Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND APPARATUSES FOR ANALYZING
POLYNUCLEOTIDE SEQUENCES
S CROSS-REFERENCES TO RELATED APPLICATIONS
This nonprovisional patent application claims the benefit of the previously
filed patent applications: U.S. provisional patent application no. 60/163,742,
filed November
4, 1999; and U.S. patent application no. 09/605,520, filed June 27, 2000,
which in turn claims
the benefit of U.S. provisional patent application no. 60/141,503 filed June
28, 1999, U.S.
provisional patent application no. 60/147,199 filed August 3, 1999, and U.S.
provisional
patent application no. 60/186,856 filed March 3, 2000. The text of these
previously filed
patent applications is hereby incorporated by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Work described herein has been supported, in part, by NIH grants HG-01642-
02. The U. S. Government may therefore have certain rights in the invention.
TECHNICAL FIELD
The present invention relates to methods for high speed, high throughput
analysis of polynucleotide sequences and apparatuses for carrying out such
methods.
BACKGROUND OF THE INVENTION
Traditional DNA sequencing techniques share three essential steps in their
approaches to sequence determination. First, a multiplicity of DNA fragments
are generated
from a DNA species which it is intended to sequence. These fragments are
incomplete copies
of the DNA species to be sequenced. The aim is to produce a ladder of DNA
fragments, each
a single base longer than the previous one. For example, with the Sanger
method (Sanger et
al., Proc. Natl. Acad. Sci. USA 74:5463, 1977), the target DNA is used as a
template for a
DNA polymerase to produce a number of incomplete clones. These fragments,
which differ
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in respective length by a single base, are then separated on an apparatus
which is capable of
resolving single-base differences in size. The third and final step is the
determination of the
nature of the base at the end of each fragment. When ordered by the size of
the fragments
which they terminate, these bases represent the sequence of the original DNA
species.
Automated systems for DNA sequence analysis have been developed, such as
discussed in Toneguzzo et al., 6 Biotechniques 460, 1988; Kanbara et al., 6
Biotechnology
816, 1988; and Smith et al., 13 Nuc. Acid. Res. 13: 2399, 1985; U.S. Pat. No.
4,707,237
(1987). However, all these methods still require separation of DNA products by
a gel
permeation procedure and then detection of their locations relative to one
another along the
axis of permeation or movement through the gel. These apparatuses used in
these methods
are not truly automatic sequencers. They are merely automatic gel readers,
which require the
standard sequencing reactions to be carried out before samples are loaded onto
the gel.
The disadvantages of the above methods are numerous. The most serious
problems are caused by the requirement for the DNA fragments to be size-
separated on a
polyacrylamide gel. This process is time-consuming, uses large quantities of
expensive
chemicals, and severely limits the number of bases which can be sequenced in
any single
experiment, due to the limited resolution of the gel. Sanger dideoxy
sequencing has a read
length of approximately 500 bp, a throughput that is limited by gel
electrophoresis
(appropriately 0.2%).
Other methods for analyzing polynucleotide sequences have been developed
more recently. In some of these methods broadly termed sequencing by
synthesis, template
sequences are determined by priming the template followed by a series of
single base primer
extension reactions (e.g., as described in WO 93/21340, WO 96/27025, and WO
98/44152).
While the basic scheme in these methods no longer require separation of
polynucleotides on
the gel, they encounter various other problems such as consumption of large
amounts of
expensive reagents, difficulty in removing reagents after each step,
misincorporation due to
long exchange times, the need to remove labels from the incorporated
nucleotide, and
difficulty to detect further incorporation if the label is not removed. Many
of these
difficulties stem directly from limitations of the macroscopic fluidics
employed. However,
small-volume fluidics have not been available. As a result, these methods have
not replaced
the traditional gel-based sequencing schemes in practice. The skilled artisans
are to a large
extent still relying on the gel-based sequencing methods.
Thus, there is a need in the art for methods and apparatuses for high speed
and
high throughput analysis of longer polynucleotide sequences which can be
automated
a2
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utilizing the available scanning and detection technology. The present
invention fulfills this
and other needs.
SUMMARY OF THE INVENTION
In one aspect of the present invention, methods for analyzing the sequence of
a
target polynucleotide are provided. The methods include the steps of (a)
providing a primed
target polynucleotide linked to a microfabricated synthesis channel; (b)
flowing a first
nucleotide through the synthesis channel under conditions whereby the first
nucleotide
attaches to the primer, if a complementary nucleotide is present to serve as
template in the
target polynucleotide; (c) determining presence or absence of a signal, the
presence of a
signal indicating that the first nucleotide was incorporated into the primer,
and hence the
identity of the complementary base that served as a template in the target
polynucleotide; (d)
removing or reducing the signal, if present; and (e) repeating steps (b)-(d)
with a further
nucleotide that is the same or different from the first nucleotide, whereby
the further
nucleotide attaches to the primer or a nucleotide previously incorporated into
the primer.
In some methods, step (a) comprises providing a plurality of different primed
target polynucleotides linked to different synthesis channels; step (b)
comprises flowing the
first nucleotide through each of the synthesis channels; and step (c)
comprises determining
presence or absence of a signal in each of the channels, the presence of a
signal in a synthesis
channel indicating the first nucleotide was incorporated into the primer in
the synthesis
channel, and hence the identity of the complementary base that served as a
template in the
target polynucleotide in the synthesis channel. In some methods, a plurality
of different
primed target polynucleotides are linked to each synthesis channels.
Some methods include the further steps of flushing the synthesis channel to
remove unincorporated nucleotides. In some methods, steps (b)-(d) are
performed at least
four times with four different types of nucleotides. In some methods, steps
(b)-(d) are
performed until the identity of each base in the target polynucleotide has
been identified.
In some methods, the nucleotides are labeled. The label can be a fluorescent
dye, and the signal can be detected optically. The label can also be a
radiolabel, and the
signal can be detected with a radioactivity detector. In some methods,
incorporation of
nucleotides is detected by measuring pyrophosphate release.
In some methods, the synthesis channel is formed by bonding a microfluidic
chip to a flat substrate. In some of these methods, the target polynucleotides
are immobilized
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to the interior surface of the substrate in the synthesis channel. In some of
these methods, the
interior surface is coated with a polyelectrolyte multilayer (PEM). In some of
these methods,
the microfluidic chip is fabricated with an elastomeric materia such as RTV
silicone.
In another aspect of the present invention, methods for analyzing a target
polynucleotide entails (a) pretreating the surface of a substrate to create
surface chemistry
that facilitates polynucleotide attachment and sequence analysis; (b)
providing a primed
target polynucleotide attached to the surface; (c) providing a labeled first
nucleotides to the
attached target polynucleotide under conditions whereby the labeled first
nucleotide attaches
to the primer, if a complementary nucleotide is present to serve as template
in the target
polynucleotide; (d) determining presence or absence of a signal from the
primer, the presence
of a signal indicating that the labeled first nucleotide was incorporated into
the primer, and
hence the identity of the complementary base that served as a template in the
target
polynucleotide; and (e) repeating steps (c)-(d) with a labeled further
nucleotide that is the
same or different from the first labeled nucleotide, whereby the labeled
further nucleotide
attaches to the primer or a nucleotide previously incorporated into the
primer.
In some of these methods, the substrate is glass and the surface is coated
with
a polyelectrolyte multilayer (PEM). In some methods, the PEM is terminated
with a
polyanion. In some methods, the polyanion is terminated with carboxylic acid
groups. In
some methods, the target polynucleotide is biotinylated, and the PEM-coated
surface is
further coated with biotin and then streptavidin.
In still another aspect of the present invention, methods of analyzing a
target
polynucleotide are provided which include the steps of (a) providing a primed
target
polynucleotide; (b) providing a first type of nucleotide of which a fraction
is labeled under
conditions whereby the first nucleotide attaches to the primer, if a
complementary nucleotide
is present to serve as template in the target polynucleotide; (c) determining
presence or
absence of a signal from the primer, the presence of a signal indicating the
first nucleotide
was incorporated into the primer, and hence the identity of the complementary
base that
served as a template in the target polynucleotide; and (d) repeating steps (b)-
(c) with a
further type of nucleotide of which a fraction is labeled the same and which
is the same or
different from the first type of nucleotide, whereby the further nucleotide
attaches to the
primer or a nucleotide previously incorporated into the primer.
In some of these methods, the label used is a fluorescent label. In some of
these methods, the removing or reducing step is performed by photobleaching.
In some of
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these methods, the fraction of labeled nucleotides are less than 10%, less
than 1%, less than
0.1 %, or less than 0.01 %.
In another aspect of the present invention, apparatuses for analyzing the
sequence of a polynucleotide are provided. The apparatuses have (a) a flow
cell with at least
one microfabricated synthesis channel; and (b) an inlet port and an outlet
port which are in
fluid communication with the flow cell and which flowing fluids such as
deoxynucleoside
triphosphates and nucleotide polymerase into and through the flow cell. Some
of the
apparatuses additionally have (c) a light source to direct light at a surface
of the synthesis
channel; and (d) a detector to detect a signal from the surface.
In some of the apparatuses, the synthesis channel is formed by bonding a
microfluidic chip to a flat substrate. In some apparatuses, the microfluidic
chip also contain
microfabricated valves and microfabricated pumps in an integrated system with
the synthesis
channel. In some of these apparatuses, a plurality of reservoirs for storing
reaction reagents
are also present, and the microfabricated valve and pump are connected to the
reservoirs. In
some apparatuses, the detector is a photon counting camera. In some of the
apparatuses, the
microfluidic chip is fabricated with an elastomeric material such as RTV
silicone. The
substrate of some of the apparatuses is a glass cover slip. The cross section
of the synthesis
channel in some of the apparatuses has a linear dimension of less than 1 OOp,m
x 1 OOp.m, less
than 1 Oprn x 1 OOpm, less than 1 p.m x 1 Opm, or less than 0.1 pm x 1 Eun.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is an illustration of a first elastomeric layer formed on top of a
micromachined mold.
Fig. 2 is an illustration of a second elastomeric layer formed on top of a
micromachined mold.
Fig. 3 is an illustration of the elastomeric layer of Fig. 2 removed from the
micromachined mold and positioned over the top of the elastomeric layer of
Fig. 1
Fig. 4 is an illustration corresponding to Fig. 3, but showing the second
elastomeric layer positioned on top of the first elastomeric layer.
Fig. S is an illustration corresponding to Fig. 4, but showing the first and
second elastomeric layers bonded together.
Fig. 6 is an illustration corresponding to Fig. 5, but showing the first
micromachine mold removed and a planar substrate positioned in its place.
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Fig. 7A is an illustration corresponding to Fig. 6, but showing the
elastomeric
structure sealed onto the planar substrate.
Figs. 7B is a front sectional view corresponding to Fig. 7A, showing an open
flow channel.
Fig. 7C corresponds to Fig. 7A, but shows a first flow channel closed by
pressurization in second flow channel.
Fig. 8 is an illustration of a first elastomeric layer deposited on a planar
substrate.
Fig. 9 is an illustration showing a first sacrificial layer deposited on top
of the
first elastomeric layer of Fig. 8.
Fig. 10 is an illustration showing the system of Fig. 9, but with a portion of
the
first sacrificial layer removed, leaving only a first line of sacrificial
layer.
Fig. 11 is an illustration showing a second elastomeric layer applied on top
of
the first elastomeric layer over the first line of sacrificial layer of Fig.
10, thereby encasing
the sacrificial layer between the first and second elastomeric layers.
Fig. 12 corresponds to Fig. 11, but shows the integrated monolithic structure
produced after the first and second elastomer layers have been bonded
together.
Fig. 13 is an illustration showing a second sacrificial layer deposited on top
of
the integral elastomeric structure of Fig. 12.
Fig. 14 is an illustration showing the system of Fig. 13, but with a portion
of
the second sacrificial layer removed, leaving only a second line of
sacrificial layer.
Fig. 15 is an illustration showing a third elastomer layer applied on top of
the
second elastomeric layer and over the second line of sacrificial layer of Fig.
14, thereby
encapsulating the second line of sacrificial layer between the elastomeric
structure of Fig. 12
and the third elastomeric layer.
Fig. 16 corresponds to Fig. 15, but shows the third elastomeric layer cured so
as to be bonded to the monolithic structure composed of the previously bonded
first and
second elastomer layers.
Fig. 17 corresponds to Fig. 16, but shows the first and second lines of
sacrificial layer removed so as to provide two perpendicular overlapping, but
not intersecting,
flow channels passing through the integrated elastomeric structure.
Fig. 18 is an illustration showing the system of Fig. 17, but with the planar
substrate thereunder removed.
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Fig. 19 illustrates valve opening vs. applied pressure for various flow
channels.
Fig. 20 illustrates time response of a 1 OO~mx 1 OO~,mx l Op.m RTV microvalve.
Fig. 21 is a schematic illustration of a multiplexed system adapted to permit
flow through various channels.
Fig. 22A is a plan view of a flow layer of an addressable reaction chamber
structure.
Fig. 22B is a bottom plan view of a control channel layer of an addressable
reaction chamber structure.
Fig. 22C is an exploded perspective view of the addressable reaction chamber
structure formed by bonding the control channel layer of Fig. 22B to the top
of the flow layer
of Fig. 22A.
Fig. 22D is a sectional elevation view corresponding to Fig. 22C, taken along
line 28D-28D in Fig. 22C.
Fig. 23 is a schematic of a system adapted to selectively direct fluid flow
into
any of an array of reaction wells.
Fig. 24 is a schematic of a system adapted for selectable lateral flow between
parallel flow channels.
Fig. 25 is a schematic of an integrated system for analyzing polynucleotide
sequences.
Fig. 26 is a schematic of a further integrated system for analyzing
polynucleotide sequences.
Fig. 27 is a schematic diagram of a sequencing apparatus.
DETAILED DESCRIPTION
I. Overview
The present invention provides methods and apparatuses for analyzing
polynucleotide sequences.
In some methods, the sequencing apparatuses comprise a microfabricated flow
channel to which polynucleotide templates are attached. Optionally, the
apparatuses
comprise a plurality of microfabricated channels, and diverse polynucleotide
templates can be
attached to each channel. The apparatuses can also have a plurality of
reservoirs for storing
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various reaction reagents, and pumps and valves for controlling flow of the
reagents. The
flow cell can also have a window to allow optical interrogation.
In these methods, single stranded polynucleotide templates with primers are
immobilized to the surface of the microfabricated channel or to the surface of
reaction
chambers that are disposed along a microfabricated flow channel, e.g., with
streptavidin
biotin links. After immobilization of the templates, a polymerise and one of
the four
nucleotide tt-iphosphates are flowed into the flow cell, incubated with the
template, and
flowed out. If no signal is detected, the process is repeated with a different
type of
nucleotide.
These methods are advantageous over the other sequencing by synthesis
methods discussed previously. First, use of microfabricated sequencing
apparatuses reduces
reagent consumption. It also increases reagent exchange rate and the speed of
sequence
analysis. In addition, the microfabricated apparatuses provides
parallelization: many
synthesis channels can be built on the same substrate. This allows analysis of
a plurality of
diverse polynucleotide sequences simultaneously. Further, due to the reduction
of time and
dead volume for exchanging reagents between different steps during the
analysis, mismatch
incorporation is greatly reduced. Moreover, the read length is also improved
because there is
less time for the polymerise to incorporate a wrong nucleotide and it is less
likely that the
polymerise falls off the template. All these advantages result in high speed
and high
throughput sequence analysis regimes.
In some methods of the present invention, the surface of a substrate (e.g., a
glass cover slip) is pretreated to create optimal surface chemistry that
facilitates
polynucleotide template attachment and subsequent sequence analysis. In some
of these
methods, the substrate surface is coated with a polyelectrolyte multilayer
(PEM). Following
the PEM coating, biotin can be applied to the PEM, and followed by application
of
streptavidin. The substrate surface can then be used to attach biotinylated-
templates. The
PEM-coated substrate provides substantial advantages for immobilizing the
template
polynucleotides and for polymerise extension reaction. First, because PEM can
easily be
terminated with polymers bearing carboxylic acids, it is easy to attach
polynucleotides.
Second, the attached template is active for extension by polymerises - most
probably, the
repulsion of like charges prevents the template from "laying down" on the
surface. Finally,
the negative charge repels nucleotides, and nonspecific binding is low.
In some other methods of the present invention, only a small percentage of
each type of nucleotides present in the extension reaction is labeled, e.g.,
with fluorescent
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dye. As a result, relatively small numbers of incorporated nucleotides are
fluorescently
labeled, interference of energy transfer is minimized, and the polymerise is
less likely to fall
off the template or be "choked" by incorporation of two labeled nucleotides
sequentially.
Optionally, the incorporated fluorescent signals are extinguished by
photobleaching.
Employment of photobleaching strategy can reduce the number of steps (e.g., it
may not be
necessary to perform the removal of label after every extension cycle). These
advantages
lead to more accurate detection of incorporated signals, more efficient
consumption of
polymerise, and a fast sequencing method.
II. Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by those of ordinary skill in the art
to which this
invention pertains. The following references provide one of skill with a
general definition of
many of the terms used in this invention: Singleton et al., DICTIONARY OF
MICROBIOLOGY
AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND
TECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINS
DICTIONARY
of BIOLOGY (1991). Although any methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, the preferred
methods and materials are described. The following definitions are provided to
assist the
reader in the practice of the invention.
The terms "nucleic acid" or "nucleic acid molecule" refer to a
deoxyribonucleotide or ribonucleotide polymer in either single- or double-
stranded form, and
unless otherwise limited, can encompass known analogs of natural nucleotides
that can
function in a similar manner as naturally occurring nucleotides.
"Nucleoside" includes natural nucleosides, including ribonucleosides and 2'-
deoxyribonucleosides, as well as nucleoside analogs having modified bases or
sugar
backbones.
A "base" or "base-type" refers to a particular type of nucleosidic base, such
as
adenine, cytosine, guanine, thymine, uracil, 5-methylcytosine, 5-bromouracil,
2-aminopurine,
deoxyinosine, N4 -methoxydeoxycytosine, and the like.
"Oligonucleotide" or "polynucleotide" refers to a molecule comprised of a
plurality of deoxyribonucleotides or nucleoside subunits. The linkage between
the nucleoside
subunits can be provided by phosphates, phosphonates, phosphoramidates,
phosphorothioates, or the like, or by nonphosphate groups as are known in the
art, such as
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peptoid-type linkages utilized in peptide nucleic acids (PNAs). The linking
groups can be
chiral or achiral. The oligonucleotides or polynucleotides can range in length
from 2
nucleoside subunits to hundreds or thousands of nucleoside subunits. While
oligonucleotides
are preferably 5 to 100 subunits in length, and more preferably, 5 to 60
subunits in length, the
length of polynucleotides can be much greater (e.g., up to 100 kb).
Specific hybridization refers to the binding, duplexing, or hybridizing of a
molecule only to a particular nucleotide sequence under stringent conditions.
Stringent
conditions are conditions under which a probe can hybridize to its target
subsequence, but to
no other sequences. Stringent conditions are sequence-dependent and are
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures.
Generally, stringent conditions are selected to be about 5° C lower
than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength and pH. The
Tm is the
temperature (under defined ionic strength, pH, and nucleic acid concentration)
at which 50%
of the probes complementary to the target sequence hybridize to the target
sequence at
equilibrium. Typically, stringent conditions include a salt concentration of
at least about 0.01
to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least
about 30°C for short probes (e.g., 10 to 50 nucleotides). Stringent
conditions can also be
achieved with the addition of destabilizing agents such as formamide or
tetraalkyl ammonium
salts. For example, conditions of SX SSPE (750 mM NaCI, 50 mM Na Phosphate, 5
mM
EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-
specific probe
hybridizations. (See Sambrook et al., Molecular Cloning 1989).
By "analysis of polynucleotide sequence of a template" is meant determining a
sequence of at least 3 contiguous base subunits in a sample fragment, or
alternatively, where
sequence information is available for a single base-type, the relative
positions of at least 3
subunits of identical base-types occurnng in sequential order in the fragment.
An example of
the latter meaning is a determined sequence "AXXAXA" (5'>3'), where a series
of 3 adenine
(A) bases are found to be separated by two and then one other base-type in the
sample
fragment.
The term "primer" refers to an oligonucleotide, whether occurring naturally as
in a purified restriction digest or produced synthetically, which is capable
of acting as a point
of initiation of synthesis when placed under conditions in which synthesis of
a primer
extension product which is complementary to a nucleic acid strand is induced,
(i.e., in the
presence of nucleotides and an inducing agent such as DNA polymerase and at a
suitable
temperature and pH). The primer is preferably single stranded for maximum
efficiency in
/0
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amplification, but can alternatively be double stranded. If double stranded,
the primer is first
treated to separate its strands before being used to prepare extension
products. Preferably, the
primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the
synthesis of extension products in the presence of the inducing agent. The
exact lengths of the
primers depend on many factors, including temperature, source of primer and
the use of the
method.
A primer is selected to be "substantially" complementary to a strand of
specific sequence of the template. A primer must be sufficiently complementary
to hybridize
with a template strand for primer elongation to occur. A primer sequence need
not reflect the
exact sequence of the template. For example, a non-complementary nucleotide
fragment can
be attached to the 5' end of the primer, with the remainder of the primer
sequence being
substantially complementary to the strand. Non-complementary bases or longer
sequences
can be interspersed into the primer, provided that the primer sequence has
sufficient
complementarity with the sequence of the template to hybridize and thereby
form a template
primer complex for synthesis of the extension product of the primer.
The term "probe" refers to an oligonucleotide (i.e., a sequence of
nucleotides),
whether occurring naturally as in a purified restriction digest or produced
synthetically,
recombinantly or by PCR amplification, which is capable of hybridizing to
another
oligonucleotide of interest. A probe can be single-stranded or double-
stranded. Probes are
useful in the detection, identification and isolation of particular gene
sequences. It is
contemplated that any probe used in the present invention can be labeled with
any "reporter
molecule," so that is detectable in any detection system, including, but not
limited to
fluorescent, enzyme (e.g., ELISA, as well as enzyme-based histochemical
assays),
radioactive, quantum dots, and luminescent systems. It is not intended that
the present
invention be limited to any particular detection system or label.
The term "template," refers to nucleic acid that is to acted upon, such as
nucleic acid that is to be mixed with polymerase. In some cases "template" is
sought to be
sorted out from other nucleic acid sequences. "Substantially single-stranded
template" is
nucleic acid that is either completely single-stranded (having no double-
stranded areas) or
single-stranded except for a proportionately small area of double-stranded
nucleic acid (such
as the area defined by a hybridized primer or the area defined by
intramolecular bonding).
"Substantially double-stranded template" is nucleic acid that is either
completely double-
stranded (having no single-stranded region) or double-stranded except for a
proportionately
small area of single-stranded nucleic acid.
/t
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III. Sequencing Apparatuses
A. Basic features of the apparatuses
The apparatuses comprise microfabricated channels to which polynucleotide
S templates to be sequenced are attached. Optionally, the apparatuses comprise
plumbing
components (e.g., pumps, valves, and connecting channels) for flowing reaction
reagents.
The apparatuses can also comprise an array of reservoirs for storing reaction
reagents (e.g.,
the polymerase, each type of nucleotides, and other reagents can each be
stored in a different
reservoir).
The microfabricated components of the apparatuses all have a basic "flow
channel" structure. The term "flow channel" or "microfabricated flow channel"
refers to
recess in a structure which can contain a flow of fluid or gas. The
polynucleotide templates
are attached to the interior surface of microfabricated channels in which
synthesis occurs.
For consistency and clarity, the flow channels are termed "synthesis channel"
when referring
to such specific use. The microfabricated flow channels can also be actuated
to function as
the plumbing components (e.g., micro-pumps, micro-valves, or connecting
channels) of the
apparatuses.
In some applications, microfabricated flow channels are cast on a chip (e.g.,
a
elastomeric chip). Synthesis channels are formed by bonding the chip to a flat
substrate (e.g.,
a glass cover slip) which seals the channel. Thus, one side of the synthesis
channel is
provided by the flat substrate. Typically, the polynucleotide templates are
attached to the
interior surface of the substrate within the synthesis channel.
The plumbing components can be microfabricated as described in the present
invention. For example, the apparatuses can contain in an integrated system a
flow cell in
which a plurality of synthesis channels are present, and fluidic components
(such as micro-
pumps, micro-valves, and connecting channels) for controlling the flow of the
reagents into
and out of the flow cell. Alternatively, the sequencing apparatuses of the
present invention
utilize plumbing devices described in, e.g., Zdeblick et al., A Microminiature
Electric-to-
Fluidic Valve, Proceedings of the 4th International Conference on Solid State
Transducers
and Actuators, 1987; Shoji et al., Smallest Dead Volume Microvalves for
Integrated
Chemical Analyzing Systems, Proceedings of Transducers '91, San Francisco,
1991; Vieider
et al., A Pneumatically Actuated Micro Valve with a Silicon Rubber Membrane
for
Integration with Fluid Handling Systems, Proceedings of Transducers '95,
Stockholm, 1995.
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As noted above, at least some of the components of the apparatuses are
microfabricated. Employment of microfabricated synthesis channels and/or
microfabricated
plumbing components significantly reduce the dead volume and decrease the
amount of time
needed to exchange reagents, which in turn increase the throughput.
Microfabrication refers
to feature dimensions on the micron level, with at least one dimension of the
microfabricated
structure being less than 1000 Vim. In some apparatuses, only the synthesis
channels are
microfabricated. In some apparatuses, in addition to the synthesis channels,
the valves,
pumps, and connecting channels are also microfabricated. Unless otherwise
specified, the
discussion below of microfabrication is applicable to production of all
microfabricated
components of the sequencing apparatuses (e.g., the synthesis channels in
which sequencing
reactions occur, and the valves, pumps, and connecting channels for
controlling reagents flow
to the synthesis channels).
Various materials can be used to fabricate the microfabricated components
(see, e.g., Unger et al., Science 288:113-116, 2000). Preferably, elastomeric
materials are
used. Thus, in some apparatuses, the integrated (i.e., monolithic)
microstructures are made
out of various layers of elastomer bonded together. By bonding these various
elastomeric
layers together, the recesses extending along the various elastomeric layers
form flow
channels through the resulting monolithic, integral elastomeric structure.
In general, the microfabricated structures (e.g., synthesis channels, pumps,
valves , and connecting channels) have widths of about 0.01 to 1000 microns,
and a width-to-
depth ratios of between 0.1:1 to 100:1. Preferably, the width is in the range
of 10 to 200
microns, a width-to-depth ratio of 3:1 to 15:1.
B. Microfabrication with elastomeric materials
1. Basic methods of microfabrication
Various methods can be used to produce the microfabricated components of
the sequencing apparatuses of the present invention. Fabrication of the
microchannels,
valves, pumps can be performed as described in Unger et al., Science 288:113-
116, 2000,
which is incorporated herein by reference. In some methods (Figs. 1 to 7B, pre-
cured
elastomer layers are assembled and bonded to produce a flow channel. As
illustrated in Fig.
1, a first micro-machined mold 10 is provided. Micro-machined mold 10 can be
fabricated
by a number of conventional silicon processing methods, including but not
limited to
photolithography, ion-milling, and electron beam lithography. The micro-
machined mold 10
has a raised line or protrusion 11 extending therealong. A first elastomeric
layer 20 is cast on
t3
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top of mold 10 such that a first recess 21 can be formed in the bottom surface
of elastomeric
layer 20, (recess 21 corresponding in dimension to protrusion 11), as shown.
As can be seen in Fig. 2, a second micro-machined mold 12 having a raised
protrusion 13 extending therealong is also provided. A second elastomeric
layer 22 is cast on
top of mold 12, as shown, such that a recess 23 can be formed in its bottom
surface
corresponding to the dimensions of protrusion 13.
As can be seen in the sequential steps illustrated in Figs. 3 and 4, second
elastomeric layer 22 is then removed from mold 12 and placed on top of first
elastomeric
layer 20. As can be seen, recess 23 extending along the bottom surface of
second elastomeric
layer 22 forms a flow channel 32.
Refernng to Fig. 5, the separate first and second elastomeric layers 20 and 22
(Fig. 4) are then bonded together to form an integrated (i.e.: monolithic)
elastomeric structure
24.
As can been seen in the sequential step of Figs. 6 and 7A, elastomeric
structure 24 is then removed from mold 10 and positioned on top of a planar
substrate 14. As
can be seen in Fig. 7A and 7B, when elastomeric structure 24 has been sealed
at its bottom
surface to planar substrate 14, recess 21 forms a flow channel 30.
The present elastomeric structures form a reversible hermetic seal with nearly
any smooth planar substrate. An advantage to forming a seal this way is that
the elastomeric
structures can be peeled up, washed, and re-used. In some apparatuses, planar
substrate 14 is
glass. A further advantage of using glass is that glass is transparent,
allowing optical
interrogation of elastomer channels and reservoirs. Alternatively, the
elastomeric structure
can be bonded onto a flat elastomer layer by the same method as described
above, forming a
permanent and high-strength bond. This can prove advantageous when higher back
pressures
are used.
In some methods, microfabrication involves curing each layer of elastomer "in
place" (Figs. 8 to 18). In these methods, flow and control channels are
defined by first
patterning sacrificial layer on the surface of an elastomeric layer (or other
substrate, which
can include glass) leaving a raised line of sacrificial layer where a channel
is desired. Next, a
second layer of elastomer is added thereover and a second sacrificial layer is
patterned on the
second layer of elastomer leaving a raised line of sacrificial layer where a
channel is desired.
A third layer of elastomer is deposited thereover. Finally, the sacrificial
layer is removed by
dissolving it out of the elastomer with an appropriate solvent, with the voids
formed by
removal of the sacrificial layer becoming the flow channels passing through
the substrate.
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Referring first to Fig. 8, a planar substrate 40 is provided. A first
elastomeric
layer 42 is then deposited and cured on top of planar substrate 40. Refernng
to Fig. 9, a first
sacrificial layer 44A is then deposited over the top of elastomeric layer 42.
Referring to Fig.
10, a portion of sacrificial layer 44A is removed such that only a first line
of sacrificial layer
44B remains as shown. Referring to Fig. 11, a second elastomeric layer 46 is
then deposited
over the top of first elastomeric layer 42 and over the first line of
sacrificial layer 44B as
shown, thereby encasing first line of sacrificial layer 44B between first
elastomeric layer 42
and second elastomeric layer 46. Refernng to Fig. 12, elastomeric layers 46 is
then cured on
layer 42 to bond the layers together to form a monolithic elastomeric
substrate 45.
Refernng to Fig. 13, a second sacrificial layer 48A is then deposited over
elastomeric structure 45. Referring to Fig. 14, a portion of second
sacrificial layer 48A is
removed, leaving only a second sacrificial layer 48B on top of elastomeric
structure 45 as
shown. Refernng to Fig. 15, a third elastomeric layer 50 is then deposited
over the top of
elastomeric structure 45 (comprised of second elastomeric layer 42 and first
line of sacrificial
1 S layer 44B) and second sacrificial layer 48B as shown, thereby encasing the
second line of
sacrificial layer 48B between elastomeric structure 45 and third elastomeric
layer S0.
Referring to Fig. 16, third elastomeric layer 50 and elastomeric structure 45
(comprising first elastomeric layer 42 and second elastomeric layer 46 bonded
together) is
then bonded together forming a monolithic elastomeric structure 47 having
sacrificial layers
44B and 48B passing therethrough as shown. Referring to Fig. 17, sacrificial
layers 44B and
48B are then removed (for example, by an solvent ) such that a first flow
channel 60 and a
second flow channel 62 are provided in their place, passing through
elastomeric structure 47
as shown. Lastly, refernng to Fig. 18, planar substrate 40 can be removed from
the bottom of
the integrated monolithic structure.
2. Multilayer construction
Soft lithographic bonding can be used to construct an integrated system which
contains multiple flow channels. A heterogenous bonding can be used in which
different
layers are of different chemistries. For example, the bonding process used to
bind respective
elastomeric layers together can comprise bonding together two layers of RTV
615 silicone.
RTV 61 S silicone is a two-part addition-cure silicone rubber. Part A contains
vinyl groups
and catalyst; part B contains silicon hydride (Si-H) groups. The conventional
ratio for RTV
615 is 10A:1B. For bonding, one layer can be made with 30A:1B (i.e. excess
vinyl groups)
and the other with 3A:1B (i.e. excess Si-H groups). Each layer is cured
separately. When the
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two layers are brought into contact and heated at elevated temperature, they
bond irreversibly
forming a monolithic elastomeric substrate.
A homogenous bonding can also be used in which all layers are of the same
chemistry. For example, elastomeric structures are formed utilizing Sylgard
182, 184 or 186,
or aliphatic urethane diacrylates such as (but not limited to) Ebecryl 270 or
Irr 245 from UCB
Chemical. For example, two-layer elastomeric structures were fabricated from
pure acrylated
Urethane Ebe 270. A thin bottom layer was spin coated at 8000 rpm for 15
seconds at 170°C.
The top and bottom layers were initially cured under ultraviolet light for 10
minutes under
nitrogen utilizing a Model ELC 500 device manufactured by Electrolite
corporation. The
assembled layers were then cured for an additional 30 minutes. Reaction was
catalyzed by a
0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals. The
resulting
elastomeric material exhibited moderate elasticity and adhesion to glass.
In some applications, two-layer elastomeric structures were fabricated from a
combination of 25% Ebe 270 / 50% Irr245 / 25% isopropyl alcohol for a thin
bottom layer,
and pure acrylated Urethane Ebe 270 as a top layer. The thin bottom layer was
initially cured
for 5 min, and the top layer initially cured for 10 minutes, under ultraviolet
light under
nitrogen utilizing a Model ELC 500 device manufactured by Electrolite
corporation. The
assembled layers were then cured for an additional 30 minutes. Reaction was
catalyzed by a
0.5% vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals. The
resulting
elastomeric material exhibited moderate elasticity and adhered to glass.
Where encapsulation of sacrificial layers is employed to fabricate the
elastomer structure as described above in Figs. 8-18, bonding of successive
elastomeric layers
can be accomplished by pouring uncured elastomer over a previously cured
elastomeric layer
and any sacrificial material patterned thereupon. Bonding between elastomer
layers occurs
due to interpenetration and reaction of the polymer chains of an uncured
elastomer layer with
the polymer chains of a cured elastomer layer. Subsequent curing of the
elastomeric layer
creates a bond between the elastomeric layers and create a monolithic
elastomeric structure.
Referring to the first method of Figs. 1 to 7B, first elastomeric layer 20 can
be
created by spin-coating an RTV mixture on microfabricated mold 12 at 2000
rpm's for 30
seconds yielding a thickness of approximately 40 microns. Second elastomeric
layer 22 can
be created by spin-coating an RTV mixture on microfabricated mold 11. Both
layers 20 and
22 can be separately baked or cured at about 80°C for 1.5 hours. The
second elastomeric
layer 22 can be bonded onto first elastomeric layer 20 at about 80°C
for about 1.5 hours.
/ ~O
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Micromachined molds 10 and 12 can be patterned sacrificial layer on silicon
wafers. In an exemplary aspect, a Shipley SJR 5740 sacrificial layer was spun
at 2000 rpm
patterned with a high resolution transparency film as a mask and then
developed yielding an
inverse channel of approximately 10 microns in height. When baked at
approximately 200°C
for about 30 minutes, the sacrificial layer reflows and the inverse channels
become rounded.
In preferred aspects, the molds can be treated with trimethylchlorosilane
(TMCS) vapor for
about a minute before each use in order to prevent adhesion of silicone
rubber.
3. Suitable materials
Allcock et al, Contemporary Polymer Chemistry, 2"a Ed. describes elastomers
in general as polymers existing at a temperature between their glass
transition temperature
and liquefaction temperature. Elastomeric materials exhibit elastic properties
because the
polymer chains readily undergo torsional motion to permit uncoiling of the
backbone chains
in response to a force, with the backbone chains recoiling to assume the prior
shape in the
1 S absence of the force. In general, elastomers deform when force is applied,
but then return to
their original shape when the force is removed. The elasticity exhibited by
elastomeric
materials can be characterized by a Young's modulus. Elastomeric materials
having a
Young's modulus of between about 1 Pa - 1 TPa, more preferably between about
10 Pa - 100
GPa, more preferably between about 20 Pa - 1 GPa, more preferably between
about 50 Pa -
10 MPa, and more preferably between about 100 Pa - 1 MPa are useful in
accordance with
the present invention, although elastomeric materials having a Young's modulus
outside of
these ranges could also be utilized depending upon the needs of a particular
application.
The systems of the present invention can be fabricated from a wide variety of
elastomers. For example, elastomeric layers 20, 22, 42, 46 and 50 can
preferably be
fabricated from silicone rubber. In some applications, microstructures of the
present systems
are fabricated from an elastomeric polymer such as GE RTV 615 (formulation), a
vinyl-silane
crosslinked (type) silicone elastomer (family). An important requirement for
the preferred
method of fabrication is the ability to bond multiple layers of elastomers
together. In the case
of multilayer soft lithography, layers of elastomer are cured separately and
then bonded
together. This scheme requires that cured layers possess sufficient reactivity
to bond together.
Either the layers can be of the same type, and are capable of bonding to
themselves, or they
can be of two different types, and are capable of bonding to each other. Other
possibilities
include the use an adhesive between layers and the use of thermoset
elastomers.
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Given the tremendous diversity of polymer chemistries, precursors, synthetic
methods, reaction conditions, and potential additives, there are a huge number
of possible
elastomer systems that could be used to make monolithic elastomeric
microstructures.
Variations in the materials used most likely are driven by the need for
particular material
properties, i.e. solvent resistance, stiffness, gas permeability, or
temperature stability.
Common elastomeric polymers include, but are not limited to, polyisoprene,
polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-
styrene), the
polyurethanes, and silicones. The following is a non-exclusive list of
elastomeric materials
which can be utilized in connection with the present invention: polyisoprene,
polybutadiene,
polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), the
polyurethanes, and
silicone polymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),
poly(carborane-
siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(1-
butene),
poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),
poly(ethyl vinyl ether),
poly(vinylidene fluoride), poly(vinylidene fluoride - hexafluoropropylene)
copolymer
(Viton), elastomeric compositions of polyvinylchloride (PVC), polysulfone,
polycarbonate,
polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon).
In addition, polymers incorporating materials such as chlorosilanes or methyl-
,
ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) such as Dow
Chemical Corp.
Sylgard 182, 184 or 186, or aliphatic urethane diacrylates such as (but not
limited to) Ebecryl
270 or Irr 245 from UCB Chemical can also be used.
In some methods, elastomers can also be "doped" with uncrosslinkable
polymer chains of the same class. For instance RTV 615 can be diluted with GE
SF96-50
Silicone Fluid. This serves to reduce the viscosity of the uncured elastomer
and reduces the
Young's modulus of the cured elastomer. Essentially, the crosslink-capable
polymer chains
are spread further apart by the addition of "inert" polymer chains, so this is
called "dilution".
RTV 615 cures at up to 90% dilution, with a dramatic reduction in Young's
modulus.
Other examples of doping of elastomer material can include the introduction
of electrically conducting or magnetic species. Should it be desired, doping
with fine
particles of material having an index of refraction different than the
elastomeric material (i.e.
silica, diamond, sapphire) is also contemplated as a system for altering the
refractive index of
the material. Strongly absorbing or opaque particles can be added to render
the elastomer
colored or opaque to incident radiation. This can conceivably be beneficial in
an optically
addressable system.
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4. Dimensions of the microfabricated structures
Some flow channels (30, 32, 60 and 62) preferably have width-to-depth ratios
of about 10:1. A non-exclusive list of other ranges of width-to-depth ratios
in accordance
with the present invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1,
more preferably 2:1
S to 20:1, and most preferably 3:1 to 15:1. In an exemplary aspect, flow
channels 30, 32, 60
and 62 have widths of about 1 to 1000 microns. A non-exclusive list of other
ranges of
widths of flow channels in accordance with the present invention is 0.01 to
1000 microns,
more preferably 0.05 to 1000 microns, more preferably 0.2 to S00 microns, more
preferably 1
to 250 microns, and most preferably 10 to 200 microns. Exemplary channel
widths include
0.1 ~,m, 1 ~.m, 2 Vim, 5 pm, 10 pm, 20 Vim, 30 p,m, 40 pm, 50 ~,m, 60 Vim, 70
Vim, 80 Vim, 90
Vim, 100 ~,m, 110 Vim, 120 Vim, 130 pm, 140 Vim, 150 p.m, 160 Vim, 170 pm, 180
Vim, 190
p,m, 200 pm, 210 p.m, 220 pm, 230 Vim, 240 Vim, and 250 p,m.
Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns. A
non-exclusive list of other ranges of depths of flow channels in accordance
with the present
invention is 0.01 to 1000 microns, more preferably 0.05 to 500 microns, more
preferably 0.2
to 250 microns, and more preferably 1 to 100 microns, more preferably 2 to 20
microns, and
most preferably S to 10 microns. Exemplary channel depths include including
0.01 pm, 0.02
p.m, 0.05 Vim, 0.1 pm, 0.2 pm, 0.5 ~.m, 1 pm, 2 Vim, 3 pm, 4 Vim, 5 p,m, 7.5
Vim, 10 p.m, 12.5
pm, 15 pm, 17.5 Vim, 20 ~.m, 22.5 Vim, 25 ~.m, 30 p.m, 40 Vim, 50 ~,m, 75 pm,
100 p.m, 150
pm, 200 p,m, and 250 pm.
The flow channels are not limited to these specific dimension ranges and
examples given above, and can vary in width in order to affect the magnitude
of force
required to deflect the membrane as discussed at length below in conjunction
with Fig. 21.
For example, extremely narrow flow channels having a width on the order of
0.01 ~m can be
useful in optical and other applications, as discussed in detail below.
Elastomeric structures
which include portions having channels of even greater width than described
above are also
contemplated by the present invention, and examples of applications of
utilizing such wider
flow channels include fluid reservoir and mixing channel structures.
Elastomeric layer 22 can be cast thick for mechanical stability. In an
exemplary embodiment, layer 22 is 50 microns to several centimeters thick, and
more
preferably approximately 4 mm thick. A non-exclusive list of ranges of
thickness of the
elastomer layer in accordance with other embodiments of the present invention
is between
about 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100 microns
to 10 mm.
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Accordingly, membrane 25 of Fig. 7B separating flow channels 30 and 32 has
a typical thickness of between about 0.01 and 1000 microns, more preferably
0.05 to 500
microns, more preferably 0.2 to 250, more preferably 1 to 100 microns, more
preferably 2 to
50 microns, and most preferably 5 to 40 microns. As such, the thickness of
elastomeric layer
22 is about 100 times the thickness of elastomeric layer 20. Exemplary
membrane
thicknesses include 0.01 Vim, 0.02 Vim, 0.03 Vim, 0.05 p,m, 0.1 ~,m, 0.2 Vim,
0.3 Vim, 0.5 ~.m, 1
Vim, 2 Vim, 3 ~.m, 5 Vim, 7.5 Vim, 10 Vim, 12.5 ~,m, 15 Vim, 17.5 Vim, 20 Vim,
22.5 Vim, 25 ~.m,
30 Vim, 40 Vim, 50 Vim, 75 Vim, 100 Vim, 150 Vim, 200 p,m, 250 Vim, 300 p.m,
400 pm, 500 Vim,
750 Vim, and 1000 p.m
Similarly, first elastomeric layer 42 can have a preferred thickness about
equal
to that of elastomeric layer 20 or 22; second elastomeric layer 46 can have a
preferred
thickness about equal to that of elastomeric layer 20; and third elastomeric
layer 50 can have
a preferred thickness about equal to that of elastomeric layer 22.
C. Operation of the microfabricated components
Figs. 7B and 7C together show the closing of a first flow channel by
pressurizing a second flow channel, with Fig. 7B (a front sectional view
cutting through flow
channel 32 in corresponding Fig. 7A), showing an open first flow channel 30;
with Fig. 7C
showing first flow channel 30 closed by pressurization of the second flow
channel 32.
Referring to Fig. 7B, first flow channel 30 and second flow channel 32 are
shown. Membrane
separates the flow channels, forming the top of first flow channel 30 and the
bottom of
second flow channel 32. As can be seen, flow channel 30 is "open".
As can be seen in Fig. 7C, pressurization of flow channel 32 (either by gas or
liquid introduced therein) causes membrane 25 to deflect downward, thereby
pinching off
25 flow F passing through flow channel 30. Accordingly, by varying the
pressure in channel 32,
a linearly actuable valuing system is provided such that flow channel 30 can
be opened or
closed by moving membrane 25 as desired.
It is to be understood that exactly the same valve opening and closing methods
can be achieved with flow channels 60 and 62. Since such valves are actuated
by moving the
roof of the channels themselves (i.e., moving membrane 25), valves and pumps
produced by
this technique have a truly zero dead volume, and switching valves made by
this technique
have a dead volume approximately equal to the active volume of the valve, for
example about
100 x 100 x 10 ~m = 100pL. Such dead volumes and areas consumed by the moving
~2 0
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membrane are approximately two orders of magnitude smaller than known
conventional
microvalves. Smaller and larger valves and switching valves are contemplated
in the present
invention, and a non-exclusive list of ranges of dead volume includes 1 aL to
1 uL, 100 aL to
100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to 100 pL
The extremely small volumes capable of being delivered by pumps and valves
in accordance with the present invention represent a substantial advantage.
Specifically, the
smallest known volumes of fluid capable of being manually metered is around
0.1 ~l. The
smallest known volumes capable of being metered by automated systems is about
ten-times
larger (1 ~,1). Utilizing pumps and valves of the present invention, volumes
of liquid of 10 n1
or smaller can routinely be metered and dispensed. The accurate metering of
extremely small
volumes of fluid enabled by the present invention would be extremely valuable
in a large
number of biological applications, including diagnostic tests and assays.
Figs. 21 a and 21 b illustrate valve opening vs. applied pressure for a 100 ~m
wide first flow channel 30 and a 50 ~m wide second flow channel 32. The
membrane of this
device was formed by a layer of General Electric Silicones RTV 615 having a
thickness of
approximately 30pm and a Young's modulus of approximately 750 kPa. Figs. 21a
and 21b
show the extent of opening of the valve to be substantially linear over most
of the range of
applied pressures.
Air pressure was applied to actuate the membrane of the device through a 10
cm long piece of plastic tubing having an outer diameter of 0.025" connected
to a 25 mm
piece of stainless steel hypodermic tubing with an outer diameter of 0.025"
and an inner
diameter of 0.013". This tubing was placed into contact with the control
channel by insertion
into the elastomeric block in a direction normal to the control channel. Air
pressure was
applied to the hypodermic tubing from an external LHI~A miniature solenoid
valve
manufactured by Lee Co.
The response of valves of the present invention is almost perfectly linear
over
a large portion of its range of travel, with minimal hysteresis. While valves
and pumps do
not require linear actuation to open and close, linear response does allow
valves to more
easily be used as metering devices. In some applications, the opening of the
valve is used to
control flow rate by being partially actuated to a known degree of closure.
Linear valve
actuation makes it easier to determine the amount of actuation force required
to close the
valve to a desired degree of closure. Another benefit of linear actuation is
that the force
required for valve actuation can be easily determined from the pressure in the
flow channel.
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If actuation is linear, increased pressure in the flow channel can be
countered by adding the
same pressure (force per unit area) to the actuated portion of the valve.
D. Schematic illustration of the elastomeric apparatuses
An exemplary sequencing system is illustrated in Fig. 25. Four reservoirs
150A, 150B, 150C and 150D have labeled nucleotides A, C, T and G respectively
disposed
therein. Four flow channels 30A, 30B, 30C and 30D are connected to reservoirs
150A, 150B,
150C and 150D. Four control lines 32A, 32B, 32C and 32D (shown in phantom) are
disposed thereacross with control line 32A permitting flow only through flow
channel 30A
(i.e.: sealing flow channels 30B, 30C and 30D), when control line 32A is
pressurized.
Similarly, control line 32B permits flow only through flow channel 30B when
pressurized.
As such, the selective pressurization of control lines 32A, 32B, 32C and 32D
sequentially
selects a desired nucleotide (A, C, T or G) from a desired reservoir (150A,
150B, 150C or
1 SOD). The fluid then passes through flow channel 120 into a multiplexed
channel flow
controller 125, which in turn directs fluid flow into one or more of a
plurality of synthesis
channels or reaction chambers 122A, 122B, 122C, 122D or 122E in which solid
phase
synthesis can be carried out.
Fig. 26 illustrates a further extension of the system shown in Fig. 25. It has
a
plurality of reservoirs R1 to R13. These reservoirs can contain the labeled
nucleotides,
nucleotide polymerase, or reagents for coating the surface of the synthesis
channel and
attaching polynucleotide templates (see below for further discussion). The
reservoirs are
connected to systems 200 as set forth in Figs. 25. Systems 200 are connected
to a
multiplexed channel flow controller 125, which is in turn connected to a
plurality of synthesis
channels or reaction chambers. An advantage of this system is that both of
multiplexed
channel flow controllers 125 and fluid selection systems 200 can be controlled
by the same
pressure inputs 170 and 172 , provided a "close horizontal" and a "close
vertical" control
lines (160 and 162, in phantom) are also provided.
Some apparatuses comprise a plurality of selectively addressable reaction
chambers that are disposed along a flow channel. In these apparatuses, the
polynucleotide
templates can be attached to the surface of the reaction chambers instead of
the surface of
flow channels. An exemplary embodiment of such apparatuses is illustrated in
Figs. 22A,
22B, 22C and 22D. It is a system for selectively directing fluid flow into one
or more of a
plurality of reaction chambers disposed along a flow channel.
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Fig. 22A shows a top view of a flow channel 30 having a plurality of reaction
chambers 80A and 80B disposed therealong. Preferably flow channel 30 and
reaction
chambers 80A and 80B are formed together as recesses into the bottom surface
of a first layer
100 of elastomer.
Fig. 22B shows a bottom plan view of another elastomeric layer 110 with two
control lines 32A and 32B each being generally narrow, but having wide
extending portions
33A and 33B formed as recesses therein.
As seen in the exploded view of Fig. 22C, and assembled view of Fig. 22D,
elastomeric layer 110 is placed over elastomeric layer 100. Layers 100 and 110
are then
bonded together, and the integrated system operates to selectively direct
fluid flow F (through
flow channel 30) into either or both of reaction chambers 80A and 80B, as
follows.
Pressurization of control line 32A will cause the membrane 25 (i.e.: the thin
portion of
elastomer layer 100 located below extending portion 33A and over regions 82A
of reaction
chamber 80A) to become depressed, thereby shutting off fluid flow passage in
regions 82A,
effectively sealing reaction chamber 80 from flow channel 30. As can also be
seen, extending
portion 33A is wider than the remainder of control line 32A. As such,
pressurization of
control line 32A will not result in control line 32A sealing flow channel 30.
As can be appreciated, either or both of control lines 32A and 32B can be
actuated at once. When both control lines 32A and 32B are pressurized
together, sample flow
in flow channel 30 will enter neither of reaction chambers 80A or 80B.
The concept of selectably controlling fluid introduction into various
addressable reaction chambers disposed along a flow line (Figs. 22) can be
combined with
concept of selectably controlling fluid flow through one or more of a
plurality of parallel flow
lines (Fig. 21) to yield a system in which a fluid sample or samples can be
sent to any
particular reaction chamber in an array of reaction chambers. An example of
such a system is
provided in Fig. 23, in which parallel control channels 32A, 32B and 32C with
extending
portions 34 (all shown in phantom) selectively direct fluid flows F 1 and F2
into any of the
array of reaction wells 80A, 80B, 80C or 80D as explained above; while
pressurization of
control lines 32C and 32D selectively shuts off flows F2 and F1, respectively.
In yet another embodiment, fluid passage between parallel flow channels is
possible. Refernng to Fig. 24, either or both of control lines 32A or 32D can
be depressurized
such that fluid flow through lateral passageways 35 (between parallel flow
channels 30A and
30B) is permitted. In this aspect of the invention, pressurization of control
lines 32C and 32D
would shut flow channel 30A between 35A and 35B, and would also shut lateral
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passageways 35B. As such, flow entering as flow F1 would sequentially travel
through 30A,
35A and leave 30B as flow F4.
E. Non-elastomer based apparatuses
As discussed above, while elastomers are preferred materials for fabricating
the sequencing apparatuses of the present invention, non-elastomer based
microfluidic
devices can also be used in the apparatuses of the present invention. In some
applications,
the sequencing apparatuses utilize microfluidics based on conventional micro-
electro-
mechanical system (MEMS) technology. Methods of producing conventional MEMS
microfluidic systems such as bulk micro-machining and surface micro-machining
have been
described, e.g., in Terry et al., A Gas Chromatographic Air Analyzer
Fabricated on a Silicon
Wafer, IEEE Trans. on Electron Devices, v. ED-26, pp. 1880-1886, 1979; and
Berg et al.,
Micro Total Analysis Systems, New York, Kluwer, 1994.
Bulk micro-machining is a subtractive fabrication method whereby single
crystal silicon is lithographically patterned and then etched to form three-
dimensional
structures. For example, bulk micromachining technology, which includes the
use of glass
wafer processing, silicon-to-glass wafer bonding, has been commonly used to
fabricate
individual microfluidic components. This glass-bonding technology has also
been used to
fabricate microfluidic systems.
Surface micro-machining is an additive method where layers of
semiconductor-type materials such as polysilicon, silicon nitride, silicon
dioxide, and various
metals are sequentially added and patterned to make three-dimensional
structures. Surface
micromachining technology can be used to fabricate individual fluidic
components as well as
microfluidic systems with on-chip electronics. In addition, unlike bonded-type
devices,
hermetic channels can be built in a relatively simple manner using channel
walls made of
polysilicon (see, e.g., Webster et al., Monolithic Capillary Gel
Electrophoresis Stage with
On-Chip Detector, in International Conference on Micro Electromechanical
Systems, MEMS
96, pp. 491-496, 1996), silicon nitride (see, e.g., Mastrangelo et al., Vacuum-
Sealed Silicon
Micromachined Incandescent Light Source, in Intl. Electron Devices Meeting,
IDEM 89, pp.
503-506, 1989), and silicon dioxide.
In some applications, electrokinetic flow based microfluidics can be employed
in the sequencing apparatuses of the present invention. Briefly, these systems
direct reagents
flow within an interconnected channel and/or chamber containing structure
through the
application of electrical fields to the reagents. The electrokinetic systems
concomitantly
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regulate voltage gradients applied across at least two intersecting channels.
Such systems are
described, e.g., in WO 96/04547 and U.S. Patent No. 6,107,044.
An exemplary electrokinetic flow based microfluidic device can have a body
structure which includes at least two intersecting channels or fluid conduits,
e.g.,
interconnected, enclosed chambers, which channels include at least three
unintersected
termini. The intersection of two channels refers to a point at which two or
more channels are
in fluid communication with each other, and encompasses "T" intersections,
cross
intersections, "wagon wheel" intersections of multiple channels, or any other
channel
geometry where two or more channels are in such fluid communication. An
unintersected
terminus of a channel is a point at which a channel terminates not as a result
of that channel's
intersection with another channel, e.g., a "T" intersection.
In some electrokinetic flow based apparatuses, at least three intersecting
channels having at least four unintersected termini are present. In a basic
cross channel
structure, where a single horizontal channel is intersected and crossed by a
single vertical
channel, controlled electrokinetic transport operates to direct reagent flow
through the
intersection, by providing constraining flows from the other channels at the
intersection.
Simple electrokinetic flow of this reagent across the intersection could be
accomplished by
applying a voltage gradient across the length of the horizontal channel, i.e.,
applying a first
voltage to the left terminus of this channel, and a second, lower voltage to
the right terminus
of this channel, or by allowing the right terminus to float (applying no
voltage).
In some other applications, the apparatus comprises a microfabricated flow
cell with external mini-fluidics. Such an apparatus is illustrated in Fig. 27.
The glass cover
slip can be anodically bonded to the surface of the flow cell. The
interrogation region is
100~,m x 100~,m x 100~m, while the input and output channels are 100~m x 100~m
x
100~,m. Holes for the attachment of plumbing are etched at the ends of the
channels. For
such apparatuses, the fluidics can be external. Plumbing can be performed with
standard
HPLC components, e.g., from Upchurch and Hamilton. In the interrogation
region, the
polynucleotide template is attached to the surface with standard avidin-biotin
chemistry.
Multiple copies of templates can be attached to the apparatus. For example,
for a 7 kb
template, the radius of gyration is approximately 0.2pm. Therefore, about 105
molecules can
be attached while preventing the molecules from touching. Reagent switching
can be
accomplished with, e.g., an Upchurch six port injection valve and driven by,
e.g., a Thar
Designs motor. Fluid can be pumped with a syringe pump. The detection system
can be an
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external optical microscope, with the objective being in close proximity to
the glass cover
slip.
IV Analysis of polynucleotide sequences
S A. Template preparation and attachment to surface of synthesis channel
1. The general scheme
In some applications, the polynucleotides to be analyzed are first cloned in
single-stranded M13 plasmid (see, e.g., Current Protocols In Molecular
Biology, Ausubel, et
al., eds., John Wiley & Sons, Inc. 1995; and Sambrook, et al., Molecular
Cloning. A
Laboratory Manual, Cold Spring Harbor Press, 1989). The single stranded
plasmid is primed
by 5'-biotinylated primers (see, e.g., U.S. Patent No. 5,484,701), and double
stranded plasmid
can then be synthesized. The double stranded circle is then linearized, and
the biotinylated
strand is purified. In some methods, templates of around 100 by in length are
analyzed. In
some methods, templates to be sequenced are about 1 kb in length. In other
methods,
1 S templates that can be analyzed have a length of about 3 kb, 10 kb, or 20
kb.
Primer annealing is performed under conditions which are stringent enough to
achieve sequence specificity yet sufficiently permissive to allow formation of
stable hybrids
at an acceptable rate. The temperature and length of time required for primer
annealing
depend upon several factors including the base composition, length and
concentration of the
primer, and the nature of the solvent used, e.g., the concentration of DMSO,
formamide, or
glycerol, and counter ions such as magnesium. Typically, hybridization with
synthetic
polynucleotides is carried out at a temperature that is approximately 5 to
10°C below the
melting temperature of the target-primer hybrid in the annealing solvent.
Preferably, the
annealing temperature is in the range of SS to 75°C. and the primer
concentration is
approximately 0.2 pM. Under these preferred conditions, the annealing reaction
can be
complete in only a few seconds.
The single stranded polynucleotide templates to be analyzed can be DNA or
RNA. They can comprise naturally occurnng and or non-naturally occurnng
nucleotides.
Templates suitable for analysis according to the present invention can have
various sizes. For
example, the template can have a length of 100 bp, 200 bp, 500 bp, 1 kb, 3 kb,
10 kb, or 20
kb.
In some methods, the templates are immobilized to the surface of the synthesis
channels (e.g., 122A-122E in Fig. 25). By immobilizing the templates,
unincorporated
nucleotides can be removed from the synthesis channels by a washing step. The
templates
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can be immobilized to the surface prior to hybridization to the primer. The
templates can
also be hybridized to the primers first and then immobilize to the surface.
Alternatively, the
primers are immobilized to the surface, and the templates are attached to the
synthesis
channels through hybridization to the primers.
Various methods can be used to immobilize the templates or the primers to the
surface of the synthesis channels or reaction chambers. The immobilization can
be achieved
through direct or indirect bonding of the templates to the surface. The
bonding can be by
covalent linkage. See, Joos et al., Analytical Biochemistry 247:96-101, 1997;
Oroskar et al.,
Clin. Chem 42:1547-1555, 1996; and Khandjian, Mole. Bio. Rep. 11:107-115,
1986. The
bonding can also be through non-covalent linkage. For example, Biotin-
streptavidin (Taylor
et al., J. Phys. D. Appl. Phys. 24:1443, 1991) and digoxigenin and anti-
digoxigenin (Smith et
al., Science 253: 1122, 1992) are common tools for attaching polynucleotides
to surfaces and
parallels. Alternatively, the bonding can be achieved by anchoring a
hydrophobic chain into
a lipidic monolayer or bilayer.
When biotin-streptavidin linkage is used to immobilize the templates, the
templates are biotinylated, and one surface of the synthesis channels are
coated with
streptavidin. Since streptavidin is a tetramer, it has four biotin binding
sites per molecule.
Thus, in order to coat a surface with streptavidin, the surface can be
biotinylated first, and
then one of the four binding sites of streptavidin can be used to anchor the
protein to the
surface, leaving the other sites free to bind the biotinylated template (see,
Taylor et al., J.
Phys. D. Appl. Phys. 24:1443, 1991). Such treatment leads to a high density of
streptavidin
on the surface of the synthesis channel, allowing a correspondingly high
density of template
coverage. Reagents for biotinylating a surface can be obtained, for example,
from Vector
laboratories.
In some applications, the substrate or synthesis channel is pretreated to
create
surface chemistry that facilitates attachment of the polynucleotide templates
and subsequent
synthesis reactions. In some methods, the surface is coated with a
polyelectrolyte multilayer
(PEM). Attachment of templates to PEM-coated surface can be accomplished by
light-
directed spatial attachment (see, e.g., U.S. Patent Nos. 5,599,695, 5,831,070,
and 5,959,837).
Alternatively, the templates can be attached to PEM-coated surface entire
chemically (see
below for detail). In some methods, non-PEM based surface chemistry can be
created prior
to template attachment.
2. Attachment of diverse templates to a single channel
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While diverse polynucleotide templates can be each immobilized to and
sequenced in a separate synthesis channel, multiple templates can also be
sequenced in a
single microfluidic synthesis channel. In the latter scenario, the templates
are attached at
different locations along the flow path of the channel. This can be
accomplished by a variety
of different methods, including hybridization of primer capture sequences to
oligonucleotides
immobilized at different points on the substrate, and sequential activation of
different points
down the channel towards template immobilization.
Methods of creation of surfaces with arrays of oligonucleotides have been
described, e.g., in U.S. Patent Nos. 5,744,305, 5,837,832, and 6,077,674. Such
a surface can
be used as a substrate that is to be bond to a microfluidic chip and form the
synthesis channel.
Primers with two domains, a priming domain and a capture domain, can be used
to anchor
templates to the substrate. The priming domain is complementary to the target
template. The
capture domain is present on the non-extended side of the priming sequence. It
is not
complementary to the target template, but rather to a specific oligonucleotide
sequence
present on the substrate. The target templates can be separately hybridized
with their
primers, or (if the priming sequences are different) simultaneously hybridized
in the same
solution. Incubation of the primer/template duplexes in the flow channel under
hybridization
conditions allows attachment of each template to a unique spot. Multiple
synthesis channels
can be charged with templates in this fashion simultaneously.
Another method for attaching multiple templates in a single channel is to
sequentially activate portions of the substrate and attach template to them.
Activation of the
substrate can be achieved by either optical or electrical means. Optical
illumination can be
used to initiate a photochemical deprotection reaction that allows attachment
of the template
to the surface (see, e.g., U.S. Patent Nos. 5,599,695, 5,831,070, and
5,959,837). For instance,
the substrate surface can be derivatized with "caged biotin", a commercially
available
derivative of biotin that becomes capable of binding to avidin only after
being exposed to
light. Templates can then be attached by exposure of a site to light, filling
the channel with
avidin solution, washing, and then flowing biotinylated template into the
channel. Another
variation is to prepare avidinylated substrate and a template with a primer
with a caged biotin
moiety; the template can then be immobilized by flowing into the channel and
illumination of
the solution above a desired area. Activated template/primer duplexes are then
attached to
the first wall they diffused to, yielding a diffusion limited spot.
Electrical means can also be used to direct template to specific points in the
channel. By positively charging one electrode in the channel and negatively
charging the
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others, a field gradient can be created which drives the template to a single
electrode, where it
can attach (see, e.g., U.S. Patent Nos. 5,632,957, 6,051,380, and 6,071,394).
Alternatively, it
can be achieved by electrochemically activating regions of the surface and
changing the
voltage applied to the electrodes.
B. Exemplary surface chemistry for attaching templates: PEM coating
In some methods, the surface of synthesis channels are coated with PEM prior
to attachment of the templates (or primers). Such attachment scheme can be
both an ex-situ
process or an in situ process. With the ex-situ protocol, the surface of the
flat substrate is
coated with PEM first, followed by attachment of the templates. The
elastomeric
microfluidic chip is then bonded to the substrate to form and seal the
synthesis channel. With
the in-situ protocol, the microfluidic chip is attached to the flat substrate
first, and a PEM is
then constructed in the channels. The templates are then attached inside the
channels. In still
some other applications, the microfluidic chip can be bonded to the flat
substrate at any point
in the template attachment process, and the remaining steps can be completed
inside the
microfluidic channels.
Preferably, the in-situ protocol is used. The method described here leads to
low nonspecific binding of labeled (e.g., with fluorescent dye) nucleotides
and good seal of
the microfluidic components and the synthesis channels. A good seal between
the
microfluidic components and the synthesis channels allows the use of higher
pressures, which
in turn increases flow rates and decreases exchange times. The various methods
for attaching
the templates to the surface of the synthesis channel are discussed in detail
below.
An exemplified scheme of the ex situ protocol is as follows. First, the
surface
of a glass cover slip is cleaned and then coated with a polyelectrolyte
multilayer (PEM).
Following biotinylation of the carboxylic acid groups, streptavidin is then
applied to generate
a surface capable of capturing biotinylated molecules. Biotinylated
polynucleotide templates
are then added to the coated glass cover slip for attachment. The surface
chemistry thus
created is particularly suited for sequencing by synthesis with fluorescent
nucleotides,
because it generates a strong negatively-charged surface which repels the
negatively-charged
nucleotides. Detailed procedures for cleaning the cover slips, coating of
polyelectrolyte
multilayer, and attachment of the templates are described below.
PEM formation proceeds by the sequential addition of polycations and
polyanions, which are polymers with many positive or negative charges,
respectively. Upon
addition of a polycation to a negatively-charged surface, the polycation
deposits on the
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surface, forming a thin polymer layer and reversing the surface charge.
Similarly, a
polyanion deposited on a positively charged surface forms a thin layer of
polymer and leaves
a negatively charged surface. Alternating exposure to poly(+) and poly(-)
generates a
polyelectrolyte multilayer structure with a surface charge determined by the
last
polyelectrolyte added; in the case of incompletely-charged surfaces, multiple-
layer deposition
also tends to increase surface charge to a well defined and stable level. PEM
formation has
been described by Decher et al.(Thin Solid Films, 210:831-835, 1992).
Carboxylic acid groups are negatively charged at pH 7, and are a common
target for covalent bond formation. By terminating the surface with carboxylic
acid groups, a
surface which is both strongly negatively-charged and chemically reactive can
be generated
In particular, amines can link to them to form amide bonds, a reaction that
can be catalyzed
by carbodiimides. A molecule with biotin at one end, a hydrophilic spacer, and
an amine at
the other end is used to terminate the surface with biotin.
An avidin molecule is capable of binding up to four biotin molecules. This
means that avidin, and its derivative Streptavidin, is capable of converting a
biotin-terminated
surface to a surface capable of capturing biotin. Streptavidin, which carries
a slight negative
charge, is used to attach the polynucleotide templates to be analyzed to the
surface by using a
biotinylated primer. A buffer with a high concentration of multivalent salt is
used in order to
screen the repulsion of the negatively charged surface for the negatively-
charged DNA.
To coat the polyelectrolyte multilayer, the glass cover slips are first
cleaned
with HP H20 (H20 deionized to 18.3 MOhm-cm and filtered to 0.2 Vim) and a RCA
Solution
(6:4:1 mixture of HP H20, (30% NH40H), and (30% H202)). The cover slips are
then
sonicated in 2% Micro 90 detergent for 20 minutes. After rinse thoroughly with
HP HzO, the
cover slips are stirred in gently boiling RCA solution for at least 1 hour,
and rinsed again
with HP H20.
After cleaning, the glass cover slips are submerged in PAII solution
(Poly(allylamine) (PAIL, +): 2 mg/ml in HP H20, adjusted to pH 7.0) and
agitate for at least
10 minutes. The cover slips are then removed from PAII and washed with HP H20
by
submerging in HP H20 with agitation for at least three times. The treatment
continues by
agitation in a PAcr solution (Poly(acrylic acid) (PAcr, -): 2 mg/ml in HP H20,
adjusted to pH
7.0) for at least 10 minutes and washing with HP H20. The treatment steps are
then repeated
once.
After PEM coating, the PEM coated glass is incubated with a EDCBLCPA
solution for 30 minutes. The EDCBLCPA solution is prepared by mixing equal
amounts of
3p
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50 mM EDC solution (in MES buffer) and 50 mM BLCPA (in MES buffer) and
diluting to
SmM in MES buffer. The glass is then rinsed with 10 mM Tris-NaCI and incubated
with 0.1
mg/ml streptavidin solution for 1 hour. After washing with 10 mM Tris-NaCI,
the glass is
incubated with a solution containing the polynucleotide template (10-~ M in
Tris 100 mM
MgCl2) for 30 minutes. The glass is again rinsed thoroughly with 10 mM Tris-
NaCI.
For in-situ attachment, the microfluidic substrate is bonded to the glass
cover
slip by HCl-assisted bonding. Essentially, the chips are first washed with a
surfactant (e.g.,
first with HP H20, then in 0.1 % Tween 20, then rinse again with HP H20). The
washed
microfluidic chips are then put on the glass cover slips with a few
microliters of dilute HCl
(e.g., 1% HCl in HP H20), followed by baking at 37° C for 1-2 hours.
Such treatment
enhances the bond strength to glass (e.g., >20 psi pressure) without
increasing nonspecific
adsorption.
Following HCl treatment, PEM formation, biotinylation, streptavidinylation,
and template attachment can be performed using essentially the same reagents
and methods
as described above for ex-situ attachment, except the solutions are injected
through the
channels by pressure instead of just being aliquoted onto the substrate
surface.
Coating the microchannel surface with the PEM technique is significant for
analyzing polynucleotide sequences according to the present invention. In
general, the
method used to attach the template to the surface should fulfill several
requirements in order
to be useful in a sequencing-by-synthesis application. First, it must be
possible to attach
reasonable quantities of polynucleotide templates. In addition, the attached
templates should
remain active for polymerase action. Further, nonspecific binding of
fluorescent nucleotides
should be very low.
If insufficient numbers of template molecules are bound, the signal-to-noise
ratio of the technique is too low to allow useful sequencing. Binding large
quantities of
templates is insufficient, however, if the primer/target duplex cannot be
extended by a
polymerase. This is a problem for surface chemistry based on building off
amine-bearing
surfaces: amines are positively charged at normal pH. This means that the
negatively-
charged DNA backbone can non-specifically stick to the surface, and that the
polymerase is
sterically impeded from adding nucleotides. Finally, if there is significant
nonspecific
binding of fluorescent nucleotides to the surface, it becomes impossible to
distinguish
between signal due to incorporation and signal due to nonspecific binding.
When the nucleotides are fluorescently labeled, they generally have relatively
strong nonspecific binding to many surfaces because they possess both a
strongly polar
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moiety (the nucleotide, and in particular the triphosphate) and a relatively
hydrophobic
moiety (the fluorescent dye). A surface bearing positively-charged groups
(i.e. amines)
invariably has a very high nonspecific binding due to the attraction of the
triphosphate group
(which is strongly negatively charged) to the positively-charged amines.
Neutral surfaces
generally have strong nonspecific binding due to the action of the fluorescent
nucleotide as a
surfactant (i.e. assembling with nonpolar moiety towards the uncharged (more
hydrophobic)
surface and polar end in the aqueous phase). A surface bearing negative
charges can repel
the negatively charged fluorescent nucleotides, so it has the lowest
nonspecific binding.
Glass is such a surface, but the surface silanols that give it its negative
charge in water are a
difficult target to attach DNA to directly. Typical DNA attachment protocols
use silanization
(often with aminosilanes) to attach template; as discussed earlier amino
groups lead to
unacceptable levels of nonspecific binding.
A polyelectrolyte multilayer terminated with carboxylic acid-bearing polymer
fulfills all three criteria. First, it is easy to attach polynucleotide to
because carboxylic acids
are good targets for covalent bond formation. Second, the attached template is
active for
extension by polymerases - most probably, the repulsion of like charges
prevents the
template from "laying down" on the surface. Finally, the negative charge
repels the
fluorescent nucleotides, and nonspecific binding is low.
The attachment scheme described here is easy to generalize on. Without
modification, the PEM/biotin/streptavidin surface that is produced can be used
to capture or
immobilize any biotinylated molecule. A slight modification can be the use of
another
capture pair, i.e. substituting digoxygenin (dig) for biotin and labeling the
molecule to be
immobilized with anti-digoxygenin (anti-dig). Reagents for biotinylation or
dig-labeling of
amines are all commercially available.
Another generalization is that the chemistry is nearly independent of the
surface chemistry of the support. Glass, for instance, can support PEMs
terminated with
either positive or negative polymer, and a wide variety of chemistry for
either. But other
substrates such as silicone, polystyrene, polycarbonate, etc, which are not as
strongly charged
as glass, can still support PEMs. The charge of the final layer of PEMs on
weakly-charged
surfaces becomes as high as that of PEMs on strongly-charged surfaces, as long
as the PEM
has sufficiently-many layers. For example, PEM formation on 02-plasma treated
silicone
rubber has been demonstrated by the present inventors. This means that all the
advantages of
the glass/PEM/biotin/Streptavidin/biotin-DNA surface chemistry can be applied
to other
substrates.
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Although the above discussion describes the immobilization of polynucleotide
templates by attachment to the surface of flow channels or the surface of
reaction chambers
disposed along flow channels, other methods of template immobilization can
also be
employed in the methods of the present invention. In some methods, the
templates can be
attached to microbeads, which can be arranged within the microfluidic system.
For instance,
commercially-available latex microspheres with pre-defined surface chemistry
can be used.
The polynucleotide templates can be attached either before or after the
microbeads are
inducted into the microfluidic system. Attachment of template before beads are
added allows
a reduction in system complexity and setup time (as many templates can be
attached to
different aliquots of beads simultaneously). Attachment of template to beads
in situ can
allow easier manipulation of surface chemistry (as bead surface chemistry can
be
manipulated in bulk and externally to the microfluidic device). Beads should
be held in place
within the flow system for this technique to be effective. Methods to achieve
this include,
e.g., flowing the beads into orifices too small for them to flow through
(where they would
become "wedged in"), the creation of "microscreens" (i.e. barners in the
channel with
apertures too small for beads to pass through), and insertion of the beads
into hollows in the
channels where they are affixed by simple Van der Waals forces.
C. Primer extension reaction
Once templates are immobilized to the surfaces of synthesis channels, primer
extension reactions are performed (E. D. Hyman, Anal. Biochem., 174, p. 423,
1988). If part
of the template sequence is known, a specific primer can be constructed and
hybridized to the
template. Alternatively, a linker can be ligated to the template of unknown
sequence in order
to allow for hybridization of a primer. The primer can be hybridized to the
template before or
after immobilization of the template to the surface of the synthesis channel.
In some methods, the primer is extended by a nucleic acid polyrnerase in the
presence of a single type of labeled nucleotide. Label is incorporated into
the
template/primer complex only if the labeled nucleotide added to the reaction
is
complementary to the nucleotide on the template adjacent the 3' end of the
primer. The
template is subsequently washed to remove any unincorporated label, and the
presence of any
incorporated label is determined. A radioactive label can be determined by
counting or any
other method known in the art, while fluorescent labels can be induced to
fluoresce, e.g., by
excitation.
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In some applications of the present invention, a combination of labeled and
non-labeled nucleotides are used in the analysis. Because there are multiple
copies of each
template molecule immobilized on the surface of the synthesis channel, a small
percentage of
labeled nucleotides is sufficient for detection by a detection device (see
below). For example,
for fluorescently labeled nucleotides, the percentage of labeled nucleotide
can be less than
20%, less than 10%, less than 5%, less than 1 %, less than 0.1 %, less than
0.01 %, or even less
than 0.001 % of the total labeled and unlabeled nucleotides for each type of
the nucleotides.
1. Labeled Nucleotides
In some methods, at least one and usually all types of the
deoxyribonucleotides (dATP, dTTP, dGTP, dCTP, dUTP/dTTP) or nucleotides (ATP,
UTP,
GTP, and CTP) are labeled. Various labels which are easily detected include
radioactive
labels, optically detectable labels, spectroscopic labels and the like.
Preferably, fluorescent
labels are used. The different types of nucleotides can be labeled with the
same kind of
labels. Alternatively, a different kind of label can be used to label each
different type of
nucleotide.
In some methods, fluorescent labels are used. the fluorescent label can be
selected from any of a number of different moieties. The preferred moiety is a
fluorescent
group for which detection is quite sensitive. For example, fluorescein- or
rhodamine-labeled
nucleotide triphosphates are available (e.g., from NEN DuPont).
Fluorescently labeled nucleotide triphosphates can also be made by various
fluorescence-labeling techniques, e.g., as described in Kambara et al. (1988)
"Optimization of
Parameters in a DNA Sequenator Using Fluorescence Detection," Bio/Technol.
6:816-821;
Smith et al. (1985) Nucl. Acids Res, 13:2399-2412; and Smith et al. (1986)
Nature 321:674-
679. Fluorescent labels exhibiting particularly high coefficients of
destruction can also be
useful in destroying nonspecific background signals.
2. Blocking agents:
In some methods during the primer extension step, a chain elongation inhibitor
can be employed in the reaction (see, e.g., Dower et al., U.S. Patent No.
5,902,723. Chain
elongation inhibitors are nucleotide analogues which either are chain
terminators which
prevent further addition by the polymerase of nucleotides to the 3' end of the
chain by
becoming incorporated into the chain themselves. In some methods, the chain
elongation
inhibitors are dideoxynucleotides. Where the chain elongation inhibitors are
incorporated
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CA 02388528 2002-04-22
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into the growing polynucleotide chain, they should be removed after
incorporation of the
labeled nucleotide has been detected, in order to allow the sequencing
reaction to proceed
using different labeled nucleotides. Some 3' to S' exonucleases, e.g.,
exonuclease III, are able
to remove dideoxynucleotides.
Other than chain elongation inhibitors, a blocking agent or blocking group can
be employed on the 3' moiety of the deoxyribose group of the labeled
nucleotide to prevent
nonspecific incorporation. Optimally, the blocking agent should be removable
under mild
conditions (e.g., photosensitive, weak acid labile, or weak base labile
groups), thereby
allowing for further elongation of the primer strand with a next synthetic
cycle. If the
blocking agent also contains the fluorescent label, the dual blocking and
labeling functions
are achieved without the need for separate reactions for the separate
moieties. For example,
the labeled nucleotide can be labeled by attachment of a fluorescent dye group
to the 3'
moiety of the deoxyribose group, and the label is removed by cleaving the
fluorescent dye
from the nucleotide to generate a 3' hydroxyl group. The fluorescent dye is
preferably linked
to the deoxyribose by a linker arm which is easily cleaved by chemical or
enzymatic means.
Examples of blocking agents include, among others, light sensitive groups
such as 6-nitoveratryloxycarbonyl (NVOC), 2-nitobenzyloxycarbonyl (NBOC),
.a,.a-
dimethyl-dimethoxybenzyloxycarbonyl (DDZ), 5-bromo-7-nitroindolinyl, o-hydroxy-
2-
methyl cinnamoyl, 2-oxymethylene anthraquinone, and t-butyl oxycarbonyl
(TBOC). Other
blocking reagents are discussed, e.g., in U.S. Ser. No. 07/492,462; Patchornik
(1970) J.
Amer. Chem. Soc. 92:6333; and Amit et al. (1974) J. Org. Chem. 39:192.
Nucleotides
possessing various labels and blocking groups can be readily synthesized.
Labeling moieties
are attached at appropriate sites on the nucleotide using chemistry and
conditions as
described, e.g., in Gait (1984) Oligonucleotide Synthesis: A Practical
Approach, IRL Press,
Oxford.
3. Polymerases
Depending on the template, either RNA polymerase or DNA polymerases can
be used in the primer extension. For analysis of DNA templates, many DNA
polymerases are
available. Examples of suitable DNA polymerases include, but are not limited
to, Sequenase
2.O®, T4 DNA polymerase or the Klenow fragment of DNA polymerase 1, or
Vent
polymerase. In some methods, polymerases which lack 3' -~ S' exonuclease
activity can be
used (e.g., T7 DNA polymerase (Amersham) or Klenow fragment of DNA polymerase
I
3~
CA 02388528 2002-04-22
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(New England Biolabs)). In some methods, when it is desired that the
polymerise have
proof reading activity, polymerises lacking 3' --~ 5' exonuclease activity are
not used. In
some methods, thermostable polymerises such as ThermoSequenaseT"" (Amersham)
or
TaquenaseT"" (ScienTech, St Louis, MO) are used.
The nucleotides used in the methods should be compatible with the selected
polymerise. Procedures for selecting suitable nucleotide and polymerise
combinations can
be adapted from Ruth et al. (1981) Molecular Pharmacology 20:415-422;
Kutateladze, T., et
al. (1984) Nuc. Acids Res., 12:1671-1686; Chidgeavadze, Z., et al. (1985) FEBS
Letters,
183:275-278.
The polymerise can be stored in a separate reservoir in the apparatus and
flowed into the synthesis channels prior to each extension reaction cycle. The
enzyme can
also be stored together with the other reaction agents (e.g., the nucleotide
triphosphates).
Alternatively, the polymerise can be immobilized onto the surface of the
synthesis channel
along with the polynucleotide template.
4. Removal of blocking group and labels
By repeating the incorporation and label detection steps until incorporation
is
detected, the nucleotide on the template adjacent the 3' end of the primer can
be identified.
Once this has been achieved, the label should be removed before repeating the
process to
discover the identity of the next nucleotide. Removal of the label can be
effected by removal
of the labeled nucleotide using a 3'-5' exonuclease and subsequent replacement
with an
unlabeled nucleotide. Alternatively, the labeling group can be removed from
the nucleotide.
In a further alternative, where the label is a fluorescent label, it is
possible to neutralize the
label by bleaching it with radiation. Photobleaching can be performed
according to methods,
e.g., as described in Jacobson et al., "International Workshop on the
Application of
Fluorescence Photobleaching Techniques to Problems in Cell Biology",
Federation
Proceedings, 42:72-79, 1973; Okabe et al., J Cell Biol 120:1177-86, 1993; and
Close et al.,
Radiat Res 53:349-57, 1973.
If chain terminators or 3' blocking groups have been used, these should be
removed before the next cycle can take place. 3' blocking groups can be
removed by
chemical or enzymatic cleavage of the blocking group from the nucleotide. For
example,
chain terminators are removed with a 3'-5' exonuclease, e.g., exonuclease III.
Once the label
3
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and terminators/blocking groups have been removed, the cycle is repeated to
discover the
identity of the next nucleotide.
Removal of the blocking groups can be unnecessary if the labels are
removable. In this approach, the chains incorporating the blocked nucleotides
are
permanently terminated and no longer participate in the elongation processes.
So long as
these blocked nucleotides are also removed from the labeling process, a small
percentage of
permanent loss in each cycle can also be tolerated.
In some methods, other than labeled nucleotides, nucleotide incorporation is
monitored by detection of pyrophosphate release (see, e.g., W098/13523,
W098/28440, and
Ronaghi et al., Science 281:363, 1998). For example, a pyrophosphate-detection
enzyme
cascade is included in the reaction mixture in order to produce a
chemoluminescent signal.
Also, instead of deoxynucleotides or dideoxynucleotides, nucleotide analogues
are used
which are capable of acting as substrates for the polymerase but incapable of
acting as
substrates for the pyrophosphate-detection enzyme. Pyrophosphate is released
upon
incorporation of a deoxynucleotide or dideoxynucleotide, which can be detected
enzymatically. This method employs no wash steps, instead relying on continual
addition of
reagents.
D. Detection of incorporated signals and scanning system
1. Optical detection
Methods for visualizing single molecules of DNA labeled with an intercalating
dye include, e.g., fluorescence microscopy as described in Houseal et al.,
Biophysical Journal
56: 507, 1989. While usually signals from a plurality of molecules are to be
detected with the
sequencing methods of the present invention, fluorescence from single
fluorescent dye
molecules can also be detected. For example, a number of methods are available
for this
purpose (see, e.g., Nie et al., Science 266: 1013, 1994; Funatsu et al.,
Nature 374: 555, 1995;
Mertz et al., Optics Letters 20: 2532, 1995; and Unger et al., Biotechniques
27:1008, 1999).
Even the fluorescent spectrum and lifetime of a single molecule before it
photobleaches can
be measured (Macklin et al., Science 272: 255, 1996). Standard detectors such
as a
photomultiplier tube or avalanche photodiode can be used. Full field imaging
with a two
stage image intensified CCD camera can also used (Funatsu et al., supra).
The detection system for the signal or label can also depend upon the label
used, which can be defined by the chemistry available. For optical signals, a
combination of
an optical fiber or charged couple device (CCD) can be used in the detection
step. In those
37
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circumstances where the matrix is itself transparent to the radiation used, it
is possible to have
an incident light beam pass through the substrate with the detector located
opposite the
substrate from the polynucleotides. For electromagnetic labels, various forms
of
spectroscopy systems can be used. Various physical orientations for the
detection system are
available and discussion of important design parameters is provided, e.g., in
Jovin, Adv. in
Biochem. Bioplyms.
Incorporated signals can be detected by scanning the synthesis channels. The
synthesis channels can be scanned simultaneously or serially, depending on the
scanning
method used. The signals can be scanned using a CCD camera (TE/CCDS 12SF,
Princeton
Instruments, Trenton, N.J.) with suitable optics (Ploem, J. S., in Fluorescent
and Luminescent
Probes for Biological Activity, Mason, T. W., Ed., Academic Press, London, pp.
1-11, 1993),
such as described in Yershov et al. (Proc. Natl. Acad. Sci. 93:4913, 1996), or
can be imaged
by TV monitoring (Khrapko et al., DNA Sequencing 1:375, 1991). For radioactive
signals
(e.g., 32 P), a phosphorimager device can be used (Johnston et al., Johnston,
R. F., et al.,
Electrophoresis 11:355, 1990; and Drmanac et al., Drmanac, R., et al.,
Electrophoresis
13:566, 1992). These methods are particularly useful to achieve simultaneous
scanning of
multiple probe-regions.
For fluorescence labeling, the synthesis channels can be serially scanned one
by one or row by row using a fluorescence microscope apparatus, such as
described in U.S.
Patent Nos. 6,094,274, 5,902,723, 5,424,186, and 5,091,652. In some methods,
standard low-
light level cameras, such as a SIT and image intensified CCD camera, are
employed (see,
Funatsu et al., Nature 374, 555, 1995). An ICCD can be preferable to a cooled
CCD camera
because of its better time resolution. These devices are commercially
available (e.g., from
Hammamatsu).
Alternatively, only the intensifier unit from Hammamatsu or DEP are used and
incorporated into other less expensive or home built cameras. If necessary,
the intensifier can
be cooled. For example, CCD camera can be purchased from Phillips, who offer a
low
priced, low noise (40 electron readout noise per pixel) model. A home built
camera allows
greater flexibility in the choice of components and a higher performance
device. The
advantage of using a camera instead of an avalanche photodiode is that one can
image the
whole field of view. This extra spatial information allows the development of
new noise
reduction techniques. For example, one can use the fact that signals are
expected from
certain spatial locations (i.e. where the polynucleotide template is attached)
in order to reject
noise.
3 c~
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In some applications, fluorescent excitation is exerted with a Q-switched
frequency doubled
Nd YAG laser, which has a KHz repetition rate, allowing many samples to be
taken per
second. For example, a wavelength of 532 nm is ideal for the excitation of
rhodamine. It is a
standard device that has been used in the single molecule detection scheme
(Smith et al.,
Science 253:1122, 1992). A pulsed laser allows time resolved experiments,
which are useful
for rejecting extraneous noise. In some methods, excitation can be performed
with a mercury
lamp and signals from the incorporated nucleotides can be detected with an
inexpensive CCD
camera (see, e.g., Unger et al., Biotechniques 27:1008, 1999.
The scanning system should be able to reproducibly scan the synthesis
channels in the apparatuses. Where appropriate, e.g., for a two dimensional
substrate where
the synthesis channels are localized to positions thereon, the scanning system
should
positionally define the synthesis channels attached thereon to a reproducible
coordinate
system. It is important that the positional identification of synthesis
channels be repeatable in
successive scan steps.
Various scanning systems can be employed in the apparatuses of the present
invention. For example, electrooptical scanning devices described in, e.g.,
U.S. Pat. No.
5,143,854, are suitable for use with the apparatuses of the present invention.
The system
could exhibit many of the features of photographic scanners, digitizers or
even compact disk
reading devices. For example, a model no. PM500-A1 x-y translation table
manufactured by
Newport Corporation can be attached to a detector unit. The x-y translation
table is connected
to and controlled by an appropriately programmed digital computer such as an
IBM PC/AT
or AT compatible computer. The detection system can be a model no. 8943-02
photomultiplier tube manufactured by Hamamatsu, attached to a preamplifier,
e.g., a model
no. SR440 manufactured by Stanford Research Systems, and to a photon counter,
e.g., an
SR430 manufactured by Stanford Research System, or a multichannel detection
device.
Although a digital signal can usually be preferred, there can be circumstances
where analog
signals would be advantageous.
The stability and reproducibility of the positional localization in scanning
determine, to a large extent, the resolution for separating closely positioned
polynucleotide
clusters on a 2 dimensional substrate. Since the successive monitoring at a
given position
depends upon the ability to map the results of a reaction cycle to its effect
on a positionally
mapped cluster of polynucleotides, high resolution scanning is preferred. As
the resolution
increases, the upper limit to the number of possible polynucleotides which can
be sequenced
on a single matrix also increases. Crude scanning systems can resolve only on
the order of
3g
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1000 ~,m, refined scanning systems can resolve on the order of 100 Vim, more
refined systems
can resolve on the order of about 10 pm, and with optical magnification
systems a resolution
on the order of 1.0 ~,m is available. The limitations on the resolution can be
diffraction
limited and advantages can arise from using shorter wavelength radiation for
fluorescent
S scanning steps. However, with increased resolution, the time required to
fully scan a matrix
can increased and a compromise between speed and resolution can be selected.
Parallel
detection devices which provide high resolution with shorter scan times are
applicable where
multiple detectors are moved in parallel.
In some applications, resolution often is not so important and sensitivity is
emphasized. However, the reliability of a signal can be pre-selected by
counting photons and
continuing to count for a longer period at positions where intensity of signal
is lower.
Although this decreases scan speed, it can increase reliability of the signal
determination.
Various signal detection and processing algorithms can be incorporated into
the detection
system. In some methods, the distribution of signal intensities of pixels
across the region of
signal are evaluated to determine whether the distribution of intensities
corresponds to a time
positive signal.
2. Non-optical detection
Other than fluorescently labeled nucleotides and optical detection devices,
other methods of detecting nucleotide incorporation are also contemplated in
the present
invention, including the use of mass spectrometry to analyze the reaction
products, the use of
radiolabeled nucleotides, and detection of reaction products with "wired
enzymes".
In some methods, mass spectrometry is employed to detect nucleotide
incorporation in the primer extension reaction. A primer extension reaction
consumes a
nucleotide triphosphate, adds a single base to the primer/template duplex, and
produces
pyrophosphate as a by-product. Mass spectrometry can be used to detect
pyrophosphate in
the wash stream after a nucleotide has been incubated with the template and
polymerase.
The absence of pyrophosphate indicates that the nucleotide was not
incorporated, whereas the
presence of pyrophosphate indicates incorporation. Detection based on
pyrophosphate
release have been described, e.g., in W098/13523, W098/28440, and Ronaghi et
al., Science
281:363, 1998.
In some methods, radiolabeled nucleotides are used. Nucleotides can be
radiolabeled either in the sugar, the base, or the triphosphate group. To
detect radioactivity,
small radioactivity sensor can be incorporated in the substrate on which the
microfluidic chip
y' 0
CA 02388528 2002-04-22
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is mounted. A CCD pixel, for instance, serves as a good detector for some
radioactive decay
processes. Radiolabeling of the sugar or base produces an additive signal:
each incorporation
increases the amount of radiolabel in the primer-template duplex. If the
nucleotide is labeled
in the portion that is released as pyrophosphate (e.g. dNTP with (3- or y-
32P), the radioactive
pyrophosphate can be detected in the wash stream. This radioactivity level is
not additive,
but rather binary for each attempted nucleotide addition, so subsequent
addition poses no read
length limit. Due to the small reagent consumption and contained nature of
microfluidics, the
total radioactivity used in such a system is relatively minimal, and
containment is relatively
simple.
In some methods, non-optical detection of pyrophosphate release makes use of
"wired redox enzymes" as described, e.g., in Heller et al., Analytical
Chemistry 66:2451-
2457, 1994; and Ohara et al., Analytical Chemistry 65:3512-3517, 1993.
Briefly, enzymes
are covalently linked to a hydrogel matrix containing redox active groups
capable of
transporting charge. The analyte to be detected is either acted on directly by
a redox enzyme
(either releasing or consuming electrons) or consumed as a reagent in an
enzymatic cascade
that produces a substrate that is reduced or oxidized by a redox enzyme. The
production or
consumption of electrons is detected at a metal electrode in contact with the
hydrogel. For
the detection of pyrophosphate, an enzymatic cascade using pyrophosphatase,
maltose
phosphorylase, and glucose oxidase can be employed. Pyrophosphatase converts
pyrophosphate into phosphate; maltose phosphorylase converts maltose (in the
presence of
phosphate) to glucose 1-phosphate and glucose. Then, glucose oxidase converts
the glucose
to gluconolactone and H2O2; this final reaction is the redox step which gives
rise to a
detectable current at the electrode. Glucose sensors based on this principle
are well known in
the art, and enzymatic cascades as described here have been demonstrated
previously. Other
enzymatic cascades besides the specific example given here are also
contemplated the present
invention. This type of detection scheme allows direct electrical readout of
nucleotide
incorporation at each reaction chamber, allowing easy parallelization.
E. Fluorescent photobleaching sequencing
In some methods, polynucleotide sequences are analyzed with a fluorescent
photobleaching method. In this methods, fluorescently labeled nucleotides are
used in the
primer extension. Signals from the incorporated nucleotides are removed by
photobleaching
before next extension cycle starts.
CA 02388528 2002-04-22
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The polynucleotide templates can be prepared as described above (e.g.,
cloning in single-stranded M13 plasrriid). Biotinylated templates are attached
to surface of
the synthesis channel that has been pretreated with the PEM technique as
discussed above.
After the primed, single stranded DNA is immobilized to the synthesis channel
in the flow
cell. A polymerise and one nucleotide triphosphate, e.g. dATP, are flowed into
the flow cell.
A high fidelity polymerise with no exonuclease proofreading ability is
preferred. In some
methods, only a fraction (e.g., less than 10%, 5%, 1 %, 0.1 %, 0.01 %, or
0.001 %) of each type
of the nucleotide triphosphates is fluorescently labeled (e.g., rhodamine-
labeled nucleotide
triphosphates from NEN DuPont). For example, if the first base of DNA sequence
following
the primer is T, then the polymerise incorporates the dATP's and some fraction
of the DNA
molecules become fluorescently labeled. If the first base is anything else, no
fluorescent
molecules become incorporated. The reagents are then flowed out of the flow
cell, and the
fluorescence of the DNA is measured. If no fluorescence is detected, the
procedure is
repeated with one of the other nucleotide triphosphates. If fluorescence is
detected, the
identity of the first base in the sequence has been determined. The
fluorescence signal is
photobleached and extinguished before the procedure is then repeated for the
next base in the
template sequence.
The fluorescence can be excited with, e.g., a Q-switched frequency doubled
Nd YAG laser (Smith et al., Science 253: 1122, 1992). This is a standard
device used in the
single molecule detection scheme that measures the fluorescent spectrum and
lifetime of a
single molecule before it photobleached. It has a kHz repetition rate,
allowing many samples
to be taken per second. The wavelength can be. e.g., 532 nm that is ideal for
the excitation of
rhodamine. A pulsed laser allows time resolved experiments and is useful for
rejecting
extraneous noise.
Detection of the incorporated label can be performed with a standard low-light
level cameras, such as a SIT or a image intensified CCD camera (Funatsu et al,
supra). An
Intensified CCD (ICCD) camera is preferable to a cooled CCD camera because of
its better
time resolution. These devices are available from, e.g., Hammamatsu. However,
because
these cameras are extremely expensive, a detection device can be made by
building just the
intensifier unit from Hammamatsu into a CCD camera. Optionally, the
intensifier can be
cooled. The CCD camera is available from Phillips, e.g., a low priced, low
noise model (40
electron readout noise per pixel). A customarily built camera allows greater
flexibility in the
choice of components and a higher performance device. The advantage of using a
camera
instead of an avalanche photodiode is that the whole field of view can be
imaged. This extra
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CA 02388528 2002-04-22
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spatial information allows the development of new noise reduction techniques.
For example,
the fact that signals are expected from certain spatial locations (i.e. where
the DNA is
attached) can be used to reject noise.
F. Other considerations
A combination of factors affect the read length and throughput of the
sequencing analysis according to the present invention. First, all of the
unincorporated
labeled nucleotides should be removed from the synthesis channel or reaction
chamber after
each cycle. Since only relatively small number of incorporated dye molecules
are to be
detected, the reagent exchange should be leave substantially fewer
unincorporated labeled
nucleotides than the number of nucleotides to be detected. Second, the rate of
reagent
exchange is limited by fluid mechanic considerations. Turbulent flow should be
avoided in
order to preserve effective reagent exchange, and the fluid flow shear forces
should be small
enough in order to not break the DNA or dislocate the enzyme. Third, the
kinetics of
nucleotide incorporation and enzyme-DNA complex formation should be
considered.
The present invention teaches how to determine acceptable flow rate of fluids
in the apparatuses. According to the invention, flow rate in the apparatuses
with
microfabricated flow channels having a depth of 100 ~,m is typically 0.1-1
cm/sec. For
microfabricated flow channels with a depth of 10 ~,m, the flow rate is usually
in the range of
1-10 cm/sec. Fluid flow in the apparatuses remains laminar as long as the
Reynolds number
R=pw/r)«1, where p is the density of the fluid, a is the velocity, v is the
dimension of the
chamber, and r1 is the viscosity (see, e.g., Landau et al., Fluid Mechanics,
Pergamon Press,
New York, 1989). The limiting velocity is in the order of 1 cm/sec for a
100~.m channel
depth. For microchannels with a depth of 10 ~,m, the limit is 10 cm/sec.
The ultimate limit on the rate at which fluid can be exchanged is determined
by the effect of drag and shear flows on the polynucleotide template and the
polymerise. The
velocity profile of constrained flow is parabolic (v(i)-va,,e(1-(i/R)2)),
causing a shear force.
Single molecule experiments with double stranded DNA have shown that one can
apply
forces of up to F=50 pN without breaking or causing irreversible damage to DNA
(see, e.g.,
Smith et al., Science 271: 795, 1996; and Cluzel et al., Science 271: 792,
1996), and a similar
order of magnitude is expected for single stranded DNA. The drag coefficient
of DNA
a=6~R5 can be estimated from the radius of gyration RS=0.3~.m. Then the
maximum fluid
velocity allowed is determined by solving the equation:
'/
CA 02388528 2002-04-22
WO 01/32930 PCT/US00/30591
V",~(R-R~ =Flcz
The maximum average velocity before shearing of DNA becomes a problem is 140
cm/sec.
Another consideration is to prevent the polymerase from falling off the
template or becoming damaged. With RNA polymerase, it has been shown that the
stalling
force for RNA polymerase, at which it might receive irreversible damage, is 14
pN (Yin et
al., Science 270:1653, 1995). Since one the drag coefficient of a DNA
polymerase can be
estimated from its size, a similar calculation as for the DNA shear leads to a
maximum
velocity of 500 cm/sec.
The time to remove all of the free nucleotides can be calculated by including
the effects of diffusion into hydrodynamic calculation of the fluid flow.
There are a great
variety of products available, including electronic switching valves with very
small dead
volumes. For example, a six port valve from Upchurch with electric motor from
Thar
Designs has a dead volume of 2 p1 and switching time of 166 msec. Combined
with 0.0025"
LD. tubing and the estimated 1 ~,1 capacity of the microfabricated flow cell,
4~1 of material
should be exchanged for each step in the process. A syringe or peristaltic
pump can give very
high flow rates, the limiting factor is low Reynolds number. The inverse rate
constant to get
rid of all of the nucleotides is
'L=(LR~Vave)Z/3 (D)-1/3
where L is the linear dimension of the device and D is the diffusion constant
of the
nucleotides. Plugging in approximate numbers gives a time of i=1 S sec. To
reduce the
nucleotide concentration from in the order of millimolar to 1 labeled
nucleotide per detection
region, which is a reduction of approximately 10-'. The amount of time to
completely flush
the device is ln(10'~)T=4 minutes.
For apparatuses with microfabricated flow channel depth of 10~m and
microfabricated valves incorporated on chip, the dead volume is reduced and
throughput
increased. The valves can provide an essentially zero dead volume and 10 msec
switching
time. This and the reduced dimensions of the device leads to a drastic
increase of throughput:
the time to flush the reagents (e.g., nucleotides) from the system is reduced
to 0.8 sec. The
overall throughput is approximately 1 base per second. Table 1 summarizes the
various
factors affecting throughput of apparatuses with microfabricated flow channels
having a
depth of 100 ~,m or 10 ~.m.
TABLE I. Parameters affecting throughput of the sequencing apparatuses
CA 02388528 2002-04-22
WO 01/32930 PCT/LTS00/30591
I II
Channel de th m 100 10
Dead Volume 1 4 10-
Turbulence vel. cm/sec 1 10
DNA Shear cm/sec 140 14
Pol erase stall cm/sec 1000 100
Reagent exchange (sec) 240 0.8
Note that in apparatuses I, the limiting factor is the fluid velocity that
causes turbulent flow. In apparatuses II,
shear forces on the DNA also becoming limiting. The reagent exchange time is
expected to improve by a factor
of 100 in apparatuses II.
The DNA polymerases can fall off of the DNA. If enzyme is replenished, it
takes time for the enzyme to find and bind to a free DNA site. This could
affect throughput
of the apparatuses. The attrition rate of the polymerase can be determined
according to
methods described in the art. For example, using the kinetics of the T4 DNA
polymerase as
nominal values (Taylor et al., J. Phys. D. Appl. Phys. 24:1443, 1991), an on-
rate of 11 ~M-~
sec 1 was obtained. Hence a 1 ~M concentration of enzyme gives an on rate of
11 sec-1, and
after 1 second, 99.3% of the DNA have polymerase bound. In the absence of
nucleotides (for
example, during fluorescence measurement) the polymerase falls off of the DNA
with a time
constant of 0.2 sec 1 (Yin et al., Science 270:1653, 1995). In other words,
after 5 seconds
without nucleotides, this can become a source of attrition. It can be
compensated for by the
addition of fresh polymerase with every sequencing cycle of the device.
For the high throughput device (e.g., apparatus II in Table I), the reagent
exchange is fast enough that polymerase falling off has no significant effect
on the
throughput. Also, the rate of incorporation of nucleotides by the polymerase
is typically
about 300 bases per second. This is not a rate limiting factor for the device
throughput.
Read length of the sequencing analysis can be affected by various factors.
However, photobleaching is unlikely to cause any chemical changes to the
polynucleotide
template that prevent the attachment of the next base. During the
photobleaching, the dye
molecule is held off from the DNA on a linker arm, and it gives off so few
photons that the
interaction cross section is negligible. Any attrition of the labeled
nucleotides also does not
present any significant problem. The statistics of the photobleaching scheme
are robust
enough to allow sequencing to continue in spite of any attrition of the
labeled nucleotides.
For example, if 0.1% of the bases are labeled, then after 3000 bases the
attrition is 95% if
incorporation of a labeled nucleotide terminates strand extension completely.
In other word,
if one starts with 105 molecules, then on the first base one expects to get a
fluorescent signal
ys
CA 02388528 2002-04-22
WO 01/32930 PCT/US00/30591
from 100 dye molecules. By the 3000th base, the signal is reduced to only 5
dye molecules.
This is still detectable, since the lower limit of detection is one dye
molecule.
It should also be noted that the attrition are discussed above is an extreme
scenario because there is little reason to expect total attrition for each
incorporated base.
Attrition is more likely to occur when the polymerise incorporates two
successive labeled
nucleotides. If 1 % of the bases are labeled, the chance of incorporating two
labeled
nucleotides next to each other is 1 °/ 2=0.01 %. Then the attrition
rate after 3000 bases is 25%.
In other words, the signal only decreases by 25% by the 3000th base. Thus,
attrition does not
cause a problem in this sequencing scheme.
Another factor that can affect read length is misincorporation. If the DNA
polymerise is starved for the proper nucleotide, it can incorporate the wrong
nucleotide.
Misincorporation efficiencies have been measured to be three to five orders of
magnitude
below the efficiency for proper nucleotide incorporation (Echols et al., Ann.
Rev. Biochem
60:477, 1991). Misincorporation can be minimized by only exposing the DNA
polymerase-
DNA complexes to nucleotides for as much time as is needed to incorporate the
proper
nucleotide. For a high fidelity DNA polymerise, misincorporation happens with
a frequency
of about 10~. If dephasing due to misincorporation is treated as total
attrition, the attrition is
only 25% after 3 kb, i.e, the signal is reduced to 75% of its original. Thus,
misincorporation
does not hinder a 3 kb or perhaps longer read length.
Many modifications and variations of this invention can be made without
departing from its spirit and scope. The specific embodiments described herein
are for
illustration only and are not intended to limit the invention in any way.
All publications, figures, patents and patent applications cited herein are
hereby expressly incorporated by reference for all purposes to the same extent
as if each was
so individually denoted.
'~ 6
