and confocal detection. The zero-mode waveguide
photonic structure allows a simpler and less expensive optical
system configuration than either of these techniques, and vastly
outperforms both from the perspective of confinement of sensitivity
to the surface.
[0069] The fill fraction is important in bioassays, because the
effective probe area is limited to the surface area of the bottoms
of the zero-mode waveguide in the detection region. The amount of
signal detectable in such an assay will be directly proportional to
the available area, and having a larger fraction of the available
surface occupied by zero-mode waveguides will thus increase the
signal strength of measurements of such assays. A high fill
fraction structure would be generally useful in any surface
sensitivity application, not limited to the ELISA assay.
[0070] The cutoff wavelength is the wavelength above which the
waveguide is essentially incapable of propagating electromagnetic
energy along the waveguide under the illumination geometry used.
Given the geometry of the core, and the properties of the cladding
material, as well as the wavelength of the incident electromagnetic
radiation, one skilled in the art can readily derive the cutoff
wavelength by solving the Maxwell's equations (see, e.g., John D.
Jackson, CLASSICAL ELECTRODYNAMICS, second edition, John Willey and
Sons). The choice of the incident wavelength will depend on the
particular application in which the subject array is to be
employed. In certain aspects, the incident wavelength may be
selected from a range of about 10 nm to about 1 mm. For detecting
fluorescent signals, the incident wavelength is typically selected
from the range of about 380 nm to about 800 nm. Polarized (linearly
or preferably circularly polarized) or unpolarized incident
radiation is generally employed to illuminate the array in order to
create a desired observation volume.
[0071] In a separate embodiment, the present invention provides an
alternative optical confinement termed external reflection
confinement (ERC). In contrast to the conventional total internal
reflection confinement (IRC), the low index medium is the
electromagnetic radiation carrier, and the high index (and opaque)
medium is the reflector. As such, the roles of the refractive
indices are reversed as compared to the IRC situation. ERC
generally requires some kind of means to provide the analyte (i.e.,
the molecules under investigation) in the opaque phase.
[0072] IRC relies on reflection of an electromagnetic radiation
incident on an interface between high index of refraction and low
index of refraction. When light is incident above the critical
angle of total internal reflection (known in the art), all of the
incident electromagnetic radiation is reflected and none is
transmitted into the low index phase. A thin region of evanescent
radiation is established proximal to the interface on the low index
side. This radiation field is typically an exponentially decaying
field with an attenuation length in the range from about 100 nm to
about 200 nm, depending on the angle of incidence and the indices
of refraction of the two phases. If the low index phase is a
solution containing an analyte, then the evanescent radiation can
be used to probe the analyte in the solution with a high degree of
surface sensitivity.
[0073] In ERC, the carrier of the propagating electromagnetic
radiation is a transparent low index film, and the analyte-bearing
medium is a high-index metallic opaque film. In this case, most of
the radiation is reflected irrespective of the angle of incidence,
and non-reflected light is rapidly attenuated according to the skin
depth of the metal. Typically, means is provided to convey the
analyte within the metal phase. Theses means can take the form of a
nanocapillary tube constructed within the metal layer. When
sufficiently small, the presence of such a tube will have little
effect on the distribution of energy in the two media, but can be
amply large enough to convey biomolecules. To be small enough, any
defects in the metal film must be small compared with the
wavelength of the illumination. This can be achieved because of the
large ratio between the wavelength of visible light, and the
typical size of biomolecules of interest. While visible light is
typically between 400 nm and 750 nm in wavelength, biomolecules of
interest are generally in the vicinity of 1-30 nm in diameter. The
attenuation of the radiation at the interface can be used to
confine illumination to a very small region of the analyte. A small
hole in an index matched (to water) film on a high index substrate
could provide lateral confinement beyond what is possible with
diffraction limited optics in the TIR context. This could give 100
zeptoliter confinement in principle. In this method, a version of
total internal reflection confinement is used in which a solid
material index-matched to the analyte solution is applied to the
substrate surface and then perforated with nanoscale holes. When
used in TIR mode, these structures will provide additional
confinements above what can be obtained with TIR alone.
[0074] Other alternative confinements are index matching solids. As
an illustrative example, such optical confinement can be fabricated
starting with a high index transparent substrate such as sapphire,
spin coat 200 nm of PMMA (polymethyl methacrylate) resist resin.
Exposure to electron beam lithography will render isolated spots
soluble according to the pattern applied. After development, the
device will have nano-scale holes in the PMMA layer and are ready
to be used in a TIR setup. Axial confinement is unaffected by the
PMMA layer, as it has nearly the same index of refraction as the
solution containing the analyte, but the solution is physically
prevented from approaching near the surface except where the holes
are situated, providing a degree of lateral confinement given by
the diameter of the holes.
[0075] The optical confinements can be provided with an optical
system capable of detecting and/or monitoring interactions between
reactants at the single-molecule level. Such optical system
achieves these functions by first generating and transmitting an
incident wavelength to the reactants contained in the confinements,
followed by collecting and analyzing the optical signals from the
reactants. Such systems typically employ an optical train that
directs signals from an array of confinements onto different
locations of an array-based detector to simultaneously detect
multiple different optical signals from each of multiple different
confinements. In particular, the optical trains typically include
optical gratings or wedge prisms to simultaneously direct and
separate signals having differing spectral characteristics from
each confinement in an array to different locations on an array
based detector, e.g., a CCD. By separately directing signals from
each confinement to different locations on a detector, and
additionally separating the component signals from each confinement
to separate locations, one can simultaneously monitor multiple
confinements, and multiple signals from each confinement.
[0076] The optical system applicable for the present invention
comprises at least two elements, namely an excitation source and a
photon detector. The excitation source generates and transmits
incident light used to optically excite the reactants contained in
the optical confinement. Depending on the intended application, the
source of the incident light can be a laser, laser diode, a
light-emitting diode (LED), a ultra-violet light bulb, and/or a
white light source. Where desired, more than one source can be
employed simultaneously. The use of multiple sources is
particularly desirable in applications that employ multiple
different reagent compounds having differing excitation spectra,
consequently allowing detection of more than one fluorescent signal
to track the interactions of more than one or one type of molecules
simultaneously. A wide variety of photon detectors are available in
the art. Representative detectors include but are not limited to
optical reader, high-efficiency photon detection system, photodiode
(e.g. avalanche photo diodes (APD)), camera, charge couple device
(CCD), electron-multiplying charge-coupled device (EMCCD),
intensified charge coupled device (ICCD), and confocal microscope
equipped with any of the foregoing detectors. Where desired, the
subject arrays of optical confinements contain various alignment
aides or keys to facilitate a proper spatial placement of the
optical confinement and the excitation sources, the photon
detectors, or the optical transmission element as described
below.
[0077] The subject optical system may also include an optical
transmission element whose function can be manifold. First, it
collects and/or directs the incident wavelength to the optical
confinement containing the reactants. Second, it transmits and/or
directs the optical signals emitted from the reactants inside the
optical confinement to the photon detector. Third, it may select
and/or modify the optical properties of the incident wavelengths or
the emitted wavelengths from the reactants. Illustrative examples
of such element are diffraction gratings, arrayed waveguide
gratings (AWG), optic fibers, optical switches, mirrors, lenses
(including microlens and nanolens), collimators. Other examples
include optical attenuators, polarization filters (e.g., dichroic
filter), wavelength filters (low-pass, band-pass, or high-pass),
wave-plates, and delay lines. In some embodiments, the optical
transmission element can be planar waveguides in optical
communication with the arrayed optical confinements. For instance,
a planar waveguides can be operatively coupled to an array of
zero-mode waveguides to directly channel incident wavelengths to
the respective cores of the zero-mode waveguides so as to minimize
the loss of wave energy. The planar channel can be included as a
detachable unit located at the base of array substrate, or it can
be bonded to the substrate as an integral part of the array.
[0078] The optical transmission element suitable for use in the
present invention encompasses a variety of optical devices that
channel light from one location to another in either an altered or
unaltered state. Non-limiting examples of such optical transmission
devices include optical fibers, diffraction gratings, arrayed
waveguide gratings (AWG), optical switches, mirrors, (including
dichroic mirrors), lenses (including microlens and nanolens),
collimators, filters, prisms, and any other devices that guide the
transmission of light through proper refractive indices and
geometries.
[0079] In a preferred embodiment, the optical confinement of the
present invention is operatively coupled to a photon detector. For
instance, the arrayed optical confinement is operatively coupled to
a respective and separate photon detector. The confinement and the
respective detector can be spatially aligned (e.g., 1:1 mapping) to
permit an efficient collection of optical signals from the
waveguide. A particularly preferred setup comprises an array of
zero-mode waveguides, wherein each of the individual waveguides is
operatively coupled to a respective microlens or a nanolens,
preferably spatially aligned to optimize the signal collection
efficiency. Alternatively, a combination of an objective lens, a
spectral filter set or prism for resolving signals of different
wavelengths, and an imaging lens can be used in an optical train,
to direct optical signals from each confinement to an array
detector, e.g., a CCD, and concurrently separate signals from each
different confinement into multiple constituent signal elements,
e.g., different wavelength spectra, that correspond to different
reaction events occurring within each confinement.
[0080] An exemplary optical setup is shown in FIG. 7, in which an
array of ZMWs is optically linked to an optical system. This system
comprises a ZMW array film (81), a glass cover slip (82) through
which light transmits and further converges through set of integral
lenses (83) made of a material having a different index of
refraction than that of the glass. In particular, 84 shows a ZMW
structure, 85 indicates a ray of light being focused onto the ZMW
by the integral lenses such as the embedded microlens.
[0081] FIG. 11 depicts one alignment strategy and optical system.
The system comprises a photodetector 131, an optional lens 132 for
collecting light, a ZMW 133 having a metal film 134 coupled to a
substrate 135, and an objective lens 136 that is aligned with the
incident light beam 137. FIG. 13 depicts an exemplary alignment
detection system and the associated components. The illustrative
system 13A comprises an optical confinement such as a zero-mode
waveguide 111 having a metal film 113 coupled to a substrate 114.
The zero-mode waveguide 111 typically contains signal generating
molecules 112, and is optically linked to the associated components
including an objective lens 115, a beam splitter/dichroic cube 117,
optically a telen lens 120 (used in infinity corrected systems),
and a photodetector 122 (e.g., a quadrant photodetector). 116
depicts rays propagating through system. 118 depicts the incident
illumination rays. 119 depicts the return rays moving towards the
detector 122. FIG. 13B depicts a front view of the quadrant
photodiode. Shown in the center of the figure is a beam mis-aligned
on the center of the quadrant detector. The four voltages generated
by the four quadrants V1, V2, V3 and V4 can be processed to
determine the degree and direction of mis-alignment of the beam and
thus the optical confinement such as ZMW 111. The subject arrays
may comprise a single row or a plurality of rows of optical
confinements on the surface of a substrate, where a plurality of
lanes are present, for example, usually at least 2, more commonly
more than 10, and more commonly more than 100. The subject array of
optical confinements may align horizontally or diagonally long the
x-axis or the y-axis of the substrate. The individual confinements
can be arrayed in any format across or over the surface of the
substrate, such as in rows and columns so as to form a grid, or to
form a circular, elliptical, oval, conical, rectangular,
triangular, or polyhedral pattern. To minimize the nearest-neighbor
distance between adjacent optical confinements, a hexagonal array
is preferred.
[0082] The array of optical confinements may be incorporated into a
structure that provides for ease of analysis, high throughput, or
other advantages, such as in a microtiter plate and the like. Such
setup is also referred to herein as an "array of arrays." For
example, the subject arrays can be incorporated into another array
such as microtiter or multi-well plate wherein each micro well of
the plate contains a subject array of optical confinements.
Typically, such multi-well plates comprise multiple reaction
vessels or wells, e.g., in a 48 well, 96 well, 384 well or 1536
well format. In such cases, the wells are typically disposed on 18
mm, 9 mm, 4.5 mm, or 2.25 mm centers, respectively.
[0083] An illustrative array of arrays is depicted in FIG. 5 in
which a subarray 71 is part of a super array 72. Arrays can also be
arranged in lattices. For example, FIG. 4 depicts a top view of an
illustrative regular disposition of ZMWs 51. In this configuration,
there is a lattice defined by the parameters d1 52, d2 54, and the
angle 53. In addition to a ZMW at each lattice point, there is a
complex unit cell that comprises a plurality of ZMWs in an
arrangement that is defined by a list of angles and distances with
one angle and one distance for each element of the unit cell. In
particular, 52 represents the first lattice distance, 53 represents
the lattice angle, 54 represents the second lattice distance, 55
represents the unit cell first distance d3, and 56 represents unit
cell first angle. While this figure shows an array with a unit cell
of two components, the unit cell can have any plurality of
elements.
[0084] As described above, the subject arrays comprise a plurality
of optical confinements. In some embodiments, the arrays have at
least about 20.times.10.sup.4 distinct optical confinements,
preferably at least about 20.times.10.sup.6 distinct confinements,
and more preferably at least about 20.times.10.sup.8 confinements.
The density of the spots on the solid surface in certain
embodiments is at least above 4.times.10.sup.4 confinements per
mm.sup.2, and usually at least about 8.times.10.sup.4, at least
about 1.2.times.10.sup.5, or at least about 4.times.10.sup.6
confinements per mm.sup.2, but does not exceed 4.times.10.sup.12
confinements per mm.sup.2, and usually does not exceed about
4.times.10.sup.10 confinements per mm.sup.2. The overall size of
the array generally ranges from a few nanometers to a few
millimeters in thickness, and from a few millimeters to 50
centimeters in width or length. Preferred arrays have an overall
size of about few hundred microns in thickness and may have any
width or length depending on the number of optical confinements
desired.
[0085] In one example as shown in FIG. 1, the array of optical
confinements, e.g. zero-mode waveguides, are arranged in a square
format. The array comprises a representative zero-mode waveguide
21, separated from an adjacent waveguide by a distance "d" (22
represents the inter-zero mode waveguide spacing). In another
example as shown in FIG. 2, the array of optical confinements, e.g.
zero-mode waveguides, are arranged in a non-square format. The
array comprises a representative zero-mode waveguide 31, separated
from an adjacent waveguide by a distance "d" (32 represents the
inter-zero mode waveguide spacing). 33 shows the angle formed
between any three adjacent ZMWs (e.g., 60 degrees). FIG. 3 depicts
a top view of another illustrative 2-dimentional array. The
adjacent optical confinements are separated in one dimension by a
distance of "d1" 41 and in another dimension by a distance of "d2"
42, with a unit vector angle 43.
[0086] The spacing between the individual confinements can be
adjusted to support the particular application in which the subject
array is to be employed. For instance, if the intended application
requires a dark-field illumination of the array without or with a
low level of diffractive scattering of incident wavelength from the
optical confinements, then the individual confinements are
typically placed close to each other relative to the incident
wavelength.
[0087] Accordingly, in one aspect, the present invention provides
an array of zero-mode waveguides comprising at least a first and at
least a second zero-mode waveguide, wherein the first zero-mode
waveguide is separated from the second zero-mode waveguide by a
distance such that upon illumination with an incident wavelength,
intensity of diffractive scattering observed from the first
zero-mode waveguide at a given angle is less than that if the first
zero-mode waveguide were illuminated with the same incident
wavelength in the absence of the second zero-mode waveguide.
Diffractive scattering can be reduced or significantly eliminated
if an array comprises zero-mode waveguides spaced in a regular
spaced lattice where the separation of zero-mode waveguides from
their nearest neighbors is less than half the wavelength of the
incident wavelength. In this regime, the structure behaves as a
zero-order grating. Such gratings are incapable of scattering
incident light despite having a large number of elements that by
themselves would scatter very effectively. This arrangement is
highly desirable for illumination approaches such as dark field
illumination, where surface scattering would cause excitation
radiation to be collected by the objective lens, thus increasing
background noise. Useful wavelengths for illumination range from
250 nm up to 8 microns, meaning that an array of zero-mode
waveguides with a spacing of less than 4000 nm would still be
useful for application in this manner. A spacing of less than 2000
nm is more preferable, while a spacing of less than 1000 nm is even
more preferable in this respect. Some configurations with spacing
larger than one half of the wavelength can have the same advantage
if the illumination is applied asymmetrically, or if the collection
cone angle is configured to be less than 90 degrees. In addition to
the benefit of reduced diffractive scattering, narrow spacing
between the individual confinements decreases the illumination area
and thus lowers the power demand.
[0088] Arrays having the optical confinements spaced far apart
relative to the incident wavelength also have desirable properties.
While the angle-dependent scattering raises the background signal
that could be disadvantageous for certain applications, it provides
a means particularly suited for characterizing the size and shape
of the optical confinements. It also readily permits ensemble bulk
measurements of molecule interactions, involving especially
unlabelled molecules. Arrays suited for such applications generally
contain individual confinements separated by more than one
wavelength of the incident radiation, usually more than 1.5 times
the incident wavelength, but usually does not exceed 150 times the
incident wavelength. FIG. 12 depicts an alternative optical
confinement made of porous film 91 on a substrate 93. 92 represents
the pores in the film.
[0089] Kits:
[0090] The present invention also encompasses kits containing the
optical confinement arrays of this invention. Kits embodied by this
invention include those that allow characterizing molecules and/or
monitoring chemical reactions at a single-molecule level. Each kit
usually comprises the devices and reagents which render such
characterization and/or monitoring procedure possible. Depending on
the intended use of the kit, the contents and packaging of the kit
will differ. Where the kit is for DNA sequencing, the kit typically
comprises: (a) an array of optical confinements, preferably
zero-mode waveguides of the present invention, that permits
resolution of individual molecules or the reaction of individual
molecules, such as those that are present at a concentration higher
than about 1 micromolar; (b) sequencing reagents typically
including polymerases, aqueous buffers, salts, primers, and
nucleotides or nucleotide analogs. Where desired a, `control`
nucleic acids of known sequence can be included to monitor the
accuracy or progress of the reaction.
[0091] The reagents can be supplied in a solid form, immobilized
form, and/or dissolved/suspended in a liquid buffer suitable for
inventory storage, and later for exchange or addition into the
reaction medium when the test is performed. Suitable individual
packaging is normally provided. The kit can optionally provide
additional components that are useful in the procedure. These
optional components include, but are not limited to, buffers,
capture reagents, developing reagents, labels, reacting surfaces,
control samples, instructions, and interpretive information.
Diagnostic or prognostic procedures using the kits of this
invention can be performed by clinical laboratories, experimental
laboratories, practitioners, or private individuals.
[0092] Preparation of the Optical Confinements:
[0093] The array of the present invention can be manufactured using
nanofabrication techniques provided by the present invention, as
well as those known in the fields of Integrated Circuit (IC) and
Micro-Electra-Mechanical System (MEMS). The fabrication process
typically proceeds with selecting an array substrate, followed by
using appropriate IC processing methods and/or MEMS micromachining
techniques to construct and integrate the optical confinement and
other associated components.
[0094] Array Substrate:
[0095] In some embodiments, the array of optical confinements is
present on a rigid substrate. In other embodiments concerning,
e.g., porous films with reflective index media, flexible materials
can be employed. In general, a rigid support does not readily bend.
Examples of solid materials which are not rigid supports with
respect to the present invention include membranes, flexible metal
or plastic films, and the like. As such, the rigid substrates of
the subject arrays are sufficient to provide physical support and
structure to optical confinements present thereon or therein under
the assay conditions in which the array is employed, particularly
under high throughput handling conditions.
[0096] The substrates upon which the subject patterns of arrays are
disposed, may take a variety of configurations ranging from simple
to complex, depending on the intended use of the array. Thus, the
substrate could have an overall slide or plate configuration, such
as a rectangular or disc configuration, where an overall
rectangular configuration, as found in standard microliter plates
and microscope slides, is preferred. Generally, the thickness of
the rigid substrates will be at least about 0.01 mm and may be as
great as 1 cm or more, but will usually not exceed about 5 cm. Both
the length and the width of rigid substrate will vary depending on
the size of the array of optical confinements that are to be
fabricated thereon or therein.
[0097] The substrates of the subject arrays may be fabricated from
a variety of materials. The materials from which the substrate is
fabricated is preferably transparent to visible and/or UV light.
Suitable materials include glass, semiconductors (e.g., silicate,
silicon, silicates, silicon nitride, silicon dioxide, quartz, fused
silica, and gallium arsenide), plastics, and other organic
polymeric materials. In preferred aspects, silica based substrates
like glass, quartz and fused silica are used as the underlying
transparent substrate material.
[0098] The substrate of the subject arrays comprise at least one
surface on which a pattern of optical confinements is present,
where the surface may be smooth or substantially planar, or have
irregularities, such as depressions or elevations. The surface may
be modified with one or more different layers of compounds that
serve to modulate the properties of the surface in a desirable
manner. Modification layers of interest include: inorganic and
organic layers such as metals, metal oxides, polymers, small
organic molecules, functional moieties such as avidin/biotin and
the like. The choice of methods for applying the coating materials
will depend on the type of coating materials that is used. In
general, coating is carried out by directly applying the materials
to the zero-mode waveguide followed by washing the excessive
unbound coating material from the surface. Alternatively or
additionally, coating materials may be deposited using other
conventional techniques, such as chemical vapor deposition (CVD),
sputtering, spin coating, in situ synthesis, and the like. Certain
coating materials can be cross-linked to the surface via heating,
radiation, and/or by chemical reactions. In preferred aspects,
suitable coating materials are coupled to substrate surfaces either
covalently or through ionic or hydrophobic/hydrophilic
interactions. In the case of silica based substrates, for example,
silane chemistries are particularly suited for covalently attaching
coating materials to surfaces, e.g., coupling groups, specific
binding moieties, and the like. Such chemistries are well known to
those of ordinary skill in the art and can be practiced without
undue experimentation.
[0099] Fabrication Process:
[0100] Fabrication of the subject array substrates can be performed
according to the methods described as follows or other standard
techniques of IC-processing and/or MEMS micromachining. The
standard techniques known in the art include but are not limited to
electron-beam lithography, photolithography, chemical vapor or
physical vapor deposition, dry or wet etching, ion implantation,
plasma etching, bonding, and electroplating. Additional fabrication
processes are detailed in the U.S. Patent Application Publication
No. 2003/0174992, the content of which is incorporated by reference
in its entirety.
[0101] In a preferred embodiment, the present invention provides a
negative tone fabrication process, which provides for the creation
of optical confinements having more uniform and consistent
dimensions than conventional positive tone fabrication processes
that can yield optical confinements of varying dimensions. A
comparison of the two fabrication processes is shown in Table 1
below.
TABLE-US-00001 TABLE 1 Positive and Negative Tone Process Steps in
Fabrication of Zero-Mode Waveguides Step # Positive Tone Process
Negative Tone Process 1 Clean fused silica substrates Same in
heated solution of hydrogen peroxide and ammonium hydroxide. 2
Cascade rinse substrates in Same deionized water. 3 Clean
substrates in oxygen Same plasma cleaner. 4 Coat substrates with
metal Spin-coat substrates with film by either thermal
electron-beam resist. evaporation or sputtering. 5 Spin-coat
substrates with Bake casting solvent out of film. electron-beam
resist over the metal layer. 6 Bake casting solvent out of film.
Expose resist with electron beam lithography. 7 Expose resist with
electron beam Develop resist in chemical bath lithography. to
reveal array of small pillars with large empty gaps in resist. 8
Develop resist in chemical bath Rinse developer away and dry to
reveal holes. chips. 9 Rinse developer away and dry Coat chips with
metal film by chips. either thermal evaporation or sputtering. 10
Use reactive-ion etching to Dissolving underlying negative transfer
resist pattern into resist using Microposit 1165 metal film.
Stripper. 11 Strip resist using oxygen plasma. Same
[0102] In a negative tone process, a negative resist is applied to
the substrate. A resist is negative if it is rendered insoluble by
application of some agent, wherein the case of photoresists or
e-beam resists, the agent is optical energy or electron beam
energy, respectively. Alternatively, a positive tone resist can be
used with a negative pattern. A negative tone pattern is
characterized by the application of the agent in all areas except
the location of the optical confinement, e.g., zero-mode waveguide,
contrasted with a positive tone image in which the agent is
confined only to the optical confinement area. In either case,
after development of the resist, resist remains only in the areas
where the optical confinement is intended to lie. It is useful in
many cases to use means to achieve an undercut sidewall profile of
these remaining resist features. Many techniques exist in the art
to obtain undercut sidewalls, for example, in electron beam
lithography. For instance, when using negative tone resists, one
method is to apply to layers of electron beam resist to the surface
sequentially, the upper film having a higher sensitivity to the
energy delivered to it by the electron beam. Because the beam has a
tendency to spread, a larger area of the upper film will be
rendered insoluble than in the lower layer, resulting in an
overhang beneath the upper layer as desired.
[0103] After development and appropriate cleaning procedures known
in the art such as a plasma cleaning procedure, the metal film
comprising the optical confinement can be applied by one of several
methods, including metal evaporation, molecular beam epitaxy and
others. In the case that the resist profile is undercut as
discussed above, the metal that is deposited in the regions still
occupied by the resist will rest on top of the resist rather than
resting on the device surface. The resist layer is subsequently
removed by any of several techniques including solvent dissolution
either with or without ultrasonication or other mechanical
agitation, reactive plasma etching, vaporization or others. The
metal which rested on the resist features is removed as the resist
is removed ("lifted off"), while the resist resting directly on the
substrate remains to form the walls of the optical confinement.
[0104] The advantage of this process is that the size of the
optical confinement is determined by the size of the resist
feature, and does not rely on the fidelity of reactive ion etch
pattern transfer mechanisms, which can be highly variable for metal
films, especially aluminum a desirable metal for these devices. The
positive tone process is subject to the inherent variation in
resist feature sizes plus the variation due to pattern transfer,
while the negative tone process is subject to the first variability
but not the second. Metal thin film techniques suffer from much
less lateral variation, and so the overall accuracy is better. This
method also does not rely on the availability of a suitable etch
for the metal in question, allowing the application of the process
to a much wider selection of metals than the positive tone
process.
[0105] FIG. 6 is a schematic presentation of an illustrative
negative tone process to make zero-mode waveguides. In this
process, the substrate 11 is first coated with a layer of negative
resist 12. Optionally, the substrate can be coated with a second
resist layer 13. Exposure of the resist to the same pattern
electron beam lithography tool used in the positive tone process,
generates the opposite pattern as previously observed, namely one
of a periodic array of small pillars of remaining resist, and empty
gaps between the pillars 15. The final zero-mode waveguide
structures are created by coating this pattern with a thin metal
layer such as an aluminum layer 17, and then dissolving the
underlying negative resist pillars 18. Because this process is not
dependent on the thickness of the alumina layer or the crystal
structure or morphology of the metal film, it produces a far more
consistent configuration, and provides much finer control over the
critical feature size. FIG. 8 depicts a scanning electron
micrographs of ZMW structures fabricated by positive tone resist
(left panels) or negative tone resist (right panels). The grain
structure of the polycrystalline film is visible in the image as
flecks, and the ZMWs as dark round structures.
[0106] A variant negative tone process is termed nanocasting. The
steps of nanocasting are similar except that the use of bi-layer
resist is avoided. The process first involves depositing on the
surface of a substrate (in this case a single-layer resist would be
used). The electron beam exposure and development follow, leaving a
cylindrical feature for each dot in the exposure pattern. For this
process, it is desirable to allow the metal deposition technique to
apply material not just on the top of the resist structure but also
on the sidewalls of the resist feature. This process is inherently
three dimensional, in that a negative replica of the exterior
surface of the three-dimensional resist feature is reproduced in
the interior surface of the metal films that forms the optical
confinement walls. In this case, the undercut resist profile and
the various methods used to produce this are not necessary, as in
the negative tone process, they are used specifically to prevent
contact of the deposited film with the sides of the resist feature.
In the nanocasting approach, the deposited film faithfully
reproduces the exterior surface of the resist feature, so an
undercut figure would only be used if a non-cylindrical confinement
is desired.
[0107] In practicing nanocasting, caution is typically employed to
removed the metal from above the nanocasting "master" (the resist
feature), as the resist feature can in some instances be entirely
buried and unavailable for removal. This, however, can be remedied
in a number of ways.
[0108] Where the deposition technique has a high degree of
anisotropy in the deposition (such as metal evaporation), the
sidewalls will be very thin near the top of the resist feature,
which in some instances can be a cylindrical pillar. This weak
point can be subject to direct mechanical disruption allowing the
removal of the metal above the resist feature and hence the ZMW
location. An isotropic etch, either solution phase or plasma can be
used to further thin the film until this weak point separates,
achieving the same effect. If the metal deposition step has a low
degree of anisotropy (such as sputtering or electroplating), then
the resist material can be exposed through chemical mechanical
polishing, or ion milling.
[0109] Simultaneous with or subsequent to the removal of the metal
cap over the resist feature, the resist material is then removed by
solvent dissolution, or reactive ion etching. This completes the
fabrication steps, provided the appropriate pattern is applied and
the other parameters are correctly chosen.
[0110] Uses of the Subject Optical Confinements and Other
Devices:
[0111] The subject devices including optical confinements and
associated optical systems provide a effective means for analyzing
molecules and monitoring chemical reactions in real time. The
subject device and detection/monitoring methods may be used in a
wide variety of circumstances including analysis of biochemical and
biological reactions for diagnostic and research applications. In
particularly preferred aspects, the present invention is applied in
the elucidation of nucleic acid sequences for research
applications, and particularly in sequencing individual human
genomes as part of preventive medicine, rapid hypothesis testing
for genotype-phenotype associations, in vitro and in situ
gene-expression profiling at all stages in the development of a
multi-cellular organism, determining comprehensive mutation sets
for individual clones and profiling in various diseases or disease
stages. Other applications include measuring enzyme kinetics, and
identifying specific interactions between target molecules and
candidate modulators of the target molecule. Further applications
involve profiling cell receptor diversity, identifying known and
new pathogens, exploring diversity towards agricultural,
environmental and therapeutic goals.
[0112] In certain embodiments, the subject devices and methods
allow high-throughput single-molecule analysis. Single-molecule
analysis provides several compelling advantages over conventional
approaches to studying biological events. First, the analysis
provides information on individual molecules whose properties are
hidden in the statistically averaged information that is recorded
by ordinary ensemble measurement techniques. In addition, because
the analysis can be multiplexed, it is conducive to high-throughput
implementation, requires smaller amounts of reagent(s), and takes
advantage of the high bandwidth of optical systems such as modern
avalanche photodiodes for extremely rapid data collection.
Moreover, because single-molecule counting automatically generates
a degree of immunity to illumination and light collection
fluctuations, single-molecule analysis can provide greater accuracy
in measuring quantities of material than bulk fluorescence or
light-scattering techniques. As such, single-molecule analysis
greatly improves the efficiency and accuracy in genotying, gene
expression profiling, DNA sequencing, nucleotide polymorphism
detection, pathogen detection, protein expression profiling, and
drug screening.
[0113] Single-Molecule Sequencing:
[0114] The subject devices, including various forms of optical
confinements and the associated optical systems, are particularly
suited for multiplexed single-molecule sequencing. Accordingly, the
present invention provides a method of simultaneously sequencing a
plurality of target nucleic acids. The method generally involves
(a) providing an array of optical confinements of the present
invention; (b) mixing in the confinements a plurality of target
nucleic acid molecules, primers complementary to the target nucleic
acid molecules, polymerization enzymes, and more than one type of
nucleotides or nucleotide analogs to be incorporated into a
plurality of nascent nucleotide strands each being complimentary to
a respective target nucleic acid molecule; (c) subjecting the
mixture to a polymerization reaction under conditions suitable for
formation of the nascent nucleotide strands by template-directed
polymerization; (d) illuminating the waveguides with an incident
light beam; and (e) identifying the nucleotides or the nucleotide
analogs incorporated into each nascent nucleotide strand.
[0115] The subject sequencing methods can be used to determine the
nucleic acid of any nucleic acid molecule, including
double-stranded or single-stranded, linear or circular nucleic
acids (e.g., circular DNA), single stranded DNA hairpins, DNA/RNA
hybrids, RNA with a recognition site for binding of the polymerase,
or RNA hairpins. The methods of the present invention are suitable
for sequencing complex nucleic acid structures, such as 5' or 3'
non-translation sequences, tandem repeats, exons or introns,
chromosomal segments, whole chromosomes or genomes.
[0116] In one aspect, the temporal order of base additions during
the polymerization reaction is identified on a single molecule of
nucleic acid. Such identifying step takes place while the
template-directed extension of primer or polymerization is taking
place within the optical confinement. In a preferred embodiment,
single-molecule sequencing is performed in a homogenous assay that
does not require transfer, separation, or washing away any reactant
or by-product (e.g. fluorophore cleaved from a nucleotide) after
each base addition event. In certain aspects of the homogenous
assay, single-molecule sequencing is performed without adding
reactants to the mixture prior to reading the next base sequence.
In this assay, stepwise addition of nucleotides or removal of
by-products after each base addition event is not necessary, as
diffusion of reactants from a large volume of reagents above the
confinement will not interfere with the detection of incorporation.
Sequence information is generated continuously as the polymerase
continually incorporates the appropriate nucleotides or nucleotide
analogs into the nascent DNA strand. For a detailed discussion of
such single molecule sequencing, see, e.g., Published U.S. Patent
Application No. 2003/0044781, which is incorporated herein by
reference in its entirety for all purposes and M. J. Levene, J.
Korlach, S. W. Turner, M. Foquet, H. G. Craighead, W. W. Webb,
SCIENCE 299:682-686, January 2003 Zero-Mode Waveguides for
Single-Molecule Analysis at High Concentrations. There is no loss
of synchronization because single molecules are observed
separately. This method also allows the use of target nucleic acid
molecules taken directly from a biological sample, minimizing the
need for cloning, subcloning, or amplification of the target
nucleic acids before sequencing can take place.
[0117] In a preferred embodiment, a polymerase enzyme is provided
anchored within the effective observation volume within an optical
confinement. Template dependent synthesis of a complementary strand
is then carried out while observing the volume, and using labeled
nucleotide analogs that are capable of being sequentially
incorporated into the growing strand without interruption, e.g.,
for deprotection, etc. In preferred aspects, nucleotide analogs
bearing a label on a non-incorporated phosphate group or
derivative, e.g., the beta, gamma, delta, etc. phosphate of a
nucleotide polyphosphate which is cleaved from the analog during
incorporation, are used in such methods. Such nucleotide analogs
provide an advantage of being sequentially incorporated into the
growing nucleic acid strand, and having their labeling groups
removed in the incorporation process so as to not provide
increasing signal noise during synthesis that would result if such
labels remained associated with the synthesized strand. In
addition, because the incorporation event provides for prolonged
presence of the labeled analogs within the observation volume (as
compared to random diffusion of non-incorporated analogs into the
observation volume), the signal associated within incorporation is
readily identifiable. In particularly preferred aspects, for single
molecule nucleic acid sequencing applications, a template nucleic
acid is used that provides for the redundant or iterative
reading/synthesis of tandem repeats of a particular sequence
segment of interest. In particular, the systems of the invention
typically provide for redundancy in numerous ways, to correct for
any errors that may arise in template dependant synthesis by the
polymerase enzyme. For example, because the methods of the
invention focus on single molecules, redundant processes are
employed to assure that mis-incorporation events by a polymerase
are corrected for in data analysis.
[0118] In a first aspect, such redundancy is supplied by utilizing
arrays of multiple different confinements that are being applied to
a given sequence of interest, e.g., in a single well of a
multi-well plate. In addition to this redundancy, the invention
also provides for the iterative sequencing of a given sequence
segment (or a copy thereof) multiple times within a single
confinement. In a first preferred aspect, such iterative sequencing
may be accomplished by providing the sequence segment of interest
in a circular template format, so that the polymerase processes
around the circular template (allowing the elucidation of the
sequence of such template) multiple times. Methods of
circularization of nucleic acid segments are known to those of
ordinary skill in the art, and are readily applied to template
sequences in accordance with the invention.
[0119] In another aspect, a similar result is accomplished by using
a template-dependant circular template bearing the sequence segment
of interest. In particular, such synthesis product will typically
include, in a single linear strand, multiple copies of the circular
template, again, providing for iterative sequencing of the sequence
segment of interest. Further, redundancy is additionally
accomplished by circularizing this linear, multi-copy template and
iteratively sequencing multiple copies, multiple times.
[0120] In another aspect, a similar result is obtained by
performing concatmerization of amplicons generated in a
single-molecule amplification strategy, several of which are known
to those skilled in the art. These strategies can employ dilution
to the single molecule level, or isolation of molecules in small
micelles in a two-phase emulsion during amplification. The
concatmerized strand is then sequenced as a single template, and
redundant information is generated from a single molecule in this
fashion.
[0121] In yet another aspect, a similar result is obtained by using
a long double stranded template with nicks and/or gaps at multiple
locations along it. The molecule can then be caused to initiate
single molecule sequencing at several locations along the strand,
each location comprising a confinement that independently sequences
the strand. Because the several confinements are acting on the same
strand, the result is that the same template is sequenced several
times providing redundant information from a single molecule.
[0122] Exemplary Experimental Setup:
[0123] In practicing a sequencing method of the present invention,
a reaction mixture comprising the target nucleic acid(s) of
interest, primers complementary to the target nucleic acids,
polymerization enzymes, and more than one type of nucleotides or
nucleotide analogs, is applied to an array of optical confinements.
Preferably, each optical confinement receives only one target
nucleic acid molecule that is to be sequenced. This can be achieved
by diluting a minute amount of target nucleic acids in a large
volume of solution containing the rest of the reactants required
for the sequencing process. Alternatively, a non-cylindrical
waveguide whose the opening is narrower in lateral dimension than
the base, can be used to restrict the entry of multiple target
nucleic acids.
[0124] Immobilization of the Target Nucleic Acid or the Polymerase
to an Optical Confinement:
[0125] The target nucleic acid can be immobilized to the inner
surface of the optical confinement by a number of ways. For
example, the target nucleic acid can be immobilized onto an optical
confinement by attaching (1) a primer or (2) a single-stranded
target nucleic acid or (3) double-stranded or partially
double-stranded target nucleic acid molecule. Thereafter, either
(1) the target nucleic acid molecule is hybridized to the attached
oligonucleotide primer, (2) an oligonucleotide primer is hybridized
to the immobilized target nucleic acid molecule to form a primed
target nucleic acid molecule complex, or (3) a recognition site for
the polymerase is created on the double-stranded or partially
double-stranded target nucleic acid (e.g., through interaction with
accessory proteins, such as a primase). A nucleic acid polymerizing
enzyme on the primed target nucleic acid molecule complex is
provided in a position suitable to move along the target nucleic
acid molecule and extend the oligonucleotide primer at the site of
polymerization.
[0126] In preferred aspects, as described previously, the
polymerization enzyme is first attached to a surface of the subject
optical confinement within the effective observation volume of the
confinement, and in a position suitable for the target nucleic acid
molecule complex to move relative to the polymerization enzyme.
[0127] One skilled in the art will appreciate that there are many
ways of immobilizing nucleic acids and enzymes onto an optical
confinement, whether covalently or noncovalently, via a linker
moiety, or tethering them to an immobilized moiety. These methods
are well known in the field of solid phase synthesis and
micro-arrays (Beier at al., Nucleic Acids Res. 27:1970-1-977
(1999). Non-limiting exemplary binding moieties for attaching
either nucleic acids or polymerases to a solid support include
streptavidin or avidin/biotin linkages, carbamate linkages, ester
linkages, amide, thiolester, (N)-functionalized thiourea,
functionalized maleimide, amino, disulfide, amide, hydrazone
linkages, and among others. Antibodies that specifically bind to
the target nucleic acids or polymerases can also be employed as the
binding moieties. In addition, a silyl moiety can be attached to a
nucleic acid directly to a substrate such as glass using methods
known in the art.
[0128] Where desired, the polymerases may be modified to contain
one or more epitopes such as Myc, HA (derived from influenza virus
hemagglutinin), poly-histadines, and/or FLAG, for which specific
antibodies are available commercially. In addition, the polymerases
can be modified to contain heterologous domains such as glutathione
S-transferase (GST), maltose-binding protein (MBP), specific
binding peptide regions (see e.g., U.S. Pat. Nos. 5,723,584,
5,874,239 and 5,932,433), or the Fc portion of an immunoglobulin.
The respective binding agents for these domains, namely
glutathione, maltose, and antibodies directed to the Fc portion of
an immunoglobulin are available, and can be used to coat the
surface of an optical confinement of the present invention.
[0129] The binding moieties or agents of either the polymerases or
nucleic acids they immobilize can be applied to the support by
conventional chemical techniques which are well known in the art.
In general, these procedures can involve standard chemical surface
modifications of a support, incubation of the support at different
temperature levels in different media comprising the binding
moieties or agents, and possible subsequent steps of washing and
cleaning.
[0130] Reaction Mixture: Labeled Nucleotides, Polymerases, and
Primers:
[0131] The various types of nucleotides utilized in accordance with
the single-molecule sequencing method are conjugated with
detectable labels so that a photon detector can detect and
distinguish their presence within the subject optical confinements.
Preferred labels are luminescent labels, and especially fluorescent
or chromogenic labels.
[0132] A variety of functional groups used as detectable labels in
nucleotides has been developed in the art. Table 1 lists numerous
examples of such functional groups. Additional examples are
described in U.S. Pat. No. 6,399,335, published U.S. Patent
Application No. 2003/0124576, and The Handbook--`A Guide to
Fluorescent Probes and Labeling Technologies, Tenth Edition` (2005)
(available from Invitrogen, Inc./Molecular Probes), all of which
are incorporated herein by reference.
TABLE-US-00002 TABLE 1 Exemplary detectable label functional groups
4-aminophenol 6-aminonaphthol 4-nitrophenol 6-nitronaphthol
4-methylphenol 6-chloronaphthol 4-methoxyphenol 6-bromonaphthol
4-chlorophenol 6-iodonaphthol 4-bromophenol 4,4'-dihydroxybiphenyl
4-iodophenol 8-hydroxyquinoline 4-nitronaphthol 3-hydroxypyridine
4-aminonaphthol umbelliferone 4-methylnaphthol Resorufin
4-methoxynaphthol 8-hydroxypyrene 4-chloronaphthol
9-hydroxyanthracene 4-bromonaphthol 6-nitro9-hydroxyanthracene
4-iodonaphthol 3-hydroxyflavone 6-methylnaphthol fluorescein
6-methoxynaphthol 3-hydroxybenzoflavone
[0133] Using these or other suitable functional groups known in the
art, a vast diversity of fluorophores suitable for the present
sequencing method can been generated. They include but are not
limited to 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic
acid, acridine and derivatives such as acridine and acridine
isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS),
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate
(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide,
anthranilamide, Brilliant Yellow, coumarin and derivatives such as
coumarin, 7-amino-4-methylcomarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;
4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfone acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin
and derivatives such as eosin and eosin isothiocyanate; erythrosin
and derivatives such as erythrosin B and erythrosin isothiocyanate;
ethidium; fluorescein and derivatives such as 5-carboxyfluorescein
(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC);
fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;
4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde;
pyrene and derivatives such as pyrene, pyrene butyrate and
succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.RTM.
Brilliant Red 3B-A); rhodamine and derivatives such as
6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101 and sulfonyl chloride derivative of
sulforhodamine 101 (Texas Red);
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid and terbium chelate derivatives.
Additional fluorophores applicable for the subject sequencing
methods are disclosed in U.S. Pat. No. 5,866,366 and WO 01/16375,
both of which are incorporated herein by reference.
[0134] The labels can be attached to the phosphate backbone, on the
base, on the ribose unit, or a combination thereof. Preferred
labels are those that do not substantially impede the continuous
addition of nucleotides in a sequencing reaction. Such labels
include those linked to the alpha phosphate, the beta phosphate,
the terminal phosphate, or the delta or more distal phosphates in
tetra, penta or hexa phosphate nucleotides, or the base unit of a
nucleotide.
[0135] Nucleotides comprising labeled terminal phosphates (e.g.,
the gamma phosphate as in dNTP), are particularly preferred because
no additional means is required to remove the label in the
sequencing procedure. During the process of nucleic acid
polymerization, the bond cleavage in the nucleotide occurs between
the alpha and the beta phosphate, causing the beta and terminal
phosphate (e.g., the gamma phosphate as in dNTP) to be released
from the site of polymerization. As such, the label attached to the
terminal phosphate is separated from the nascent strand once the
nucleotide is being incorporated. In general,
terminal-phosphate-linked nucleotides may comprise three or more
phosphates, typically about three to about six phosphates,
preferably about three to about five phosphates. Table 1 lists
numerous examples of nucleotides with labeled terminal phosphates.
Many other terminal-phosphate-linked nucleotides have been
developed and are detailed in U.S. patent application number
2003/0124576, which is incorporated herein by reference in its
entirety.
TABLE-US-00003 TABLE 2
Adenosine-5'-(.gamma.-4-nitrophenyl)triphosphate
Guanosine-5'-(.gamma.-4-nitrophenyl)triphosphate
Cytosine-5'-(.gamma.-4-nitrophenyl)triphosphate
Thymidine-5'-(.gamma.-4-nitrophenyl)triphosphate
Uracil-5'-(.gamma.-4-nitrophenyl)triphosphate
3'-azido-3'-deoxythymidine-5'-(.gamma.-4- nitrophenyl)triphosphate
3'-azido-2',3'-dideoxythymidine-5'-(.gamma.-4-
nitrophenyl)triphosphate
2',3'-didehydro-2',3'-dideoxythymidine-5'-(.gamma.-
4-nitrophenyl)triphosphate
Adenosine-5'-(.gamma.-4-aminophenyl)triphosphate
Adenosine-5'-(.gamma.-4-methylphenyl)triphosphate
Adenosine-5'-(.gamma.-4-methoxyphenyl)triphosphate
Adenosine-5'-(.gamma.-4-chlorophenyl)triphosphate
Adenosine-5'-(.gamma.-4-bromophenyl)triphosphate
Adenosine-5'-(.gamma.-4-iodophenyl)triphosphate
Adenosine-5'-(.gamma.-4-nitronaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-aminonaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-methylnaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-methoxynaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-chloronaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-bromonaphthyl)triphosphate
Adenosine-5'-(.gamma.-4-iodonaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-methylnaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-methoxynaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-aminonaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-nitronaphthyl triphosphate
Adenosine-5'-(.gamma.-6-chloronaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-bromonaphthyl)triphosphate
Adenosine-5'-(.gamma.-6-iodonaphthyl)triphosphate
Adenosine-5'-(.gamma.-4'-hydroxybiphenyl)triphosphate
Adenosine-5'-(.gamma.-8-quinolyl)triphosphate
Adenosine-5'-(.gamma.-3-pyridyl)triphosphate
Adenosine-5'-(.gamma.-umbelliferone)triphosphate
Adenosine-5'-(.gamma.-resorufin)triphosphate
Adenosine-5'-(.gamma.-pyrene)triphosphate
Adenosine-5'-(.gamma.-anthracene)triphosphate
Adenosine-5'-(.gamma.-6-nitroanthracene)triphosphate
Adenosine-5'-(.gamma.-flavonyl)triphosphate
Adenosine-5'-(.gamma.-fluorescein)triphosphate
Adenosine-5'-(.gamma.-benzoflavone)triphosphate
Adenosine-5'-(.gamma.-(4-nitrophenyl)-.gamma.'-
(4-aminophenyl)triphosphate
Adenosine-5'-(.gamma.-(4-nitrophenyl)-.gamma.'-
(4-nitronaphthyl)triphosphate
[0136] Nucleotides comprising modified phosphate backbones can also
be used. For example, the modified component can be a
phosphordiamidate, methylphosphonate, alkyl phosphotriester,
formacetal, phosphorodithioate, phosphothioate,
phosphoramidothioate, phosphoramidate, or an analog thereof.
[0137] In some embodiments, the nucleotides or nucleotide analogs
used in the present invention are reversible extension terminators
comprising reversible blocking groups. In some embodiments, the
blocking group on a reversible extension terminator is linked to a
detectable label. In other embodiments, the blocking group and the
detectable label are located on different positions of a
nucleotide. In yet other embodiments, the blocking group is also a
label.
[0138] An illustrative reversible extension terminator comprises a
labeled ribose unit at the 3' end. Each label on the ribose unit,
typically acts as a reversible blocking group that must be removed
before the next nucleotide addition event can take place during a
polymerization reaction. Preferred 3'-ribose labels comprise
photo-removable functional groups that can be deprotected upon
exposure to a light beam at a suitable wavelength.
[0139] In another example, the reversible blocking group is located
at the 2' or the 4' position of the ribose unit of a nucleotide. In
yet another embodiment, the reversible blocking group is linked to
or conjugated to the base (adenine, thymine, cytosine, guanine, or
uracil) a nucleotide. Non-limiting examples of reversible blocking
groups, and especially photocleavable blocking groups include but
are not limited to those molecules depicted in FIGS. 14 and 15 and
those described in the co-pending application Ser. No. 60/649,009,
which is incorporated herein by reference in its entirety.
[0140] The wavelength used to cleave the photocleavable blocking
groups will depend on the choice of the blocking group. The
wavelength may range from about 320 nm to about 800 nm. In some
embodiment, the wavelength for cleaving the blocking group is about
the same as the wavelength used to detect the label. In other
embodiments, the wavelength for cleaving the blocking group is
different from the wavelength used to detect the label.
[0141] In some embodiments, it is advantageous to use a mixture of
labeled nucleotides that is substantially free of unlabeled
nucleotides. Such composition and the uses thereof for sequencing
are detailed in co-pending application Ser. No. 60/651,846, which
is incorporated herein. Briefly, the composition is prepared by
treating a mixture comprising labeled and unlabeled nucleotides or
nucleotide analogs with an agent that specifically modifies
unlabeled or incorrectly labeled nucleotides or nucleotide analogs
to reduce their ability to be used in a hybridization or sequencing
assay. Preferably, the agent used specifically modifies unlabeled
or incorrectly labeled nucleotides analogs to render them incapable
of being used in a hybridization or sequencing assay. For example,
the nucleotides can be modified so that they no longer contain
structures generally needed for the Watson Crick base pairing in a
hybridization or template-directed sequencing assay. In some
embodiments, for example, base units of the nucleotides are
modified. In some embodiments, phosphate groups, preferably
terminal phosphate groups, of the nucleotides or nucleotide analogs
are modified to yield molecules that are incorporated to a lesser
extent into a nascent nucleic acid strand during a
template-directed polymerization reaction. In more preferred
embodiments, the terminal phosphate groups of a nucleotide or
nucleotide analogs are modified to yield molecules that cannot or
that substantially cannot be incorporated into a nascent nucleic
acid strand during a template-directed polymerization reaction.
[0142] The agents can comprise one or more enzymes. A variety of
enzymes known in the art are suitable for modifying the nucleotides
or nucleotide analogs, e.g. by cleaving or altering the
configuration of the sugar, base, or phosphates, so as to disrupt
the specific Watson Crick base pairing. Exemplary agents include
but are not limited to guanine or adenine P-ribosyl transferase,
purine nucleoside phosphorylase, AMP nucleosidase, nucleoside
deoxyribosyl transferase for purines, and orotate P-ribosyl
transferase, thymidine phosphorylase, thymidine or uridine
nucleosidase, uridine phosphorylase, pyrimidine nucleoside
phosphorylase nucleoside deoxyribosyl transferase.
[0143] Enzymes applicable for modifying the terminal phosphate
groups of nucleotides or nucleotide analogs include a wide array of
phosphatases. An example of such enzyme is Shrimp Alkaline
Phosphatase (SAP) that can remove the gamma and beta phosphates
from a deoxynucleoside triphosphate (dNTP). The enzyme can convert
specifically unlabeled dNTP into a nucleoside monophosphate dNMP
which is generally incapable of being utilized by a polymerase
enzyme in a template-directed sequencing reaction. It has been
shown, that this phosphatase selectively modify nucleotides that
are not labeled, e.g. at the terminal phosphate. Therefore, in a
mixture of terminal phosphate-labeled and unlabeled nucleotides,
the SAP will preferentially act on unlabeled nucleotides, leaving a
larger proportion of labeled nucleotides available for
incorporation in a sequencing reaction.
[0144] Other suitable phosphatases that can be used include but are
not limited to calf intestinal alkaline phosphatases, and/or
phosphatases of other mammals, crustaceans, and other animals.
Examples of phosphatases that may be useful practicing the present
invention can be found in US 20040203097, US 20040157306, US
20040132155; and US 20040110180.
[0145] Any other naturally occurring or synthetic phosphatases or
phosphatases made by recombinant DNA technology can also be used so
long as they specifically or preferentially convert unlabeled
nucleotides or analogs (as compared to labeled nucleotides), to
molecules that are substantially incapable of being utilized by a
polymerization enzyme. Directed molecular evolution can also be
used to enhance and extend the activity of related enzymes to yield
the desired property described above. A wide variety of mutagenesis
techniques, both in silicon and in situ, are available in the art.
An example of a mutagenesis or screening assay for generating such
enzymes can involve a first test for abrogation of polymerization
in the system with unlabeled nucleotides, and a second screen
checking for the retention of polymerization activity in the
presence of labeled nucleotides. Both of these screens can be
performed in the context of a highly multiplexed parallel assay.
Enzymes showing some beneficial specificity can be retained,
mutated by some method, and then re-screened. Methods such as these
have been shown to produce many orders of magnitude improvement in
specificity and performance.
[0146] Enzymes capable of selectively or preferentially modifying a
subset of unlabeled nucleotides can also be employed. For example,
creatine kinase enzyme is specific for the removal of a phosphate
from adenoside triphosphate, and will not act on other bases. Other
enzymes that selectively or preferentially act on one or more types
of unlabeled nucleotides can also be used.
[0147] The nucleotide modifying enzymes described above can be used
to pre-treat the nucleotides or nucleotide analogs, or can be used
in the hybridization and/or sequencing reaction mixture, e.g.,
along with other hybridization or sequencing reagents.
[0148] The reaction conditions under which the modification of the
nucleotides takes place will vary depending on the choice of the
modifying enzymes. In one aspect, the conditions may be set within
the following parameters: pH is between 4.0 and 12.0, more
preferably between pH 6.0 and 10.0, more preferably between 7.0 and
9.0, more preferably less than 8, more preferably between 7 and 8,
and most preferably pH 7.5 and 8.5, preferably controlled by a
buffer. The buffer can be Tris-based preferably at pH 7.5 to pH
8.5. Other buffers may be used such as, but not limited to: organic
buffers such as MOPS, HEPES, TRICINE, etc., or inorganic buffers
such as phosphate or acetate. Buffers or other agents may be added
to control the pH of the solution thereby increasing the stability
of the enzymes. Where desired, reducing agent such as but not
limited to dithiotreitol (DTT) or 2-mercaptoethanol may be added to
limit enzyme oxidation that might adversely affect stability of the
enzymes. The choice of specific reaction conditions including
various buffers and pH conditions is within the skill of
practitioners in the field, and hence is not further detailed
herein.
[0149] Upon completion of the pre-treatment, the enzymes can be
heat-inactivated by raising the reaction temperature to at least
about 65.degree. C., preferably between about 65.degree. C. to
about 80.degree. C. Alternatively, the enzymes can be depleted from
the reaction mixture by, e.g., centrifugation through a filter
(e.g., Millipore) that has a molecular weight cutoff smaller than
the size of the enzyme.
[0150] After the treatment, the mixture generally comprises less
than about 30%, preferably less than about 20%, more preferably
less than about 10%, more preferably less than about 5%, more
preferably less than about 1%, more preferably less than about
0.5%, or more preferably less than about 0.1%, and even more
preferably less than 0.01% of unlabeled nucleotides or unlabeled
nucleotide analogs. This enriched mixture of labeled nucleotides or
nucleotide analogs is particularly useful for high-resolution
detection of the labeled nucleotides in a single-molecule sequence
reaction.
[0151] Importantly, the result of the foregoing treatment is a
process for synthesis of nucleic acids, preferably for elucidating
a template sequence using substantially only nucleotides, e.g.,
substantially complete replacement of native nucleotides with
nucleotide analogs, and particularly labeled analogs. Such template
dependant synthesis in the presence of substantially only
nucleotide analogs, and particularly labeled analogs, also referred
to as substantially complete replacement, in sequencing operations
is considerably different from previously described sequencing
methods where a single nucleotide is substituted with a labeled
chain terminating nucleotide among the remaining three natural
nucleotides, or where a polymerase template complex are
interrogated with only one analog at a time to determine whether
such analog is incorporated.
[0152] Another type of suitable nucleotides for the subject
sequencing methods allows detection via fluorescence resonance
energy transfer (FRET). In FRET, an excited fluorophore (the donor)
transfers its excited state energy to a light absorbing molecule
(the acceptor) in a distance-dependent manner. The limitation on
the distance over which the energy can travel allows one to discern
the interactions between labeled molecules and entities in close
proximity. Nucleotides of this type can comprise a donor
fluorophore attached to the base, ribose or preferably the
phosphate backbone (e.g., attached to the terminal phosphate), and
an acceptor fluorophore attached to the base, ribose or the
phosphate backbone where the donor is not attached. In a preferred
embodiment, the donor fluorophore is attached to the terminal
phosphate, and an acceptor fluorophore is linked to the base or the
ribose unit of the nucleotide. Upon incorporation of this type of
nucleotide into the nascent strand, a fluorescent signal can be
detected which can be caused by the release of poly-phosphate that
is no longer quenched. By determining the order of the fluorescent
poly-phosphate that is released upon incorporating a complementary
nucleotide during the polymerization event, one can deduce the base
sequence of the target nucleic acid. Additional examples of this
type of nucleotides is disclosed in U.S. application no.
20030194740, which is incorporated herein by reference.
[0153] In another embodiment, the donor fluorophore can be present
in a nucleotide, and the acceptor is located in the polymerase, or
vice versa. Where desired, the fluorophore in the polymerase can be
provided by a green fluorescent protein (GFP) or a mutant thereof
that has a different emission and/or absorption spectrum relative
to the wildtype green fluorescent protein. For example, the GFP
mutant H9-40 (Tsien et al., Ann. Rev. Biochem. 67: 509 (1998))
which is excited at 399 nm and emits at 511 nm, may serve as a
donor fluorophore for use with BODIPY, fluorescein, rhodamine green
and Oregon green. In addition, tetramethylrhodamine, Lissamine.TM.,
Texas Read and napthofluorescein can be used as acceptor
fluorophores with this GFP mutant.
[0154] Other representative donors and acceptors capable of
fluorescence energy transfer include, but are not limited to,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonap-hthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphth-al imide-3,5disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives: coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamine-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,-2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives: eosin, eosin isothiocyanate,
erythrosin and derivatives: erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives:
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)amin-ofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein, fluorescein isothiocyanate, QFITC, (XRITC);
fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;
4-methylumbelliferoneortho cresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde;
pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl
1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron.TM.
Brilliant Red 3B-A) rhodamine and derivatives:
6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine
101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA);
tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate
(TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy
3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo
cyanine; and naphthalo cyanine.
[0155] In alternative configurations, both donor and acceptor
fluorophores may be present upon each nucleotide analog, where the
donor provides a substantially uniform excitation spectrum, but
donates energy to an acceptor that provides an emission spectrum
that is different for each type of analog, e.g., A, T, G, or C.
Such configurations provide an ability to utilize a single
excitation source for multiple different emission profiles,
reducing energy input requirements for the systems utilized.
[0156] In addition, xanthene dyes, including fluoresceins and
rhodamine dyes can be used as donor and acceptor pairs.
[0157] Many of these dyes contain modified substituents on their
phenyl moieties which can be used as the site for bonding to the
terminal phosphate or the base of a nucleotide. Where desired,
acceptors acting as quenchers capable of quenching a wide range of
wavelengths of fluorescence can be used. Representative examples of
such quenchers include 4-(4'-dimethylaminophenylaz-o)-benzoic acid
(DABCYL), dinitrophenyl (DNP) and trinitrophenyl (TNP).
[0158] The polymerization enzymes suitable for the present
invention can be any nucleic acid polymerases that are capable of
catalyzing template-directed polymerization with reasonable
synthesis fidelity. The polymerases can be DNA polymerases or RNA
polymerases, a thermostable polymerase or a thermally degradable
polymerase wildtype or modified. Non-limiting examples for suitable
thermostable polymerases include polymerases from Thermus
aquaticus, Thermus caldophilus, Thermus filiformis, Bacillus
caldotenax, Bacillus stearothermophus, Thermus thermophilus,
Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and
Thermotoga maritima. Useful thermodegradable polymersases include
E. coli DNA polymerase, the Klenow fragment of E. coli DNA
polymerase, T4 DNA polymerase, T7 DNA polymerase.
[0159] Additional examples of polymerization enzymes that can be
used to determine the sequence of nucleic acid molecules include E.
coli T7, T3, SP6 RNA polymerases and AMY, M-MLV and HIV reverse
transcriptases. The polymerase can be bound to the primed target
nucleic acid sequence at a primed single-stranded nucleic acid, an
origin of replication, a nick or gap in a double-stranded nucleic
acid, a secondary structure in a single-stranded nucleic acid, a
binding site created by an accessory protein, or a primed
single-stranded nucleic acid.
[0160] In one preferred embodiment, the polymerization enzymes
exhibit enhanced efficiency as compared to the wildtype enzymes for
incorporating unconventional or modified nucleotides, e.g.,
nucleotides linked with fluorophores. Recombinant DNA techniques
can be used to modify the wildtype enzymes. Such techniques
typically involve the construction of an expression vector or a
library of expression vector, a culture of transformed host cells
under such condition such that expression will occur. Selection of
the polymerases that are capable of incorporating unconventional or
modified nucleotides can be carried out using any conventional
sequencing methods as well as the sequencing methods disclosed
herein.
[0161] In another preferred embodiment, sequencing is carried out
with polymerases exhibiting a high degree of processivity, i.e.,
the ability to synthesize long stretches of nucleic acid by
maintaining a stable nucleic acid/enzyme complex. A processive
polymerase can typically synthesize a nascent strand over about 10
kilo bases. With the aid of accessory enzymes (e.g.,
helicases/primases), some processive polymerases can synthesize
even over 50 kilobases. For instance, it has been shown that T7 DNA
polymerase complexed with helicase/primase can synthesize several
100 kilobases of nucleotides while maintaining a stable complex
with the target nucleic acid (Kelman et al., "Processivity of DNA
Polymerases: Two Mechanisms, One Goal" Structure 6: 121-125
(1998)).
[0162] In another preferred embodiment, sequencing is performed
with polymerases capable of rolling circle replication, i.e.,
capable of replicating circular DNA templates including but not
limited to plasmids and bacteriophage DNA. A preferred rolling
circle polymerase exhibits strand-displacement activity, and
preferably has reduced or essentially no 5' to 3' exonuclease
activity. Strand displacement results in the synthesis of tandem
copies of a circular DNA template, thus allowing re-sequencing the
same DNA template more than once. Re-sequencing the same DNA
template greatly enhances the chances to detect any errors made by
the polymerase, because the same errors unlikely would be repeated
by the polymerase and the same error certainly would not be
exponentially amplified as in a polymerase chain reaction.
[0163] Non-limiting examples of rolling circle polymerases suitable
for the present invention include but are not limited to T5 DNA
polymerase (Chatterjee et al., Gene 97:13-19 (1991)), and T4 DNA
polymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157
(1995)), phage M2 DNA polymerase (Matsumoto et al., Gene 84:247
(1989)), phage PRD1 DNA polymerase (Jung et al., Proc. Natl. Aced.
Sci. USA 84:8287 (1987), and Zhu and Ito, Biochim. Biophys. Acta.
1219:267-276 (1994)), Klenow fragment of DNA polymerase I (Jacobsen
et al., Eur. J. Biochem. 45:623-627 (1974)).
[0164] A preferred class of rolling circle polymerases utilizes
protein priming as a way of initiating replication. Exemplary
polymerases of this class are modified and unmodified DNA
polymerase, chosen or derived from the phages .PHI.29, PRD1, Cp-1,
Cp-5, Cp-7, .PHI.15, .PHI.1, .PHI.21, .PHI.25, BS 32 L17, PZE, PZA,
Nf, M2Y (or M2), PR4, PR5, PR722, B103, SF5, GA-1, and related
members of the Podoviridae family. Specifically, the wildtype
bacteriophage .PHI.29 genome consists of a linear double-stranded
DNA (dsDNA) of 19,285 base pairs, having a terminal protein (TP)
covalently linked to each 5' end. To initiate replication, a
histone-like viral protein forms a nucleoprotein complex with the
origins of replication that likely contributes to the unwinding of
the double helix at both DNA ends (Serrano at al., The EMBO Journal
16(9): 2519-2527 (1997)). The DNA polymerase catalyses the addition
of the first dAMP to the hydroxyl group provided by the TP. This
protein-primed event occurs opposite to the second 3' nucleotide of
the template, and the initiation product (TP-dAMP) slides back one
position in the DNA to recover the terminal nucleotide After
initiation, the same DNA polymerase replicates one of the DNA
strands while displacing the other. The high processivity and
strand displacement ability of .PHI.29 DNA polymerase makes it
possible to complete replication of the .PHI.29 TP-containing
genome (TP-DNA) in the absence of any helicase or accessory
processivity factors (reviewed by Serrano at, The EMBO Journal
16(9): 2519-2527 (1997)).
[0165] Modified .PHI.29 DNA polymerases having reduced 5' to 3'
exonuclease activity have also been described (U.S. Pat. Nos.
5,198,543 and 5,001,050, both being incorporated herein). These
polymerases are particularly desirable for sequencing as the 5' to
3' exonucleases, if present excessively, may degrade the nascent
strand being synthesized.
[0166] Strand displacement can be enhanced through the use of a
variety of accessory proteins. They include but are not limited to
helicases (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)),
herpes simplex viral protein ICP8 (Skaliter and Lehman, Proc. Natl.
Acad. Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA
binding proteins (Bigler and Romano, J. Biol. Chem. 270:8910-8919
(1995)), adenovirus DNA-binding protein (Zijderveld and van der
Vliet, J. Virology 68(2):1158-1164 (1994)), and BMRF1 polymerase
accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653
(1993)).
[0167] In a preferred embodiment, the sequence reaction involves a
single complex of strand-displacement polymerization enzyme and a
circular target DNA, which is immobilized to an optical
confinement. Upon mixing the labeled nucleotides or nucleotide
analogs and the primers, the strand-displacement polymerization
enzyme directs the synthesis of a nascent strand and a time
sequence of incorporating the various types of labeled nucleotides
or nucleotide analogs into the nascent strand is registered. Where
desired, the strand-displacement polymerase is allowed to
synthesize multiple tandem repeats of the target DNA, and thus
effecting re-sequencing the same circular DNA target multiple
times. It is preferably to register the time sequence of the
nucleotides or nucleotide analogs incorporated into at least two
tandem repeats of the target DNA molecule, more preferably at least
about three to about ten or about three to about one hundred tandem
repeats, and preferably no more than about one million repeats.
This multiple rounds of or redundant sequencing can take place
under an isothermal condition and/or at ambient temperature.
[0168] Using the subject method, sequencing can be carried out at
the speed of at least 1 base per second, preferably at least 10
bases per second, more preferably at least 100 bases per second. It
has been reported that polymerases can polymerize 1,000 bases per
second in vivo and 750 bases per second in vitro (see, e.g. Kelman
et al., "Processivity of DNA Polymerases: Two Mechanisms, One
Goal," Structure 6: 121-125 (1998); Carter et al., "The Role of
Exonuclease and Beta Protein of Phage Lambda in Genetic
Recombination. II. Substrate Specificity and the Mode of Action of
Lambda Exonuclease," J. Biol. Chem. 246: 2502-2512 (1971); Tabor et
al., "Escherichia coli Thioredoxin Confers Processivity on the DNA
Polymerase Activity of the Gene 5 Protein of Bacteriophage T7," J.
Biol. Chem. 262: 16212-16223 (1987); and Kovall et al., "Toroidal
Structure of Lambda-Exonuclease" Science 277: 1824-1827 (1997),
which are hereby incorporated by reference).
[0169] Reaction Conditions:
[0170] The sequencing procedures of the present invention are
performed under any conditions such that template-directed
polymerization can take place using a polymerization enzyme. In one
aspect, the substrates of the polymerization enzyme, namely the
various types of nucleotides present in the sequence reaction, are
adjusted to a physiologically relevant concentration. For example,
the nucleotides used in the sequencing reaction are present at a
concentration about Michaelis constant of the polymerization
enzyme. Such concentration typically ranges from about 1 micromolar
to about 50 micromolar or about 100 micromolar.
[0171] The sequencing procedures can also be accomplished using
less than four labels employed. With three labels, the sequence can
be deduced from sequencing a nucleic acid strand (1) if the fourth
base can be detected as a constant dark time delay between the
signals of the other labels, or (2) unequivocally by sequencing
both nucleic acid strands, because in this case one obtains a
positive fluorescence signal from each base pair. Another possible
scheme that utilizes two labels is to have one base labeled with
one fluorophore and the other three bases with another fluorophore.
In this case, the other three bases do not give a sequence, but
merely a number of bases that occur between the particular base
being identified by the other fluorophore. By cycling this
identifying fluorophore through the different bases in different
sequencing reactions, the entire sequence can be deduced from
sequential sequencing runs. Extending this scheme of utilizing two
labels only, it is even possible to obtain the full sequence by
employing only two labelled bases per sequencing run.
[0172] The sequencing procedures can be performed under an
isothermal condition, at ambient temperature, or under thermal
cycling condition. The choice of buffers, pH and the like is within
the skill of practitioners in the art, and hence is not detailed
herein.
[0173] Detection:
[0174] The subject sequencing method requires the imaging of
individual molecules confined in an optical confinement. The
polymerase and/or the nucleotides are labeled with fluorophores
that emit a distinguishable optical signal when a particular type
of nucleotide is incorporated into the nascent strand. The sequence
of the distinguishable signals is detected as the nucleotides are
sequentially added to the nascent strand within the optical
confinement. In a preferred embodiment, such detection is performed
without the need to transfer, separation or washing away any
reactant or by-product (e.g. fluorophore cleaved from a nucleotide)
after each nucleotide addition event. In one aspect of this
preferred embodiment, sequence detection is performed without
adding reactants to the mixture prior to reading the next base
sequence nucleotide to be incorporated.
[0175] Imaging individual molecules confined in the subject optical
confinements is performed with the aid of an optical system. Such
system typically comprises at least two elements, namely an
excitation source and a photon detector. Numerous examples of these
elements are described above.
[0176] In a preferred embodiment, the excitation source is a laser,
preferably a polarized laser. The choice of laser light will depend
on the fluorophores attached to the different type of nucleotides
and/or the polymerases. For most of the fluorophorescent compounds,
the required excitation light is within the range of about 300 nm
to about 700 nm. For proteinaceous fluorophores such as
green-fluororescent protein and mutants thereof, the excitation
wavelength may range from about 488 nm to about 404 nm. Those
skilled in the art will know or will be able to ascertain the
appropriate excitation wavelength to excite a given fluorophore by
routine experimentation (see e.g., The Handbook--`A Guide to
Fluorescent Probes and Labeling Technologies, Tenth Edition` (2005)
(available from Invitrogen, Inc./Molecular Probes) previously
incorporated herein by reference).
[0177] Another consideration in selecting an excitation source is
the choice between one-photon and multiphoton excitation of
fluorescence. Multiphoton excitation coupled with detection, also
known as multiphoton micropscopy ("MPM"), provides enhanced
sensitivity and spatial resolution. MPM is a form of laser-scanning
microscopy that uses localized nonlinear excitation to excite
fluorescence within a thin raster-scanned plane. In MPM, as in
conventional laser-scanning confocal microscopy, a laser is focused
and raster-scanned across the sample. The image consists of a
matrix of fluorescence intensity measurements made by digitizing
the detector signal as the laser sweeps back and forth across the
sample. Two-photon excitation probabilities are extremely small,
and focusing increases the local intensity at the focal point.
Although two-photon excited fluorescence is usually the primary
signal source in MPM, three-photon or more excited fluorescence and
second or third-harmonic generation can also be used for imaging.
See, e.g., a review of multiphoton micropscopy in Webb et al.
Nature Biotechnology (2003) 21: (11) 1251-1409. A preferred MPM
setup comprises MPM laser scanning microscopes and second-harmonic
imaging, equipped with femtosecond mode-locked titanium sapphire
lasers operating at wavelengths from about 700 to 1,000 mm. Such
setup can capture more than about 100 photons per pixel in most of
the conventional imaging multiphoton microscope.
[0178] The sequence of the distinguishable signals can also be
detected by other optical systems comprising elements such as
optical reader, high-efficiency photon detection system, photo
multiplier tube, gate sensitive FET's, nano-tube FET's, photodiode
(e.g. avalanche photo diodes (APD)), camera, charge couple device
(CCD), electron-multiplying charge-coupled device (EMCCD),
intensified charge coupled device (ICCD), and confocal
microscope.
[0179] A preferred combination comprises wide field CCD or ICCD and
intensified video imaging microscopes with digital image processing
capability, as well as Fluorescence Photobleaching Recovery (FPR)
and Fluorescence Correlation Spectroscopy (FCS) coupled with
confocal multiphoton capability and continuous data acquisition and
control. Such set up may further comprise modular instrument for
quasi-elastic light scattering, laser DIC interferometry,
correlation spectroscopy instrumentation, components of optical
force microscopy, and Time Correlated Single Photon Counting
(TCSPC).
[0180] These optical systems may also comprise optical transmission
elements such as diffraction gratings, arrayed waveguide gratings
(AWG), optic fibers, optical switches, mirrors, lenses (including
microlens and nanolens), collimators. Other examples include
optical attenuators, polarization filters (e.g., dichroic filter),
wavelength filters (low-pass, band-pass, or high-pass),
wave-plates, and delay lines. In some embodiments, the optical
transmission element can be planar waveguides in optical
communication with the arrayed optical confinements.
[0181] These and other optical components known in the art can be
combined and assembled in a variety of ways to effect detection of
the distinguishable signals emitted from the sequencing reaction.
Preferred devices allow parallel data collection using arrays
having a large number of optical confinements, where simultaneous
and independent sequencing of nucleic acids takes place. In one
aspect, the preferred system can collect and process signals from
more than 10.sup.4 optical confinements, more than 2.times.10.sup.4
optical confinements, or more than 10.sup.5 optical confinements,
or more than 2.times.10.sup.5 optical confinements, or preferably
more than 10.sup.6, or preferably more than 2.times.10.sup.6
optical confinements, and even more preferably more than 10.sup.7
or 2.times.10.sup.7 optical confinements. In another aspect, the
preferred setup can monitor in real time the simultaneous and
independent sequencing of nucleic acids at a speed of about 1 base
per second, preferably at a speed of about 10 bases per second,
more preferably at a speed of about 100 bases per second and even
more preferably at 1,000 bases per second. As such, the massive
parallelism coupled with the rapid sequencing reaction can provide
an overall sequencing output greater than 100,000 bases per second.
The overall output can be scaled up to at least 1 megabase per
second, preferably 10 or more megabases per second. Further by
obtaining such date from multiple different sequence fragments
e.g., in from two or more different reaction volumes, one can
obtain independent sequences, e.g. from contiguous fragments of
genomic DNA, allowing the high rate of throughput that is directly
applicable to genomic sequencing.
[0182] Other Single-Molecule Applications:
[0183] The subject optical confinements and arrays of optical
confinements find utility in many other chemical and biological
applications where single molecule analyses are desired. In
general, the subject optical confinements are applicable for any
single molecule analysis involving any reagent that can be attached
to the surface and for which substrates can be labeled, including,
enzymes, nucleic acids, antibodies, antigens, and the like. Such
applications include discerning interactions involving biological
molecules such as proteins, glycoproteins, nucleic acids, and
lipids, as well as inorganic chemicals, or any combinations
thereof. The interactions may be between nucleic acid molecules,
between nucleic acid and protein, and between protein and small
molecules.
[0184] Abnormalities in interactions involving biological molecules
have long been acknowledged to account for a vast number of
diseases including, numerous forms of cancer, vascular diseases,
neuronal, and endocrine diseases. An abnormal interaction, in form
of e.g., constitutive activation and premature inactivation of a
signaling complex, are now known to lead to aberrant behavior of a
disease cell. In the case of cancer, abnormal interactions between
two signaling transduction molecules, such as growth factor
receptors and their corresponding ligands, may result in
dysfunction of cellular processes, which ultimately lead to
dysregulated growth, lack of anchorage inhibition, genomic
instability and/or propensity for cell metastasis.
[0185] A specific interaction between biological or chemical
molecules typically involves a target molecule that is being
investigated and a probe suspected to be able to specifically
interact with the target. In practicing the subject methods, the
target and the probe are placed within an optical confinement. The
target-probe complex can be a protein-protein complex, a
glycoprotein-protein complex (e.g., receptor and ligand complex), a
protein-nucleic acid complex (e.g., transcription factor and
nucleic acid complex), a protein-lipid complex, and complex of
inorganic or organic small molecules.
[0186] Preferably, each optical confinement contains only one
target that is being investigated. This can be achieved by diluting
a minute amount of target in a large volume of solution, such that
deposition over an array of confinements results in a primary
distribution, or a majority of confinements will have a single
target molecule disposed there. Alternatively, a non-cylindrical
waveguide, wherein the opening of the waveguide core is narrower in
lateral dimension than the base, can be used to restrict the entry
of multiple target proteins while permitting the entry of a number
of smaller probes.
[0187] The target or probe can be immobilized onto the inner
surface of the optical confinement by any of the methods applicable
for immobilizing and depositing the polymerases described in the
section above. Such methods encompass the uses of covalent and
noncovalent attachments effected by a variety of binding moieties.
The choice of the binding moieties will depend on the nature of the
target and/or the probe. For example, the binding moieties can be
synthetically linked to the protein target or the probe, or made as
a fusion motif or tag via a recombinant means. A preferred way to
immobilize the target protein or the proteinaceous probe involves
the use of the streptavidin or avidin/biotin binding pair, and any
other binding moieties or agents described above.
[0188] The reaction conditions will depend on the particular
interaction that is under investigation. One may vary the reaction
temperature, the duration of the reaction, the buffer strength, and
the target concentration or the probe concentration. For example,
one may vary the concentration of the probe in order to measure its
binding affinity to the target protein. To determine the thermal
stability of the target-probe complex, one may vary the reaction
temperature. Stability of the target-probe complex can also be
determined by varying the pH, or buffer salt concentration. Where
desired, the interaction can be studied under physiologically
relevant temperature and buffer conditions. A physiologically
relevant temperature ranges from approximately room temperature to
approximately 37.degree. C. A physiological buffer contains a
physiological concentration of salt at neutral pH ranging from
about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
Adjusting the reaction conditions to discern a particular
interaction in vitro between a given target and a probe is within
the skill of artisans in the field, and hence is not detailed
herein.
[0189] The target and/or the probe are generally labeled with
detectable labels so that a photon detector can detect a signal
indicative of their interaction. Suitable labels encompass all of
those labels disclosed in the Single-Molecule Sequencing section.
Preferred labels are luminescent labels, and especially fluorescent
or chromogenic labels.
[0190] In one embodiment, the target is labeled with a fluorophore
whose signal is quenched upon interaction with the corresponding
probe conjugated with an appropriate quencher. A variety of
suitable fluorophore-quencher pairs is disclosed in the section
above and hence is not detailed herein. A variation of this
embodiment is to label the target and the probe with donor and
acceptor fluorphores (or vise versa) that emit a distinguishable
signal when the two molecules bind to each other. A wide range of
applicable donor and acceptor fluorophores is also described above.
Those of skill in the art will appreciate the wide diversity of
detectable labels and the combinations thereof to generate a
distinguishable signal that is indicative of a specific interaction
between biological molecules and/or chemical compounds.
[0191] The detection of the distinguishable signal indicative of a
specific interaction is performed with the aid of the optical
systems described herein. Any of the systems applicable for
single-molecule sequencing is equally suited for detecting
interactions between other biological molecules and/or chemical
compounds. A preferred system allows parallel data collection using
arrays having a large number of optical confinements, where
simultaneous and independent target-probe interactions can take
place. In one aspect, the preferred system can collect and process
signals from more than 10.sup.4 optical confinements, more than
2.times.10.sup.4 optical confinements, more than 10.sup.5 optical
confinements, more than 2.times.10.sup.5 optical confinements,
preferably more than 10.sup.6, or preferably more than
2.times.10.sup.6 optical confinements, and even more preferably
more than 10.sup.7 or 2.times.10.sup.7 optical confinements.
[0192] Of particular significance is the application of the
aforementioned method in detecting the presence of a specific
protein-protein interaction. Such application generally employs a
proteinaceous probe and a target protein placed in an optical
confinement. In one aspect of this embodiment, the specific
protein-protein interaction is between a cell surface receptor and
its corresponding ligand. Cell surface receptors are molecules
anchored on or inserted into the cell plasma membrane. They
constitute a large family of proteins, glycoproteins,
polysaccharides and lipids, which serve not only as structural
constituents of the plasma membrane, but also as regulatory
elements governing a variety of biological functions. In another
aspect, the specific protein-protein interaction involves a cell
surface receptor and an immunoliposome or an immunotoxin. In yet
another aspect, the specific protein-protein interaction may
involve a cytosolic protein, a nuclear protein, a chaperon protein,
or proteins anchored on other intracellular membranous structures.
In yet another aspect, the specific protein-protein interaction is
between a target protein (e.g., an antigen) and an antibody
specific for that antigen.
[0193] The specific interaction between an antigen and an antibody
has been explored in the context of immunoassays. There exists a
variety of immunoassays in the art, but none of which permits
single-molecule detection. For instance, the conventional
radioimmunoassay detects the interactions between a population of
antigens and a population of radioactively labeled antibodies on an
immunoblot. Another conventional immunoassay termed ELISA (Enzyme
Linked Immunoradiometric Assay) utilizes an antigen-specific
antibody and an enzyme-lined generic antibody that binds to the
specific antibody. The specific interaction between the antigen and
the antibody is visualized upon addition of the substrate to the
linked enzyme. Such assay again is performed on an immunoblot
providing an ensemble measurement of all interactions detected.
[0194] The subject optical confinement provides an effective tool
for conducting a single-molecule immunoassay. Unlike the
conventional immunoassays, the specific interaction between the
antigen and the antibody can be resolved at the single-molecule
level. While all of the optical confinements embodied in the
present invention are applicable for conducting single-molecule
immunoassays, a particularly desirable system comprises an array of
optical confinements with a relatively high fill fraction ratio.
For example, a preferred system comprises an array of waveguides
having a fill fraction greater than 0.0001, more preferably greater
than about 0.001, more preferably greater than about 0.01, and even
more preferably greater than 0.1.
[0195] In practicing the subject immunoassays, the antibodies an be
labeled with a suitable label selected from radioactive labels,
fluorescent labels, chemiluminescent labels, enzyme tags, such as
digoxigenin, .beta.-galactosidase, urease, alkaline phosphatase or
peroxidase, avidin/biotin complex, and any of the detectable labels
disclosed herein.
[0196] The subject immunoassays can be performed to characterize
biological entities, screen for antibody therapeutics, and
determine the structural conformations of a target antigen. For
instance, immuoassays involving antibodies that are specific for
the biological entity or specific for a by-product produced by the
biological entity have been routinely used to identify the entity
by forming an antibody-entity complex. Immunoassays are also
employed to screen for antibodies capable of activating or
down-regulating the biological activity of a target antigen of
therapeutic potential. Immunoassays are also useful for determining
structural conformations by using anti-idotypic antibodies capable
of differentiating target proteins folded in different
conformations.
[0197] Another important application of the aforementioned
single-molecule analysis is to study enzyme kinetics, which may
include determining the enzymatic turnover cycle, the dynamic
behavior, folding and unfolding intermediates, and binding
affinities. The enzymes under investigation may be immobilized
within the optical confinements or present in solutions confined
within the subject optical confinements.
[0198] All of the optical confinements embodied by the present
invention can be employed to study enzyme kinetics. The choice of a
specific optical confinement will depend on the specific
characteristic that is under investigation. For instance, an
optical confinement comprising a non-cylindrical core having an
opening on the upper surface that is narrower than that of the base
of the optical confinement is preferable for measuring the
association rate constant (on-rate) of an enzymatic reaction. This
configuration significantly restricts the diffusion of reactants or
substrates, and hence increases the average residence time in the
observation volume. On the other hand, an optical confinement
comprising a core with an opening that is wider in lateral
dimension than the base imposes impose a stearic or entropic
hinderence to entering the structure, hence is useful for measuring
the accessibility for large enzymes or enzymatic complexes.
[0199] Uses of the Subject Optical Confinements in Ensemble
Measurements:
[0200] While the optical confinements of the present invention are
particularly useful in conducting single-molecule analyses, the
subject confinements are also suited for high throughput
performance of ensemble bulk measurements. Accordingly, the present
invention provides a method of detecting interactions among a
plurality of molecules, comprising: placing said plurality of
molecules in close proximity to an array of zero-mode waveguides,
wherein individual waveguides in said array are separated by a
distance sufficient to yield a detectable intensity of diffractive
scattering at multiple diffracted orders upon illuminating said
array with an incident wavelength of light beam; illuminating said
array of zero-mode waveguides with said incident wavelength; and
detecting a change in said intensity of diffractive scattering of
said incident wavelength at said multiple diffracted orders,
thereby detecting said interactions among said plurality of
molecules.
[0201] Arrays employed for this method typically comprises optical
confinements spaced far apart relative to the incident wavelength.
Such spacing of the individual optical confinements far apart
relative to the illuminating radiation (e.g., half of the
wavelength of the illuminating radiation) creates a larger effect
on the diffractive scattering of incident light at a given angle
away from the angle of specula reflection. In one aspect of this
embodiment, the arrays contain individual confinements separated by
more than one wavelength of the incident radiation, usually more
than 1.5 times the incident wavelength, but usually does not exceed
150 times the incident wavelength.
[0202] Arrays having the optical confinements spaced far apart
relative to the incident wavelength also have desirable properties.
While the angle-dependent scattering may raise the background
signal that could be disadvantageous for certain applications, it
provides a means particularly suited for characterizing the size
and shape of the optical confinements. It also readily permits
ensemble bulk measurements of molecule interactions, involving
especially unlabelled molecules. Arrays suited for such
applications generally contain individual confinements separated by
more than one wavelength of the incident radiation, usually more
than 1.5 times the incident wavelength, but usually not exceeding
150 times the incident wavelength.
[0203] The ensemble bulk measurement is typically performed with
the aid of the optical systems described herein. Any of the setup
applicable for single-molecule sequencing is equally suited for
this analysis.
[0204] Further illustrations of the fabrication of the optical
confinements of the present invention and then uses in sequencing
are provided in the Example section below. The examples are
provided as a guide to a practitioner of ordinary skill in the art,
and are not meant to be limiting in any way.
EXAMPLES
Example 1
[0205] The following provides an illustrative process of
fabricating zero-mode waveguide. The parameters described herein
are meant to be illustrative and not intended to be limiting in any
manner. [0206] 1. Substrates: Substrates are double polished, 60/40
scratch/dig surface quality, Fused Silica wafers, cut to 100
millimeters (+/-0.2 mm) diameter, and 175 micrometer (+/-25
micrometers) thick and a total thickness variation of less than 25
micrometers. [0207] 2. Clean: A mix of 5 parts deionized water, 1
part of (30% v/v Hydrogen Peroxide in water), 1 part of (30% v/v
Ammonium Hydroxide in water) is heated to 75 degree Celsius on a
hotplate. The wafers are immersed in the mix using a Teflon holder
or other chemically resistant holder for a duration of 15 minutes.
[0208] 3. Rinsing: The holder containing the wafers is removed from
the RCA clean bath and immersed in a bath of deionized water. The
wafers are left in this second bath for a 2 minutes period. The
holder still containing the wafers is removed from the bath, and
sprayed with deionized water to thoroughly finish the rinsing
process. [0209] 4. Drying: Within a minute of the final rinsing
step, the wafers are dried, while still in the holder, using a dry
clean nitrogen flow. [0210] 5. Oxygen Plasma: The wafers are then
placed in a Glenn 1000p plasma Asher, used in plasma etch mode
(wafers on a powered shelf, and under another powered shelf), with
140 mTorr pressure and 400 Watts of forward power at 40 kHz
frequency. The plasma is maintained for 10 minutes. A flow of 18
sccm of molecular oxygen is used. [0211] 6. Vapor Priming: The
wafers are loaded within 3 minutes after the Oxygen plasma in a
Yield Engineering Systems vapor priming oven where they are coated
with a layer of HexaMethylDiSilazane (HMDS) adhesion promoter.
[0212] 7. Electron beam resist coating: The wafers are coated
within 15 minutes after the Vapor Priming in a manual spinner unit
using NEB-31 electron beam resist (Sumitomo Chemical America).
About 3 ml are dispensed on the wafer, which is then spun at 4500
rpm for 60 seconds. Initial acceleration and deceleration are set
to 3 seconds [0213] 8. Resist Bake: The wafers are baked on a CEE
hotplate at a temperature of 115 degree Celsius for 2 minutes. The
plate is equipped with a vacuum mechanism that allows good thermal
contact between the wafers and the hotplate surface. [0214] 9. Gold
Evaporation: a layer of 10 nm of gold is then thermally evaporated
on the Wafers, on the side coated with the resist. A pressure of
less than 2 10e-06 Ton must be reached before the evaporation. The
evaporation is performed at a rate of approximately 2.5 Angstrom
per second and monitored using an Inficon controller. [0215] 10.
Electron beam exposure: a pattern consisting of Zero Mode
Waveguides is exposed on the wafers, using a high resolution
electron beam lithography tool such as a Leica VB6-HR system. Zero
mode waveguides are patterned as single exel features. At a current
of nominally 1 nanoAmpere, and a Variable Resolution Unit of 1, and
for an exel setting of 5 nanometers, doses can range from 10000
microCoulombs per square centimeters to 300000 microCoulombs per
square centimeters. [0216] 11. Post Exposure Bake: The wafers are
then submitted to a 2 minute post exposure bake on a hotplate at 95
degree Celsius, equally equipped with a vacuum mechanism. [0217]
12. Gold Etch: After removal from the electron beam system, the 10
nanometer gold layer is removed using gold etchant TFA at room
temperature (GE 8148, Transene Corporation), for 10 seconds. Wafers
are held in a Teflon holder similar to the one used in step 2:
[0218] 13. Rinsing: The holder containing the wafers is removed
from the gold etchant bath and immerse in a bath of deionized
water. The wafers are left in this second bath for a 2 minutes
period or shorter with gentle manual agitation. The holder still
containing the wafers is removed from the bath, and sprayed with
deionized water to thoroughly finish the rinsing process.
Alternatively, the holder still containing the wafer is then placed
into a new container containing fresh deionized water. [0219] 14.
Drying: Within a minute of the final rinsing step, the wafers are
dried, while still in the holder, using dry clean nitrogen flow.
[0220] 15. Post Exposure Bake: The wafers are then submitted to a 2
minute post exposure bake on a hotplate at 95 degree Celsius,
equally equipped with a vacuum mechanism. [0221] 16. Developing:
The wafers still in the chemically resistant holder are immersed in
developer MF-321 (Shipley Chemicals, Rohm-Haas) at room temperature
for duration of 30 seconds. [0222] 17. Rinsing: The holder
containing the wafers is removed from the developer etchant bath
and immerse in a bath of deionized water. The wafers are left in
this second bath for a 2 minutes period with gentle manual
agitation. The holder still containing the wafers is removed from
the bath, and sprayed with deionized water to thoroughly finish the
rinsing process. [0223] 18. Drying: Within a minute of the final
rinsing step, the wafers are dried, while still in the holder,
using dry clean nitrogen flow. [0224] 19. Surface Descum: The
wafers are loaded in a Glenn 1000p plasma asher run in ashing mode
(Wafers on a grounded plate below a powered plate), and submitted
to a 30 seconds surface descuming oxygen plasma at a pressure of
140 mTorr and a power of 100 Watts forward power at 40 kHz. A flow
of 18 sccm of molecular oxygen is used. [0225] 20. Aluminum
Evaporation: The wafers are loading in a metal evaporator within 5
minutes of the surface descum process. A layer of 100 nm of
thermally evaporated Aluminum is now deposited on the wafers.
Evaporation is made at a pressure of no less than 2 10 -6 Torr at a
rate of 25 Angstrom per seconds and monitored using an Inficon
controller. [0226] 21. Aluminum Thickness measurement: The
thickness of the aluminum is measured using a P-10 Profilometer
(Tencor). [0227] 22. Zero Mode Waveguide Decasting: The Zero Mode
Waveguide are decasted from the enclosing Aluminum film by
immersing them, in a Teflon holder or other chemically resistant
holder, in a bath of 1165 Stripper (Shipley Chemicals, Rohm-Haas),
or in a bath of AZ-300T Stripper (Shipley Chemicals, Rohm-Haas).
The bath is submitted to sonication by immersing the Container
holding both the Stripper and the wafer holder in a sonicator. The
wafers are left in the decasting bath for 30 minutes or longer for
about 45 minutes, and are provided with additional gentle
agitation. [0228] 23. Rinsing: The stripping bath is removed from
the sonicator. The wafers are removed from the stripper bath and
immerse in a bath of deionized water. The wafers are left in this
second bath for a 2 minutes period with gentle manual agitation.
The wafers are removed from the bath, and sprayed with deionized
water to thoroughly finish the rinsing process. [0229] 24. Drying:
Within a minute of the final rinsing step, the wafers are dried,
while still in the holder, using dry clean nitrogen flow [0230] 25.
Photoresist coating: The wafers are coated with Shipley 1827
photoresist spun at a speed of 1500 rpm. About 5 ml of resist is
dispensed. Acceleration and deceleration is set to 5 seconds.
[0231] 26. Resist Bake: The wafers are baked on a CEE hotplate at a
temperature of 115 degree Celsius for 15 minutes. The plate is
equipped with a vacuum mechanism that allows good thermal contact
between the wafers and the hotplate surface. [0232] 27. Dicing: The
wafer are diced using a K&S-7100 dicing saw (Kulicke &
Soffa) using a resin/diamond blade (ADT 00777-1030-010-QIP 600).
The wafers are mounted on a low-tack adhesive tape prior to dicing.
[0233] 28. Die Removal: The dies are removed from the adhesive tape
manually and stored. [0234] 29. Resist removal: The layer of 1827
photoresist is removed by immersing the dies first in an acetone
bath for 1 minute, then in a 2-propanol bath for 2 minute with
gentle manual agitation. [0235] 30. Die Drying: The die is dried
after being removed from the 2-propanol bath using dry clean air.
[0236] 31. Plasma Clean: The wafers are loaded in a Drytek 100
plasma etcher, and submitted to a 1 minute oxygen plasma at a
pressure of 140 mTorr, a molecular oxygen flow of 85 seem oxygen
and an RF power of 500 Watts forward power at 13 Mhz.
Alternatively, Harrick Plasma Cleaner PDC-32G are submitted to a 5
minute dry clean air plasma at a pressure of 2 Torr and 10.5 Watts
of power.
Example 2
Monitoring Enzymatic Synthesis of a DNA Strand by a Single DNA
Polymerase Molecule in Real Time
[0237] This experiment can be performed using the optical system
and reaction mixtures detailed below. However, the reference to any
particular optical system and parameter, buffer, reagent,
concentration, pH, temperature, or the like, is not intended to be
limiting. They are included to provide as one illustrative example
of carrying out the methods of the present invention.
[0238] Enzymatic synthesis of a DNA strand by a single DNA
polymerase molecule was tracked in real time using a fluorescently
labeled nucleotides. Individual Phi29.sup.N62D DNA polymerase
enzymes (Amersham Biosciences, Piscataway, N.J.) were immobilized
in zero-mode waveguides (ZMWs) by non-specific binding using a
dilute enzyme solution. After immobilization, the ZMW structures
were washed to remove unbound enzyme, and then exposed to a
solution containing the reaction reagents. As of the DNA template,
a 70-bp pre-primed circular DNA sequence was used that contained
two guanine bases in characteristic, asymmetric spacing (FIG. 9A).
Strand-displacement polymerizing enzymes such as Phi29 DNA
polymerase will continuously loop around the circular template and
thus generate a long and highly repetitive complementary DNA
strand.
[0239] An R110-dCTP (Amersham Biosciences, Piscataway, N.J.) was
used as the fluorescently-tagged nucleotide analog in which the
fluorophore is attached to the nucleotide via a linker to the
gamma-phosphate. In contrast to the more commonly used base-labeled
nucleotide analogs, gamma-phosphate-linked analogs are cleaved
through the enzymatic activity of DNA polymerase as the attached
nucleotide is incorporated into the growing DNA strand and the
label is then free to diffuse out of the effective observation
volume surrounding the DNA polymerase. The efficient removal of the
fluorophore ensures continuously low background levels and prevents
significant interference with DNA polymerase activity. These
features of the gamma-phosphate-linked fluorophore are preferable
for this application because they will enable replacement of all
four bases with fluorophore-tagged analogs. Binding of a nucleotide
and its subsequent incorporation into nucleic acid from a mismatch
event is distinguished because the rate constants of these two
processes are significantly different, and because nucleotide
incorporation involves several successive steps that prevent zero
delay time events.
[0240] All other nucleotides were supplied without labels. We have
established a very effective way of removing any remaining trace
amount of native dNTP in a nucleotide analog preparation to ensure
that errors are not introduced due to the incorporation of
unlabeled dNTPs by an enzymatic purification using an alkaline
phosphatase prior to the polymerization assay.
[0241] To investigate the speed and processivity of the
Phi29.sup.N62D DNA polymerase under these conditions, the
incorporation characteristics were measured using R110-dCTP
completely replacing dCTP in the reaction mixture, both in solution
and with enzyme immobilized on a glass surface. It was found that
the enzyme efficiently utilized this analog, synthesizing
complementary DNA of many thousands of base pairs in length without
interruption in a rolling circle synthesis protocol, using both
small preformed replication forks (FIG. 9A) as well as larger
circular DNA such as M13 DNA. Only two, asymmetrically spaced
R110-dCTPs were to be incorporated into this template. Similar
experiments demonstrated that DNA polymerase can be immobilized to
the bottom of ZMWs without losing this catalytic activity.
[0242] The incorporation of the fluorescently labeled dCTP
nucleotide was tracked during rolling-circle DNA synthesis by
recording the fluorescent light bursts emitted in an individual
ZMW. DNA polymerase activity was observed in many waveguides as
distinct bursts of fluorescence, which lasted several minutes. The
fluorescence time trace showed a characteristic double burst
pattern (FIG. 9B), each burst corresponding to an incorporation
event of a R110-dCTP analog into the DNA strand and subsequent
cleavage of the fluorophore. In histograms of burst intervals
derived from the full time trace, two peaks corresponding to DNA
synthesis along the short (14 bases, approximately one second) and
long (54 bases, approximately four seconds) DNA template segments
are visible, consistent with an overall average speed measured in
bulk solution under these conditions of approximately ten base
pairs per second.
[0243] It is noteworthy that this single-molecule activity at a
fluorophore concentration of 10 .mu.M was readily observable. In
conventionally created excitation volumes, the number of
fluorophores would be far too high to permit the observation of
individual enzymatic turnovers of DNA polymerase. These experiments
thus confirmed the validity of the ZMW-based single-molecule DNA
sequencing approach by verifying that (a) immobilization of DNA
polymerase in ZMWs does not affect its enzymatic activity; (b)
fluorescent gamma-phosphate-linked nucleotide analogs do not
inhibit the activity of DNA polymerase; and (c) ZMWs provide an
adequate degree of confinement to detect single-molecule DNA
polymerase activity at physiological concentrations of reagents.
More generally, these results prove that ZMWs allow single-molecule
analysis of enzyme kinetics, especially involving any enzyme that
can be attached to the surface and for which substrates can be
fluorescently labeled.
Example 3
Real Time Sequencing Using Multiple Different Labeled
Nucleotides
[0244] An experiment similar to that described in Example 2, above,
was performed using two different labeled nucleotide analogs. The
experiment can be performed using the optical setup or system and
reaction mixtures detailed as follows. However, the reference to
any particular optical setup and parameter, buffer, reagent,
concentration, pH, temperature, or the like, is not intended to be
limiting. They are included to provide as one illustrative example
of carrying out the methods of the present invention.
[0245] Preparing reaction samples: Approximately 10 .mu.l of
reaction mixture is used in one sequencing reaction. The reaction
mixture generally contains 0.5-1 mM MnCl.sub.2, 0.1-1 uM DNA
template, 10 .mu.M dATP, 10 .mu.M dGTP, 10 .mu.M SAP-treated Alexa
488-dC4P, and 10 .mu.M SAP-treated Alexa 568-dT4P, and DNA
polymerase. The labeled dC4P and dT4P can also be substituted with
labeled dA4P and dG4P.
[0246] Preparing Zero-mode waveguide: Prior to the polymerization
reaction, a zero-mode waveguide is typically refreshed in a plasma
cleaner. A PDMS gasket covering the ZMW is placed onto the
waveguide to cover the individual optical confinements. An aliquot
of the reaction mixture described above except the DNA polymerase
is applied without touching the waveguide surface. The diffusion
background is measured. If the background (i.e., fluorescence burst
from the ZMW) low and acceptable, then DNA polymerase will be
applied to ZMW and immobilized thereon. The immobilization mixture
typically contains 0.5 to 1 mM MnCl.sub.2. 0.1 to 1 uM template, 15
nM DNA polymerase, in a buffer of 25 mM Tris-HCL, pH 7.5 and 10 mM
beta-mercaptoethanol. The polymerase is allowed to stick to the
surface of ZMW after an incubation of about 15 minutes at about
0.degree. C. The immobilization reaction mixture is then removed,
replaced with the reaction mixture described above.
[0247] A microscope system equipped with an appropriate laser,
e.g., Ar/Kr laser, is used that includes an optical setup for
simultaneous collection and detection of signals from multiple
different waveguides, and for the resolution of each of the A488
and 568 fluorophores present. The system includes an objective lens
and a series of dichroics/notch-off filters for separating emitted
fluorescence from reflected excitation light. The emitted signals
are passed through a wedge filter to spatially separate the signal
component of each fluorophore, and each signal is imaged onto an
EMCCD camera.
[0248] Polymerase Activity Measurement: The ZMW is placed under the
microscope. Polymerization reaction is then monitored using a
camera for a desired period of time, e.g., two minutes or longer,
after the transillumination light is applied. The data is
automatically transmitted to a computer that stores and trace the
fluorescence burst of each reaction in the ZMW.
[0249] A circular DNA having either a block of repeating A bases
followed by a block of G bases, or a series of repeating A-G bases
was sequenced according to the aforementioned procedures. A
representative profile of the fluorescence bursts corresponding to
each incorporation event of the labeled nucleotides is depicted in
FIG. 16, which indicates that real-time and single-molecule
sequencing has been achieved with more than one type of labeled
nucleotides. Statistical analysis of pulse data from multiple
separate repeats and multiple different waveguides establishes the
sequence dependant detection of incorporation of labeled bases in
real time.
[0250] Although described in some detail for purposes of
illustration, it will be readily appreciated that a number of
variations known or appreciated by those of skill in the art may be
practiced within the scope of present invention. To the extent not
already expressly incorporated herein, all published references and
patent documents referred to in this disclosure are incorporated
herein by reference in their entirety for all purposes.
* * * * *