U.S. patent application number 12/844544 was filed with the patent office on 2011-01-27 for microfabricated integrated dna analysis system.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Robert BLAZEJ, Palani KUMARESAN, Chung LIU, Richard A. MATHIES, Stephanie H. I. YEUNG.
Application Number | 20110020920 12/844544 |
Document ID | / |
Family ID | 35463468 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110020920 |
Kind Code |
A1 |
MATHIES; Richard A. ; et
al. |
January 27, 2011 |
MICROFABRICATED INTEGRATED DNA ANALYSIS SYSTEM
Abstract
Methods and apparatus for genome analysis are provided. A
microfabricated structure including a microfluidic distribution
channel is configured to distribute microreactor elements having
copies of a sequencing template into a plurality of microfabricated
thermal cycling chambers. A microreactor element may include a
microcarrier element carrying the multiple copies of the sequencing
template. The microcarrier element may comprise a microsphere. An
autovalve at an exit port of a thermal cycling chamber, an optical
scanner, or a timing arrangement may be used to ensure that only
one microsphere will flow into one thermal cycling chamber wherein
thermal cycling extension fragments are produced. The extension
products are captured, purified, and concentrated in an integrated
oligonucleotide gel capture chamber. A microfabricated component
separation apparatus is used to analyze the purified extension
fragments. The microfabricated structure may be used in a process
for performing sequencing and other genetic analysis of DNA or
RNA.
Inventors: |
MATHIES; Richard A.; (San
Francisco, CA) ; BLAZEJ; Robert; (Berkeley, CA)
; LIU; Chung; (Albany, CA) ; KUMARESAN;
Palani; (Los Angeles, CA) ; YEUNG; Stephanie H.
I.; (Berkeley, CA) |
Correspondence
Address: |
Weaver Austin Villeneuve & Sampson LLP
P.O. BOX 70250
OAKLAND
CA
94612-0250
US
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
35463468 |
Appl. No.: |
12/844544 |
Filed: |
July 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11139018 |
May 25, 2005 |
7799553 |
|
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12844544 |
|
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60576102 |
Jun 1, 2004 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
C12Q 1/6869 20130101;
B01L 7/52 20130101; B01L 2300/0887 20130101; B01L 2200/147
20130101; B01L 2300/1827 20130101; B01L 2300/0803 20130101; B01L
3/50273 20130101; B01L 2300/0864 20130101; B01L 2300/0874 20130101;
B01L 2200/10 20130101; B01L 2400/0415 20130101; C12Q 1/686
20130101; B01L 2400/0633 20130101; B01L 3/502753 20130101; B01L
2300/087 20130101; B01L 2400/0421 20130101; C12Q 1/686 20130101;
C12Q 2565/518 20130101; C12Q 2537/143 20130101; C12Q 1/6869
20130101; C12Q 2565/629 20130101; C12Q 2565/518 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Goverment Interests
STATEMENT REGARDING GOVERNMENTAL SUPPORT
[0002] The invention was made with government support under Grant
Numbers AI056472, CA77664, and HG001399 awarded by the National
Institute of Health. The government has certain rights in this
invention.
Claims
1. A microfabricated structure comprising: a distribution channel
to distribute microreactor elements carrying multiple copies of a
clonal sequencing template into a plurality of thermal cycling
chambers such that only one microreactor element will pass into one
thermal cycling chamber wherein thermal cycling extension fragments
are produced from a microreactor element; purification chambers
connected to the thermal cycling chambers to capture and
concentrate the extension fragments; and component separation
channels connected to the purification chambers to analyze the
extension fragments.
2. The microfabricated structure of claim 1 wherein the
microreactor element includes a microcarrier element that carries
the multiple copies of the clonal sequencing template.
3. The microfabricated structure of claim 1 wherein the
microreactor element is a bolus or a microemulsion droplet.
4. The microfabricated structure of claim 3 wherein the
microreactor element includes a microsphere carrying the multiple
copies of the clonal sequencing template.
5. The microfabricated structure of claim 1 wherein the sequencing
template is a DNA or RNA sequencing template.
6. A system for performing sequencing comprising: means for
shearing DNA or RNA into fragments; means for ligating the
fragments to form a mixture of desired circular and contaminating
linear products; means for selectively removing the contaminating
linear products; means for generating microreactor elements
carrying multiple clonal copies of a single sequencing template;
means for selecting which microreactor elements have a sequencing
template; microfluidic distribution means for distributing a
selected microreactor element with a sequencing template into a
thermal cycling chamber; means for ensuring that only one
microreactor element will flow into one thermal cycling chamber;
extension means, including the thermal cycling chambers, for
producing thermal cycling extension fragments from the microreactor
elements carrying multiple copies of the sequencing template;
purification chamber means for capturing, purifying and
concentrating the extension fragments; and component separation
means for analyzing the extension fragments.
7. The system of claim 6 wherein the microreactor element includes
a microcarrier element that carries the multiple copies of the
clonal sequencing template.
8. The system of claim 6 wherein the microreactor element is a
bolus or a microemulsion droplet.
9. The system of claim 8 wherein the microreactor element includes
a microsphere carrying the multiple copies of the clonal sequencing
template.
10. A microfabricated structure comprising: a distribution channel
to distribute microspheres carrying multiple copies of a clonal
sequencing template into a plurality of thermal cycling chambers;
an autovalve at an exit port of a thermal cycling chamber to ensure
that only one microsphere will flow into one thermal cycling
chamber wherein thermal cycling extension fragments are produced
from a microsphere; purification chambers connected to the thermal
cycling chambers to capture and concentrate the extension
fragments; and component separation channels connected to the
purification chambers to analyze the extension fragments.
11. The microfabricated structure of claim 10 wherein the diameter
of a microsphere is between about 1 and 100 microns.
12. The microfabricated structure of claim 11 wherein the diameter
of a microsphere is about 10 microns.
13. The microfabricated structure of claim 10 wherein each thermal
cycling chamber is in fluid communication with a separation
channel.
14. The microfabricated structure of claim 10 wherein the
sequencing template is a DNA or RNA sequencing template.
15-44. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. application Ser. No. 11/139,018, filed May 25, 2005 (Atty.
Docket No. UCALP054), titled "MICROFABRICATED INTEGRATED DNA
ANALYSIS SYSTEM," which claims the benefit under 35 U.S.C. 119(e)
from Provisional U.S. Patent Application No. 60/576,102, filed Jun.
1, 2004 (Atty. Docket No. UCALP054P), entitled "MICROBEAD
INTEGRATED DNA ANALYSIS SYSTEM (MINDS)," all of which are
incorporated herein by reference for all purposes.
BACKGROUND
[0003] 1. Field of Invention
[0004] The present invention relates to microfabricated and
microfluidic structures. In one example, the present invention
relates to a microfabricated system and method for genome
sequencing.
[0005] 2. Description of Related Art
[0006] The genome of an organism is defined by the DNA
(deoxyribonucleic acid) or, for some viruses, the RNA (ribonucleic
acid) of the organism. Genome sequencing is figuring out the order
of the nucleotides, or bases, of a DNA or RNA strand.
[0007] A current approach to genome-scale sequencing of DNA is
shown in FIG. 1. This shotgun sequencing approach 100 uses
bacterial transformation, selection and growth to manipulate
individual genomic DNA fragments. The first three steps of this
approach, shearing, vector ligation, and transformation (steps 102,
104, and 106), need only be performed once. As such, they do not
present any particular problems. The last step, the capillary
electrophoresis (CE) (step 114), has been miniaturized through
microfabrication. This has reduced the cost and processing time
associated with this step. The plate and grow (step 108), and the
pick, grow and extract steps (step 110), however, are problematic.
These steps, which precede the Sanger extension step (step 112),
perform clone isolation and insert amplification. The amplification
may be bacterial, PCR (polymerase chain reaction) or RCA (rolling
circle amplification). These steps have remained refractory to
miniaturization and integration. The current macroscopic paradigm
has thus relied upon the use of robotics as the enabling technology
for these key steps. However, this is a problem because no less
than 30 million colonies must be picked and grown to produce a
sequencing template for a genome. Furthermore, the minimum
quantities of materials prepared by such robotic technologies are
orders of magnitude more than that required by modern
microfabricated CE analysis systems.
[0008] Therefore, it is desirable to improve the processing time,
volume scale, and level of integration of genome sequencing. It is
also desirable to reduce the cost and space requirements of genome
sequencing.
SUMMARY
[0009] In one aspect, the invention features a microfabricated
structure including a distribution channel to distribute
microreactor elements carrying multiple copies of a clonal
sequencing template into a plurality of thermal cycling chambers.
Only one microreactor element is passed into one thermal cycling
chamber wherein thermal cycling extension fragments are produced
from a microreactor element. Purification chambers are connected to
the thermal cycling chambers to capture and concentrate the
extension fragments. Component separation channels are connected to
the purification chambers to analyze the extension fragments.
[0010] Various implementations of the invention may include one or
more of the following features. The microreactor includes a
microcarrier element that carries the multiple copies of the clonal
sequencing template. The microreactor element is a bolus or
microemulsion droplet. The microreactor element includes a
microsphere carrying the multiple copies of the clonal sequencing
template. The sequencing template is a DNA or RNA sequencing
template.
[0011] In yet another aspect, the invention is directed to a system
for performing sequencing. The system includes a means for shearing
DNA or RNA into fragments and means for ligating the fragments to
form a mixture of desired circular and contaminating linear
products. The system further includes means for selectively
removing the contaminating linear products and means for generating
microreactor elements carrying multiple clonal copies of a single
sequencing template. The system also includes means for selecting
which microreactor elements have a sequencing template and
microfluidic distribution means for distributing a selected
microreactor element with a sequencing template into a thermal
cycling chamber. Additionally, the system includes means for
ensuring that only one microreactor element will flow into one
thermal cycling chamber and extension means, including the thermal
cycling chambers, for producing thermal cycling extension fragments
from the microreactor elements carrying multiple copies of the
sequencing template. Purification chamber means for capturing,
purifying and concentrating the extension fragments, and component
separation means for analyzing the extension fragments are also
part of the system.
[0012] Other implementations of the invention may include one or
more of the following features. The microreactor element includes a
microcarrier element that carries the multiple copies of the clonal
sequencing template. The microreactor element is a bolus or a
microemulsion droplet. The microreactor element includes a
microsphere carrying the multiple copies of the sequencing
template.
[0013] In another aspect, the invention features a microfabricated
structure including a distribution channel to distribute
microspheres carrying multiple copies of a clonal sequencing
template into a plurality of thermal cycling chambers. An autovalve
is located at an exit port of a thermal cycling chamber to ensure
that only one microsphere will flow into one thermal cycling
chamber wherein thermal cycling extension fragments are produced
from the microsphere. Purification chambers are connected to the
thermal cycling chambers to capture and concentrate the extension
fragments. Component separation channels are connected to the
purification chambers to analyze the extension fragments.
[0014] Various implementations of the invention may include one or
more of the following features. The diameter of a microsphere is
between about 1 and 100 microns. The diameter of a microsphere is
about 10 microns. Each thermal cycling chamber is in fluid
communication with a separation channel. The sequencing template is
a DNA or RNA sequencing template.
[0015] In still another aspect, the invention is directed to a
microfabricated structure including a microfluidic distribution
channel means for distributing a microsphere carrying a sequencing
template into a thermal cycling chamber. Autovalving means are used
to ensure that only one microsphere will flow into one thermal
cycling chamber. Extension means, including the thermal cycling
chamber, produce thermal cycling extension fragments from a
microsphere carrying a sequencing template. Integrated purification
chamber means are used for capturing, purifying and concentrating
the extension fragments. Component separation means are used to
analyze the extension fragments.
[0016] Other implementations of the invention may include one or
more of the following features. The extension means is a Sanger
extension means including a plurality of thermal cycling chambers.
The autovalving means includes an autovalve at an exit port of the
thermal cycling chambers. Purification chamber means includes
purification chambers connected to the thermal cycling chambers.
The component separation means is a capillary array electrophoresis
means including a plurality of microchannels connected to the
purification chambers. The sequencing template is a DNA or RNA
sequencing template.
[0017] In a further aspect, the invention is directed to a
microfabricated apparatus including a thermal cycling chamber. The
thermal cycling chamber is configured to receive a microsphere
carrying a clonal template. The chamber has an inlet port and an
outlet port wherein the outlet port includes a constriction that is
configured to trap a microsphere in the chamber and to
substantially block further flow into the thermal cycling
chamber.
[0018] Various implementations of the invention may include one or
more of the following features. The shape of the constriction is
substantially circular or semicircular. A first value is located in
an inlet channel in fluid communication with the inlet port and a
second valve is located in an outlet channel in fluid communication
with the outlet port. In operation, the second valve is closed
before the first valve to move a microsphere out of the
constriction and into a main body portion of the chamber before
thermal cycling. A purification chamber is in fluid communication
with the outlet port of the thermal cycling chamber and an outlet
port of the purification chamber is in fluid communication with a
component separation apparatus.
[0019] In yet another aspect, the invention features a system for
performing sequencing. The system includes means for shearing DNA
or RNA into fragments and means for ligating the fragments to form
a mixture of desired circular and contaminating linear products.
The system further includes means for selectively removing the
contaminating linear products and means for generating microspheres
carrying multiple clonal copies of a single sequencing template.
The system also includes means for selecting which microspheres
have a sequencing template and microfluidic distribution channel
means for distributing a selected microsphere with a sequencing
template into a thermal cycling chamber. Additionally, the system
includes means for ensuring that statistically only one microsphere
will flow into one thermal cycling chamber. The system also
includes extension means, including the thermal cycling chambers,
for producing thermal cycling extension fragments from the
microspheres carrying multiple copies of the sequencing template.
Purification chamber means for capturing, purifying and
concentrating the extension fragments, and component separation
means for analyzing the extension fragments are also part of the
system.
[0020] Other implementations of the invention may include one or
more of the following features. The ensuring means is at least one
of an autovalve in the thermal cycling chamber, an optical
detector, and a timing mechanism. The optical detector is an
optical scanner that detects light scattered from a microsphere.
The timing mechanism includes a pneumatic input located adjacent to
an inlet of a thermal cycling chamber.
[0021] In a further aspect, the invention features a system for
performing DNA sequencing. The system includes means for shearing
DNA into DNA fragments and means for ligating the DNA fragments to
form a mixture of desired circular and contaminating linear
products. The system also includes means for exonuclease
degredation for selectively removing the contaminating linear
products and emulsion PCR reaction means for generating
microspheres carrying multiple clonal copies of a single DNA
sequencing template. Additionally, the system includes fluorescent
activated cell sorting (FACS) means for selecting which
microspheres have a DNA sequencing template. Microfluidic
distribution channel means are used to distribute a selected
microsphere with a DNA sequencing template into a thermal cycling
chamber. Autovalving means are used to ensure that statistically
only one microsphere will flow into one thermal cycling chamber.
Sanger extension means, including the thermal cycling chambers, are
used to produce thermal cycling extension fragments from the
microspheres carrying multiple copies of the DNA sequencing
template. Integrated purification chamber means are used to
capture, purify and concentrate the extension fragments, and
capillary array electrophoresis means are used to analyze the
extension fragments.
[0022] In still another aspect, the invention is directed to a
process for performing sequencing. The process includes shearing
DNA or RNA into fragments, ligating the fragments to form a mixture
of desired circular and contaminating linear products, and
selectively removing the contaminated linear products. The process
further includes generating microreactor elements carrying multiple
clonal copies of a sequencing template, selecting which
microreactor elements have a sequencing template, and distributing
the microreactor elements with the sequencing template into thermal
cycling chambers. Thermal cycling extension fragments are produced
from the microreactor elements carrying the multiple copies of the
sequencing template. The extension fragments are captured,
concentrated, and analyzed.
[0023] Various implementations of the invention may include one or
more of the following features. The microreactor element includes a
microcarrier element which carries the multiple copies of the
clonal sequencing template. The microreactor element is a bolus or
a microemulsion droplet. The microreactor element includes a
microsphere carrying the multiple copies of the clonal sequencing
template. The distributing step is done such that only one
microreactor element will pass into one thermal cycling chamber. An
autovalve is used at an exit port of the thermal cycling chambers
to ensure that only one microreactor element will flow into one
thermal cycling chamber. The generating step includes generating
multiple clonal copies of a sequencing template by emulsion PCR
reactions or by a flow through PCR process. The selecting step is
FACS.
[0024] In yet another aspect, the invention features a process for
performing DNA sequencing. The process includes shearing DNA into
DNA fragments, ligating the DNA fragments to form a mixture of
desired circular and contaminating linear products, and selectively
removing the contaminating linear products by exonuclease
degredation. Microspheres carrying multiple clonal copies of a DNA
sequencing template are generated by emulsion PCR reactions. The
microspheres that have a DNA sequencing template are detected by
FACS. The microspheres with the DNA sequencing template are
distributed into thermal cycling chambers. An autovalve at an exit
port of a thermal cycling chamber is used to ensure that
statistically only one microsphere will flow into one thermal
cycling chamber. Thermal cycling extension fragments are produced
from the microspheres carrying multiple copies of the DNA
sequencing template. The extension fragments are captured,
purified, concentrated and analyzed.
[0025] Various implementations of the invention may include one or
more of the following features. The capturing step uses an
oligonucleotide capture matrix.
[0026] In still another aspect, the invention features a method for
sequencing. The method includes receiving a microsphere carrying a
clonal template at an inlet port of a thermal cycling chamber and
using a constriction at an outlet port of the chamber to trap the
microsphere in the chamber and substantially block further flow
into the chamber.
[0027] In a further aspect, the invention features a method for
analysis including producing a microsphere carrying a sequencing
template. The microsphere is located in a thermal cycling chamber
by use of a constriction at an outlet port in the chamber such that
further flow into the chamber is substantially blocked.
[0028] Other implementations of the invention may include one or
more of the following features. A first valve is located at an
inlet port of the thermal cycling chamber and a second valve is
located at an outlet port of the thermal cycling chamber such that
the second valve may be closed before the first valve. As such, a
microsphere is moved out of the constriction and into a main body
portion of the thermal cycling chamber before thermal cycling.
[0029] The invention can include one or more of the following
advantages. The laborious bacterial manipulations required by
shotgun genome sequencing are replaced with easily automated and
integrated in vitro steps. As such, millions of manual or robotic
colony picking operations are eliminated. All fluidic and
temperature control structures necessary to produce, purify, and
separate sequencing extension fragments are integrated on a
microfabricated device. Significant cost, time, and space savings
can be achieved. Other genetic analysis techniques can be
performed.
[0030] These and other features and advantages of the present
invention will be presented in more detail in the following
specification of the invention and the accompanying figures, which
illustrate, by way of example, the principles of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0031] The invention may best be understood by reference to the
following description taken in conjunction with the accompanying
drawings that illustrate specific embodiments of the present
invention.
[0032] FIG. 1 is a diagrammatic representation of the steps
involved in a conventional genome-scale sequencing operation.
[0033] FIG. 2 is a diagrammatic representation of the steps
involved in a genome sequencing process in accordance with the
present invention.
[0034] FIG. 3 is a diagrammatic representation of a cloning process
that may be used with the present invention.
[0035] FIG. 4A is a diagrammatic representation of a single-channel
microdevice in accordance with the present invention, FIG. 4B is an
enlarged view of a portion of the device of FIG. 4A, and FIG. 4C is
a diagrammatic exploded view of the single-channel microdevice.
[0036] FIGS. 5A, 5B, and 5C are fluorescent images of a device in
accordance with the present invention during capture, wash, and
sample injection, respectively, of extension fragments.
[0037] FIG. 6A is a diagrammatic representation of a sequencing
reaction chamber of a device in accordance with the present
invention, including an enlarged view of a portion of the reaction
chamber; FIG. 6B is a view along line 6B-6B of FIG. 6A; and FIG. 6C
is a diagrammatic representation of a constriction region of the
reaction chamber of FIG. 6A that is used to trap beads carrying the
DNA to be sequenced.
[0038] FIG. 7A is a bright field image of a constriction region of
an empty sequencing reaction chamber, FIG. 7B is a bright field
image of a constriction region of a sequencing reaction chamber
filled with solution but with no microsphere at the constriction
region, and FIG. 7C is a dark field image of a microsphere located
at a constriction region of a sequencing reaction chamber.
[0039] FIG. 8A is a diagrammatic representation of an alternate
embodiment of a constriction region of a sequencing reaction
chamber designed for bead capture, and FIG. 8B is a view along line
8B-8B of FIG. 8A.
[0040] FIG. 9A is a diagrammatic representation of one quadrant of
an integrated sequencing array system, and FIG. 9B is an enlarged
view of a portion of the system of FIG. 9A.
[0041] FIG. 10A is a diagrammatic representation of a continuous
flow through PCR microfabricated device; FIG. 10B is an enlarged
view at lines 10B-10B of FIG. 10A, illustrating a "T"-injector
region of the microfabricated device; and FIG. 10C is an enlarged
view at lines 10C-10C of FIG. 10A, illustrating a portion of the
channels of the microfabricated device.
DETAILED DESCRIPTION
[0042] Reference will now be made in detail to some specific
embodiments of the present invention including the best modes
contemplated by the inventor for carrying out the invention.
Examples of these specific embodiments are illustrated in the
accompanying drawings. While the invention is described in
conjunction with these specific embodiments, it will be understood
that it is not intended to limit the invention to the described
embodiments. On the contrary, it is intended to cover alternatives,
modifications, and equivalents as may be included within the spirit
and scope of the invention as defined by the appended claims.
[0043] In the following description, numerous specific details are
set forth in order to provide a thorough understanding of the
present invention. The present invention may be practiced without
some or all of these specific details. In other instances, well
known process operations have not been described in detail in order
not to unnecessarily obscure the present invention.
[0044] Furthermore, techniques and mechanisms of the present
invention will sometimes be described in singular form for clarity.
However, it should be noted that some embodiments can include
multiple iterations of a technique or multiple applications of a
mechanism unless noted otherwise.
[0045] The system and method of the present invention will be
described in connection with DNA sequencing. However, the system
and method may also be used for RNA sequencing. Additionally, the
system and method may be used for other genetic analysis of DNA or
RNA.
[0046] The system and method of the present invention uses
microreactor elements such as a microemulsion droplet or a bolus
which, in some embodiments, include a microcarrier element such as
a microsphere or bead. The microcarrier element, contained within a
bolus or a microemulsion droplet, is useful in capturing DNA or RNA
products; that is, amplified DNA or RNA can be chemically linked to
the microcarrier element. Alternatively, the amplified DNA or RNA
may be carried by a microreactor element without the use of a
microcarrier element. For instance, a PCR reaction may be done in a
bolus or a microemulsion droplet, and then the resulting bolui or
microemulsion droplets may be routed to the next step of the
process.
[0047] A microfabricated integrated DNA analysis system (MINDS) of
the present invention provides a system and method that are readily
compatible with microfluidic sample component separation devices.
As shown by FIG. 2, the MINDS process 200, in one embodiment,
begins with the shearing of DNA into DNA fragments (step 202). The
fragments are then ligated to form a mixture of desired circular
and contaminating linear products (step 204). The contaminating
linear products are then selectively removed, for instance, by
exonuclease degradation (step 206). A colony of microspheres
carrying multiple clonal copies of a DNA sequencing template is
next formed (step 208). The colony may be generated, for example,
by emulsion PCR reactions. The microspheres having a DNA sequencing
template are then identified (step 210). The microspheres may be
identified by a fluorescence activated cell sorting (FACS)
technique. Only one such run is required, and it may take 6 hours
or less to complete. The microspheres with the DNA sequencing
templates are then distributed into thermal cycling or sequencing
reaction chambers where extension fragments are produced;
thereafter, the fragments are purified and concentrated (step 212).
The extension fragment are then analyzed by a sample component
separation apparatus, for example, a CE device (step 214).
[0048] The MINDS method, process, and apparatus, in one embodiment,
comprises a microfabricated structure on which thermal cycling,
affinity capture, sample purification, and capillary array
electrophoresis (CAE) components are integrated. As will be
discussed, such a system includes a microfluidic distribution
channel to distribute microspheres carrying multiple copies of a
sequencing template into a plurality of thermal cycling or
sequencing reaction chambers. An autovalve may be located at an
exit port of the thermal cycling chambers to ensure that only one
microsphere will flow into each chamber wherein thermal cycling
extension fragments are produced from the microsphere. Purification
chambers are connected to the thermal cycling chambers to capture,
purify, and concentrate the extension fragments. Microfabricated CE
separation channels are connected to the purification chambers to
analyze the extension fragments.
[0049] The present invention eliminates the laborious, expensive,
and time consuming in vivo cloning, selection, colony isolation,
and amplification steps of conventional sequencing. Instead, these
steps are replaced with readily miniaturized and automated in vitro
steps.
[0050] Microspheres are ideal carriers, providing flexible control
over size, surface, fluorescent, and magnetic properties.
Miniaturization of a sequencing reaction chamber through
microfabrication and the concomitant reduction in reagent volume
makes possible the use of a single, clonal microsphere as a carrier
for sufficient DNA sequencing template. This enables the use of a
matched process flow that permits selection, amplification and
sorting of clonal templates for direct integration with a nanoliter
extension, clean-up and sequencing process.
[0051] FIG. 3 provides an overview of a cloning process 300 that
may be used with the MINDS. The process includes library creation
and selection. In one embodiment, genomic DNA is isolated from
whole blood and then sheared in a nebulizer generating DNA
fragments (steps 302 and 304). The DNA fragments are then subjected
to an enzymatic treatment to yield blunt-end DNA (step 306).
Processed fragments are ligated into a vector yielding a mixture of
desired circular and contaminating linear products (steps 308 and
310). Exonuclease degredation then selectively removes the insert
and vector, the contaminating linear products, from the ligation
product so that greater than about 95% of the remaining vectors
contain inserts, the desired circular products (step 312).
[0052] The first five steps of the cloning process 300 (steps
302-310) follow standard library creation procedures. More
specifically, Invitrogen (Carlsbad, Calif.) supplies a TOPO
Subcloning Kit (#K7000-01) which is capable of generating a 1 to 6
Kb insert genomic shotgun library in two hours. The subcloning kit
requires about 20 micrograms (.mu.g) of genomic DNA that can be
obtained from 1 mL of whole blood using Qiagen's (Oslo, Norway)
Blood & Cell Culture DNA Mini Kit (#13323). The DNA is sheared
in a nebulizer generating 1-6 Kb DNA fragments. The fragments are
then subjected to an enzymatic treatment with T4 DNA and Klenow
polymerases and calf intestinal phosphatase (CIP) to yield
dephosphorylated blunt-end DNA. Processed fragments are next
ligated into Invitrogen's pCR 4 Blunt-TOPO vector. Use of
covalently linked vaccinia virus topoisomerase I to the cloning
vector results in rapid and efficient ligation (a 5 minute room
temperature ligation yields greater than 95% transformants) and
permits dephosphorylation of the inserts prior to ligation thereby
preventing chimeric clone formation.
[0053] The final step in canonical library creation (step 312),
which has been modified to be compatible with the in vitro MINDS
process, is bacterial transformation and antibiotic selection.
Exonuclease degredation selectively removes insert and vector from
the desired ligation product. Lambda exonuclease (New England
BioLabs #M0262S) can be used for this step as it degrades
5'-phosphorylated and non-phosphorylated double-stranded DNA but is
unable to initiate DNA digestion at the nicks present in the vector
after ligation. Assuming a pessimistic 1% recovery of the starting
genomic DNA, the resulting library contains greater than 6,000 fold
molecular excess needed for 10.times. sequence coverage, though
this excess is reduced through dilution in the single-molecule PCR
amplification step described later.
[0054] A gel separation and band purification step can be performed
after DNA shearing (step 304) if strict control over insert size is
required for paired-end whole-genome de novo sequencing.
Alternatively, insert size can be restricted using limiting
extension times during PCR amplification or flow cytometric
selection of microspheres within a narrow fluorescence intensity
range. Labeling microsphere-clones with an intercalating dye, such
as thiazole orange (TO), will yield a fluorescent signal
proportional to amplicon size. Since flow cytometry is a step in
the MINDS process, this is an appealing size-selection method that
also eliminates the >5% recircularized vectors with the only
drawback being reduced yield.
[0055] The vector used in the subcloning kit may be optimized for
sequencing with the T7 and T3 priming sites 33 bp (base pairs) away
from the insertion site. Since the MINDS vector is free from
biological constraints, the sequencing priming sites can be moved
closer to the insert site and the pUC ori, fl ori, lacZ, and
antibiotic resistance genes can be removed. Optimized acrydite
capture sequences and homo-PCR priming sites can be inserted for
improved sample clean-up and reduced primer-dimer formation.
[0056] Sequencing in the MINDS process may use clonal template DNA
attached to individual microspheres or beads. The clonal attachment
is accomplished by linking one of the primers covalently to a
microsphere.
[0057] Clonal isolation of 1,000 to 100,000 DNA molecules through
single-molecule amplification (for example microemulsion polonies)
or combinatorial hybridization approaches has been demonstrated.
See, Mitra, R. D., et al., "Digital genotyping and haplotyping with
polymerase colonies", Proceedings of the National Academy of
Sciences of the United States of America, 2003, 100(10): p.
5926-5931; Dressman, D., et al., "Transforming single DNA molecules
into fluorescent magnetic particles for detection and enumeration
of genetic variations", Proceedings of the National Academy of
Sciences of the United States of America, 2003, 100(15): p.
8817-8822; and Brenner, S., et al., "Gene expression and analysis
by massively parallel signature sequencing (MPSS) on microbead
arrays", Nature Biotechnology, 2000, 18(6): p. 630-634; each of
which is hereby incorporated by reference. Conventional microchip
sequencing requires approximately 1 billion template DNA molecules
per reaction. Twenty billion extension fragments have been
generated after 20.times. linear Sanger-sequencing amplification,
representing a 20-fold excess for high signal-to-noise (S/N)
detection using a confocal radial scanner (1 million fragments per
band X 1,000 bands=1 billion molecules). This excess is necessary
because a cross-injection process requires streaming extension
fragments across the separation column until an equimolar mixture
is obtained at the intersection and then injecting about 5% of the
total sample. An affinity capture direct-injection strategy
discussed below eliminates the 20-fold excess requiring only 50
million initial template copies.
[0058] CPG Biotech (Lincoln Park, N.J.) produces a 5 micron (.mu.m)
controlled pore glass paramagnetic microsphere with a long chain
alkylamine linker for covalent attachment to an amine modified
primer that can consistently bind 80 million DNA molecules.
Starting from a single DNA molecule, 30 cycles of PCR has a
theoretical gain of 1 billion. In practice, the gain is lower due
to less than 100% efficiency for each cycle, reduced enzyme
activity in later cycles, and limiting reagents in pL scale
reactions. To generate clonal microspheres, a single DNA molecule
and a single primer-coupled microsphere must be placed in a PCR
reactor. Efficient single-molecule PCR requires extremely small
volume reactors (1-10 pL) to increase the effective concentration
of a single DNA molecule. Approximately 30 million microsphere
clones are needed to sequence a human-size genome. High-throughput
combination of a single microsphere and DNA molecule in a chamber
is possible using statistically dilute microemulsion solutions. If
the DNA molecules and microspheres are each diluted such that one
species is present in 10 times the reactor volume, there is a high
probability (greater than 99%) of concurrence in 1% of the
reactors. Because 99% of the reactors are non-productive, 100-fold
more reactions (3 billion) are required to generate 30 million
microsphere clones.
[0059] Two possible approaches for generating large numbers of
small volume reactions are microfabricated PCR devices and emulsion
PCR. PCR devices, however, require thousands of runs to achieve the
required 3 billion reactions. Emulsion PCR, on the other hand, has
the ability to thermally cycle millions to billions of separate
compartments in a single tube using a conventional thermal cycler.
Clonal PCR amplicons attached to magnetic particles using emulsion
PCR have been produced. On average, each microsphere contained
greater than 10,000 250 bp amplicons, a value below the theoretical
maximum of 600,000 amplicons based on the available nucleoside
triphosphates in each 5 .mu.diameter compartment. Fifteen micron
microemulsion PCR compartments have been demonstrated in which the
maximum number of 1,000 bp amplicons is 5 million--a value ten
times less than the minimum required 50 million initial template
copies. This problem may be solved in a variety of ways:
[0060] First, additional microemulsion PCR steps could be performed
to increase the amount of DNA linked to the beads. Second, an
additional non-reagent-limited amplification step may be necessary
after the fluorescence flow cytometry step. Secondary PCR
amplification can be performed in an on-chip, dual-use thermal
cycling chamber in which paramagnetic microspheres are routed to
individual reactors and magnetically retained. Fresh PCR mix is
passed into the chambers and thermal cycled 15.times. to saturate
each microsphere to the maximum of 80 million templates. In this
case, the magnetic field is maintained as Sanger-sequencing
reaction mix is washed into the chambers followed by solid-phase
sequencing. Finally, the S/N of the scanner may be improved up to
10-fold enabling sequencing of about a 10-fold lower template than
calculated above.
[0061] Approximately 15 million 15 .mu.m diameter compartments may
be created in a 25 .mu.L aqueous phase, 75 .mu.L oil phase
emulsion. Three billion compartments are required to generate 30
million coincident single DNA molecule and microsphere events.
Thus, approximately two 100 .mu.L 96-well plate reactions are
required. When the reactions are complete, 30 million microsphere
clones must be separated from a background of 300 million
un-labeled microspheres.
[0062] As noted, the beads having sequencing templates may be
identified by FACS. A system that may be used is the BD FACS
ArrayBioanalyzer System, available from BD Biosciences, San Jose,
Calif. The BD FACSArray flow cytometer can process up to 15,000
events per second, enabling the isolation of all clones needed for
10.times. coverage of a 3 billion base genome in less than 6 hours.
The beads will be treated with an intercalation dye, such as TO,
that is nonfluorescent until intercalated into double-stranded DNA.
The fluorescence intensity of TO is linearly proportional to the
amount of DNA allowing for easy differentiation between beads that
have amplified DNA and those that do not.
[0063] A diagrammatic representation of a single-channel
microdevice 400 in accordance with the present invention is shown
in FIGS. 4A, 4B, and 4C. The device may be fabricated as a four
layer glass-glass-PDMS (polydimethysiloxane)-glass sandwich that
incorporates microfluidic valves, heaters, resistive temperature
detectors (RTDs), and all reaction, capture and clean-up, and CE
structures. The device, in another configuration, may be fabricated
as a glass-PDMS-glass-glass stack. A four layer microfabricated
system including valves, heaters, RTDs, chambers, and CE structures
is described in U.S. patent application Ser. No. 10/750,533, filed
Dec. 29, 2003, entitled "Fluid Control Structures In Microfluidic
Devices", assigned to the Assignee of the subject application, and
which is hereby incorporated by reference.
[0064] The microdevice 400 is capable of performing all down-stream
steps in the MINDS process including extension fragment production,
reaction clean-up, and extension fragment separation (steps 212 and
214 of FIG. 2). The device 400 includes a thermal cycling or
sequencing reaction chamber 402, a capture or purification chamber
404, and a CE system 406 including separation channels 407. As
shown, these components of the device 400 are connected by various
valves and channels. A heater (not shown), for example, a kapton
heater, may be used to heat the contents of the thermal cycling
chamber. The template of the chamber is monitored by RTDs 405.
[0065] As shown in FIG. 4C, the device 400 includes three glass
layers, including a channel layer 430, a via layer 432, and a
manifold layer 434. A PDMS membrane layer 436 is provided between
the via layer 432 and the manifold layer 434. The top layer 430
contains the thermal cycling reactors, the capture chambers and the
CE features. The second layer 432 incorporates the RTDs on the top
surface of the glass wafer and etched features on the bottom to
form the valves and pumps with the membrane layer 436 below. The
last layer 434 includes the heater, and completes the valve and
pump structures with pneumatic actuation lines and displacement
chambers.
[0066] In one method of operation, a sequencing master mix is
loaded from a port 408 of the device 400 through a microvalve 410
into the thermal cycling chamber 402. The volume of the chamber 402
may be approximately 250 nano-liters (nL). The separation channels
of the CE system 406 are filled with linear polyacrylimide from a
port 412 to a port 414. The CE system may be a 16-centimeter (cm)
hyperturn system. An acrydite capture matrix is loaded from a port
416 to a port 418 to fill the capture chamber 404 and an
intervening pinched chamber 419. After thermal cycling the chamber
402, extension fragments and residual reactants are
electrokinetically driven through a valve 420 and a channel 422
into the capture chamber 404. The captured extension fragments are
then electrophoretically washed and injected into a channel 424 for
separation by the CE system 406.
[0067] An advantage of this affinity capture, sample clean-up
microdevice is that purified extension fragments may be retained in
1-10 nL at a concentration determined by the quantity of the
acrydite monomer added during synthesis. FIGS. 5A, 5B, and 5C
display fluorescence images (where relative applied potentials are
indicated by "+" and "-") from the operation of a microdevice 500
like that of the device 400 where thermal cycling, capture, wash
(purification), and concentration of a sample as well as separation
were performed. Specifically, an acrydite capture matrix was
synthesized in a 2-mL solution of 5% w/v acrylamide, 1.times.TTE
(50 mM Tris, 50 mM TAPS free acid, 1 mM EDTA, pH=8.4), and 20 nmol
of the methacrylate-modified oligo
(5'-Acrydite-ACTGGCCGTCGTTTTACAA-3' (SEQ. ID NO. 1), TM=60.4 C,
Operon Technologies, Emeryville, Calif.). The solution was sparged
with argon for 2 hours prior to adding 0.015% w/v APS and TEMED to
initiate polymerization. The polymerized capture matrix was then
loaded into a capture chamber 504 using a 1 mL syringe. A polymer
sequencing matrix (CEQ, Beckman Corp., Fullerton, Calif.) was
loaded into a CE system 506 using a high-pressure gel loader. A
C-track master mix was prepared containing 80 nM ET-primer,
1.times. C terminator mix (Amersham), and 4 nM PCR product and
injected into a 250 nL thermal cycling chamber or reactor (not
shown). Thermal cycling (35.times., 94.degree. C. 30 seconds,
45.degree. C. 40 seconds, 70.degree. C. 40 seconds) was performed
on-chip using a LabVIEW program (National Instruments, Austin,
Tex.).
[0068] Sequencing reaction clean-up was performed by first
equilibrating the microdevice on a 50.degree. C. heated stage for
30 seconds. Then, sample capture (FIG. 5A) was initiated by
applying 2000 V to the capture chamber outlet (port 416 of FIG. 4A)
while grounding the reactor inlet (port 408 of FIG. 4A). Thus, the
sample containing extension fragments and residual nucleotides,
primers, and salts was electrophoretically driven from the thermal
cycling chamber through a channel 522 into the capture chamber 504.
Extension fragments hybridize to the Acrydite matrix in the capture
chamber while residual reactants pass through. When oligonucleotide
capture was complete, the retained extension fragments were
electrophoretically washed (FIG. 5B) for 30 seconds to remove
excess primer and other contaminants still present in the capture
chamber. After electrophoretic washing, the stage was ramped to
70.degree. C. and equilibrated for 60 seconds to allow full
denaturation of the product-matrix duplex. The denatured sample is
directly injected into separation columns 507 of the CE apparatus
506 (FIG. 5C) by applying 2,500 V to the anode while grounding the
capture chamber outlet. (Images have been processed to highlight
channel structure and remove fluorescent surface
contamination.)
[0069] Alternatively, a straight cross-injector and a 30-cm
separation capillary could be used. This will improve
resolution.
[0070] The microsphere-colony creation procedure and a single
reactor microdevice can generate single-ended reads suitable for
resequencing efforts. De novo whole-genome shotgun sequencing of
complex genomes requires paired-end reads from short and long
insert clones. Long-range PCR followed by fluorescent flow
cytometry can be used on a subset of microspheres to selectively
generate long-insert clones. Traditional paired-end sequencing
requires procedural and microdevice modifications such that clonal
DNA could be released from the microspheres and routed to separate
forward and reverse sequencing reactors. An alternative strategy is
to perform forward and reverse sequencing simultaneously on a
subset of bases using an altered base labeling scheme. These
paired-end reads generate long single or double-base reads suitable
for anchoring four-base single-ended reads in the sequence
assembly.
[0071] As illustrated in FIGS. 6A, 6B and 6C, a microsphere 600 is
used to transfer a clonal template into a thermal cycling or
sequencing reaction chamber 602 where sequencing reactions are
performed. This process requires that one microsphere be introduced
in each sequencing reaction chamber. Autovalving techniques for
trapping an individual microsphere in a sequencing reaction
chamber, without the need for individually actuated valves and
sensing, are used for this purpose.
[0072] As shown, the reaction chamber 602 includes an introduction
channel or arm 604 and an exit channel or arm 606. The introduction
channel is in fluid communication with an inlet port 605 of the
reaction chamber, while the exit channel is in fluid communication
with an outlet port 607 of the reaction chamber. Self-valving is
achieved by creating a constriction or constriction region 608 at
the outlet end or port 607 of the reaction chamber. The
constriction region is semicircular in shape.
[0073] The constriction will trap a microsphere before it exits the
chamber. The trapped microsphere will produce a drop in the flow
rate, thereby preventing any further microspheres from flowing into
the chamber. The dilution of the microspheres will be chosen such
that the probability that another microsphere flows into the
reaction chamber before the first microsphere has blocked the
chamber is below about 0.5%. If this statistical approach is
unreliable or leads to low sorting rates, an on-chip sensor for
bead light scattering can be used, and two valves in the sorting
loop can be added to produce a more temporally uniform bead
distribution.
[0074] Glass fabrication processes can be used to create the device
shown in FIGS. 6A-6C. In one example, all features except the
constriction were isotropically etched to a depth of 30 .mu.m. The
sample introduction and exit channels were set to 70 .mu.m in width
and 15 mm in length. The sequencing reaction chamber had a volume
of 250 nL. After the basic fabrication process was completed for
the channels and the reaction chamber, a second mask was used to
fabricate the constriction. For this, a more viscous photoresist
(SJR 5740, Shipley, Marlborough, Mass.) was spin-coated on a wafer
at 2500 rpm for 35 seconds. This was followed by a soft bake at
70.degree. C. for 7 minutes and 90.degree. C. for 6 minutes. A
higher viscosity photoresist gives more uniform coating on the
featured wafer. The constriction pattern was transferred to the
coated wafer using a contact printer. Alignment marks were used to
align (.+-.1 .mu.m) the constriction with the already etched
channels and chamber. After development, the amorphous silicon
masking layer was removed via plasma etching, and the exposed glass
was wet etched using 5:1 BHF with an effective etch rate of 2
.mu.m/hour for 1.5 hours. This gave a constriction depth or height
of 3 .mu.m. The constriction width was set to 8 .mu.m and the
length was set to 15 .mu.m.
[0075] Testing of the self-valving concept was performed using 6
.mu.m diameter, Streptavidin Coated Fluoresbrite YG Carboxylated
Polystyrene microspheres (Polysciences Inc., Warrington, Pa.)
suspended in a solution of 1.times. Tris (pH 8.0) and 1% Triton
X-100 diluted to a final concentration of 1 microsphere/3 .mu.L.
Using the Poisson distribution, it was calculated that a 10.times.
dilution of the reaction chamber volume will insure that the
probability that another microsphere will enter the 250 nL chamber
before the first microsphere blocks the constriction is below
0.5%.
[0076] A device like that shown in FIGS. 6A-6C was pre-filled with
a solution of 1.times. Tris (pH 8.0) and 1% Triton X-100. The
microsphere solution (6 .mu.L) was pipetted into an inlet access
hole. A vacuum line was used to draw the beads through the chamber.
The pressure drop increased from -60 kPa to -70 kPa when a
microsphere was trapped at the constriction, and blockage occurred
within the first 60 seconds. The experiment was continued for 12
minutes and no other microsphere was observed to enter the
chamber.
[0077] FIGS. 7A and 7B show bright field images of a constriction
region 708 of a thermal cycling chamber 702 at 20.times.
magnification when empty and with solution (but no microsphere),
respectively. FIG. 7C shows a dark field image of the thermal
cycling chamber 702 with a fluorescent microsphere 700 trapped at
the constriction 708 and acting as a valve. The experiments were
performed for microspheres of approximately 6 .mu.m diameter. The
concept is easily scalable to larger microspheres to increase the
number of templates in the reactor. Also, smaller microsphere could
be used. The microspheres, in particular, may be between about 1
and 100 .mu.m in diameter. In one embodiment, they are about 10
.mu.m in diameter.
[0078] An alternative autovalving embodiment, which will result in
complete flow blockage, is presented in FIGS. 8A and 8B. Here, a
reaction chamber 802 has a near circular constriction or
constriction region 808 formed at a chamber outlet port 807. The
chamber and the constriction will be double-etched; that is, they
will be etched on both of two joined glass surfaces. This will
produce a near circular constriction, as opposed to a semicircular
constriction of the embodiment of FIGS. 6A-6C. This will provide
better valving for trapping a microsphere 800.
[0079] As discussed, valves will be incorporated on either side of
a sequencing reaction or thermal cycling chamber 402 (see FIGS. 4A
and 4B). Once a microsphere has been trapped at the constriction,
it needs to be pushed back into the chamber for thermal cycling so
that there is good accessibility of the clonal templates to the
polymerase, primers, dNTPs and ddNTPs. By closing the valve 420 at
the outlet of the chamber before closing the inlet valve 410, the
microsphere will be pushed back into the chamber as a result of the
finite dead volume of the valves. This principle is easily scaled
to an array system by actuating the inlet and outlet valves in
parallel.
[0080] To realize a high-throughput and fully integrated system for
DNA analysis, a microfabricated array of integrated analyzers that
incorporate thermal cycling chambers, purification chambers as well
as separation channels for CE analysis of the sequencing fragments
is provided. A schematic of such a system 900 is shown in FIGS. 9A
and 9B. This system extends the single channel device of FIGS.
4A-4C to an array structure that is able to perform highly parallel
analyses.
[0081] According to various embodiments, the system 900 includes
multiple thermal cycling chambers 902 and associated sample
purification or capture chambers 904 arranged about a circular axis
to form a radially parallel system. The thermal cycling chambers
and the sample purification chambers are all integrated with a CE
analyzer system 906 including separation channels or microchannels
908. The CE analyzer has a common central anode (A) 910, a cathode
reservoir (C) 912, and a waste reservoir (W) 914. The cathode and
anode reservoirs are associated with adjacent sets of separation
channels 908. The microchannels 908 are connected to the anode 910
for detection using a rotary confocal fluorescence scanner of the
type discussed in the article entitled: "High-throughput genetic
analysis using microfabricated 96-sample capillary array
electrophoresis microplates", Peter C. Simpson, et al., Proc. Natl.
Acad. Sci. USA, Vol. 95, pp. 2256-2261, March 1998, which is hereby
incorporated by reference.
[0082] The system 900 further includes a distribution channel 916,
integrated heaters 918a and 918b, and RTDs 920. The heaters 918a
and 918b address the thermal cycling chambers and purification
chambers, respectively, in parallel. As such, the use of simple
ring heaters to drive the thermal cycling and sample purification
reactions is more than adequate.
[0083] The temperature of these reactions are monitored by the
RTDs. In one embodiment, four-equally spaced RTDs are integrated on
the substrate to provide precise temperature sensing across the
heater 918a for optimal thermal cycling performance. Similarly,
four-equally spaced RTDs may be used to monitor the temperature of
the heater 918b.
[0084] The system 900 also includes an array of integrated valves
and ports for controlling the system flow process. The valves may
be monolithic elastomer (PDMS) membrane valves. The system may be
fabricated as described in the above-identified U.S. patent
application Ser. No. 10/750,533, which has been incorporated by
reference. As such, the system may include a four layer
glass-glass-PDMS-glass stack that incorporates the microfluidic
valves and pumps, the RTDs, the thermal cycling chambers, the
clean-up and concentration chambers, and the CE channels.
[0085] The system 900, in one embodiment, includes 24 thermal
cycling chambers, 24 purification chambers, and 24 CE channels
arranged on a quadrant of a 150-mm diameter glass substrate. Each
thermal cycling chamber (.about.250 nL) is isolated from the
distribution channel by PDMS membrane valves (V1) 922. Rigid
containment of the chamber volume with active valves is necessary
for bubble-free loading and immobilization of a sample during
thermal cycling since sample movement, bubble formation, and sample
evaporation can seriously affect the performance.
[0086] The RTDs may be fabricated of titanium (Ti) and platinum
(Pt). Different materials (Au, Al, Pt, Ni) using different metal
deposition techniques, such as sputtering and both thermal and
electron-beam evaporations, may be used to fabricate the heaters.
Nickel heaters exhibit good heating uniformity, have good scratch
resistance, show no noticeable degradation of performance even
after hundreds of thermal cycles, and are easily fabricated. The
microfabricated heaters and the thermal cycling chambers are
positioned away from the CE microchannels to avoid evaporation of
buffer in the cathode reservoirs and to minimize the heating of the
injection region during thermal cycling. The resistive ring heaters
can be fabricated on the back side of the bottom wafer to ensure
good thermal transfer between the heaters and the chambers. The
equally-spaced RTDs are integrated on the microplate to provide
precise temperature sensing ensuring temperature uniformity across
the heaters for optimal performance.
[0087] The thermal cycling chambers may be cycled with a LabVIEW
program (National Instruments, Austin, Tex.). (LabView VI)
Temperature control can be accomplished through a
proportion/integration/differentiator (PID) module within the
LabVIEW program.
[0088] In operation, in one embodiment, a sequencing separation
matrix is introduced into the CE separation channels using positive
pressure through the anode 910. A separation matrix fills the
cathode reservoirs 912 and the waste reservoirs 914, as well as
arms 924 connecting the cross-injection point to the associated
sample purification chambers. The filling rate can be modulated
through adjustment of the widths and lengths of the various
interconnecting channels. The cathode and anode reservoirs, and the
connecting arms may be filled at the same rate. Water, for example,
is loaded from a loading (L) port 926 connected to the distribution
channel 916 to fill the thermal cycling chambers 902 and the sample
purification chambers 904 ensuring continuity for subsequent
processes. An exit port (E) 936 is shared by both the thermal
cycling chambers 902.
[0089] A capture gel matrix is loaded into the sample purification
chambers through ports (F) 928. Libraries of clonal DNA beads that
have been sorted and prepared using a FACS unit are loaded from the
port 926 (valves (V1) 922 and (V2) 930 open, and valve (V3) 932
closed) using, for example, a syringe pump. A pressure transducer
is employed in the inlet to monitor the head pressure. Auto-valves
at the exit ports of the thermal cycling chambers will stop the
flow into a chamber once a bead is trapped in the auto-valve as
described above. As each auto-valve is filled with a bead in each
thermal cycling chamber, the head pressure will continue to
increase. In this manner, loading a bead in each thermal cycling
chamber is achieved by monitoring the head pressure.
[0090] Upon filling the chambers, the valves (V2) 930 and (V1) 922
are closed and thermal cycling reactions initiated. Since the beads
will be sorted directly in the thermal cycling cocktail, no
additional steps are needed before thermal cycling. Alternatively,
sorting could be performed in a buffer followed by introduction of
the thermal cycling cocktail into the thermally cycling chambers to
minimize reagent usage. The heaters 918a and 918b, as noted, drive
the thermal cycling and sample purification reactions, and the
reaction temperatures are monitored by the RTDs 920.
[0091] Thermal cycling products are driven to the purification
chambers by applying an electric field across the port (L) 926 and
a sample port or electrical contact point (S) 934 with the valves
(V1) 922 and (V3) 932 open. Flushing of the non-captured reagents
is performed by applying an electric field between the port (S) 934
and the port (F) 928. Thereafter, the sequencing products are
injected from the purification chambers into the associated
microchannels 908 for CE separation, by applying a potential
between the port (S) 934 and the waste reservoir (W) 914. The
rotary confocal fluorescence scanner is used to detect the
sequencing samples. The scanner interrogates the channels
sequentially in each rotation of the scanner head.
[0092] As shown, separations of the purified DNA sequencing
products are achieved by using cross-injection. Alternatively,
direct injection could be used.
[0093] In another embodiment, the microemulsion PCR approach of
colony formation is replaced by a continuous flow through PCR
technique in which bolui or droplets for performing single-molecule
PCR amplification of templates are formed and then amplified. The
bolui or plugs are formed, for example, by combining an aqueous
solution of PCR reagents and dilute template fragments with a
hydrophobic solution that acts as a carrier. The bolui produced by
this technique will contain statistically a small number of copies
of a sequencing template. Ideally the concentration is chosen so
that only one in 10 bolui contain a single sequencing template.
This mean that only one in 100 will simultaneously contain
undesirably two templates. A microsphere or bead carrying one of
the primers in the PCR reaction can also be entrained within a
bolus or droplet in the process of performing this technique. Thus,
a colony may comprise bolui carrying multiple clonal copies of a
single sequencing template or bolui having microspheres carrying
such copies.
[0094] As shown in FIGS. 10A, 10B, and 10C, a continuous flow
through microfabricated PCR system 1000 includes ports 1002 and
1004. An aqueous solution of microspheres, PCR reagents and
template fragments, in one embodiment, is introduced into a
"T"-injector region 1006 via the port 1002, while a hydrophobic
carrier solution is introduced into the "T"-injector region via the
port 1004. A bolus or droplet 1012 containing a microsphere is thus
formed downstream of the "T"-injector region.
[0095] The system 1000 further includes an optical sensor 1007
(FIG. 10B) located downstream of the "T"-injector near an input end
of a channel 1014. The sensor may comprise a focused laser beam and
a photodiode to detect off-axis light scattering.
[0096] The sensor is configured to select a bolus that contains a
bead and to reject bolui with no bead or with more than one bead. A
rejected bolus is passed out of the system via a valve 1009 and a
waste port and channel 1011. The valve 1009 may be a four-layer
PDMS valve that when open allows a rejected bolus to exit the
system via the waste port 1011. The valve may be activated via a
valve actuation port 1013. Since the fluidic resistance of the
continuous flow system is so great, no valve is needed in the main
channel 1014.
[0097] The system 1000 is designed for two-step PCR where the
anneal and extend steps are combined. As such, the system includes
two temperature zones 1008 and 1010 through which droplets 1012
pass via the flow through channels 1014. The device 1000 includes
RTDs 1016 and associated RTD contact pads 1018 for monitoring the
temperature of the different temperature zones. A bolus and
microsphere, after undergoing two-temperature PCR, exit the system
via an exit port 1020.
[0098] As discussed, the microsphere can be treated with an
intercalation dye, such as TO, that is nonfluorescent until
intercalated into double-stranded DNA. Therefore, the microspheres
that have amplified DNA can be identified by, for example, the FACS
technique after they pass through the port 1020. Thereafter, the
microspheres are introduced into a distribution channel of a device
like the device 900 (See FIGS. 9A and 9B). The microspheres that do
not have amplified DNA, on the other hand, will be disposed of.
[0099] The continuous flow through PCR system 1000, in one example,
uses nanoliter droplets of PCR reagents suspended in a
perfluorohydrocarbon carrier. See, Song, H., et al., "A
microfluidic system for controlling reaction networks in time",
Angewandte Chemie-International Edition 42, 768-772 (2003), which
is hereby incorporated by reference. Droplets containing
microspheres, PCR reagents, and template fragments, separated from
one another by an immiscible liquid, are formed at the
microfabricated T-injector 1006. Thereafter, the droplets flow
through the regions 1008 and 1010 of the device that are held at
anneal-extend-denature temperatures. The lengths of the channels
1014 in the different temperature zones 1008 and 1010 are selected
based on required residence times at those temperatures. The
cross-section of the channels and the desired flow rates are chosen
to achieve stable droplet formation. The parameters of the device
may be set to achieve a hot start at the denature temperature for
90 seconds, followed by anneal/extend for 45 seconds with a 1
second auto-extend for 35 cycles and a denature time of 15 seconds.
The last 15 cycles may be configured to have a constant
anneal/extend time of 80 seconds.
[0100] The device 1000 may comprise two 1.1-mm thick by 10-cm
diameter borofloat glass wafers. The channels on the patterned
wafer may have a D-shape cross-section with a width of about 210
.mu.m and a depth of about 95 .mu.m. On the other wafer, Ti/Pt RTDs
may be fabricated for monitoring temperature gradients at different
points in the device. Holes may be drilled for accessing the
channels, and the two wafers may be thermally bonded to each other
to form enclosed channels. The nanoports 1002 and 1004 are used to
interface the device with micro-liter syringes through PEEK tubing.
The glass surface of the channels may be rendered hydrophobic
through silanization with
1H,1H,2H,2H-perfluorodecyltrichlorosilane, based on a procedure
published by Srinivasan et al., "Alkyltrichlorosilane-based
self-assembled monolayer films for stiction reduction in silicon
micromachines", Journal of Microelectromechanical Systems 7,
252-260 (1998), which is hereby incorporated by reference. Two
syringe pumps may be used to dispense water and a 10:1 mixture of
perfluorodecalin (mixture of cis and trans, 95%, Acros Organics,
New Jersey, USA) and 1H,1H,2H,2H-perfluorooctanol (Acros Organics,
New Jersey, USA) at a flow rate of 0.5 .mu.l/min each. FIG. 10B
shows the droplet formation process at the T-injector at about 1
droplet/s, and FIG. 10C shows the droplets as they move through the
channel.
[0101] The technique, in one embodiment, uses microbeads in 10 nL
droplets. Throughput is maintained at one in ten droplets
containing both a single template molecule and a single bead. The
aqueous PCR mix is prepared such that there is one template
molecule per 100 nL of mix, corresponding to one molecule for every
ten droplets. Also in the PCR mix are microbeads at a concentration
of one per 10 nL of mix, corresponding to one bead for every
droplet on average. As dictated by the Poisson distribution, 37% of
the 10 nL droplets will contain only one microbead, the remaining
containing either none or two plus. Each droplet, as discussed, is
then optically scanned to determine the number of beads it
contains. If the droplet does not contain a single microbead, the
valve 1009 is opened, passing the droplet to waste. Approximately
one third of the droplets will contain a single bead and are routed
to the main channel; thus, the average flow rate is equal to about
1 droplet every 1.5 s. As such, every droplet in the main channel
contains a single microbead, and one in ten also contains a single
template molecule.
[0102] At the end of the device 1000, the droplets may be collected
at the port 1020 via a standard capillary into a microfuge tube.
The droplets are broken through centrifugation. Microbeads are
collected and washed in 1.times.TE, again through centrifugation.
The microbeads are routed into the thermal cycling chambers of the
device 900 by either autovalving or active valving with on-chip
detection. On-chip detection may comprise the use of an optical
scanner or a timing arrangement that determines when a microsphere
is located adjacent to an inlet of a thermal cycling chamber. The
optical scanner, as discussed, may use bead light scattering to
determine the location of a microsphere within the distribution
channel. The timing arrangement is based on the fact that the fluid
in the distribution channel is incompressible. As such, the
location of a microsphere within the distribution channel, for
instance, adjacent to an inlet of a thermal cycling chamber, can be
calculated from the time that the microsphere has been in the
distribution channel. This is advantageous because the valved
entrances to each of the 96 inputs from the distribution channel,
for example, can be actuated by a single pneumatic input. Also the
pneumatic input is not actuated until a detected bead is flowed
directly opposite a reactor that does not have a bead yet.
[0103] To demonstrate the feasibility of generating sequencing
templates using two-temperature PCR, high-temperature primers were
designed and tested in a conventional thermal cycler. Two primers,
M13.sub.--2T_F--5'TTCTGGTGCCGGAAACCAGGCAAAGCGCCA-3'(SEQ. ID NO. 2)
Tm=70.3.degree. C. and M13.sub.--2T_R
5'-ACGCGCAGCGTGACCGCTACACTTGCCA-3' (SEQ. ID NO. 3) Tm=70.7.degree.
C. were designed to generate a 943 bp amplicon from the M13 genome.
To approximate the concentration of a single template molecule in a
11 nL emulsion compartment, 18.75 femtograms of M13 template were
cycled in a 25 uL PCR reaction (94.degree. C. 1.5 min followed by
50 cycles of 94.degree. C. 10 s, 70.degree. C. 30 s with an
auto-extend of 1 s/cycle). The resulting amplicon was a single
clean peak at the expected size with a yield of about 40 ng/uL.
[0104] If such a system is used to amplify genomic DNA, for
1.times. coverage and average fragment size of 1000 bps, about 3
million fragments need to be amplified. The probability that two
different fragments end up in a single droplet can be reduced to
less than 0.01 by diluting the fragments in the PCR reagent such
that on an average one in ten droplets contains a fragment. Hence,
the system will have to process 30 million droplets. This device is
designed to generate one droplet about every 1.5 seconds. If 20
such devices are run in parallel, the entire genome can be
amplified and interfaced with a bank of the sequencing systems 900
to produce 1.times. coverage in only one month.
[0105] While the invention has been particularly shown and
described with reference to specific embodiments, it will also be
understood by those skilled in the art that changes in the form and
details of the disclosed embodiments may be made without departing
from the spirit or scope of the invention. For example, the
embodiments described above may be implemented using a variety of
materials. Therefore, the scope of the invention should be
determined with reference to the appended claims.
Sequence CWU 1
1
3119DNAArtificial SequenceSelected as an example in the study of
the operation of the MINDS system 1actggccgtc gttttacaa
19230DNAArtificial SequenceSelected as an example in the study of
the operation of the MINDS system 2ttctggtgcc ggaaaccagg caaagcgcca
30328DNAArtificial SequenceSelected as an example in the study of
the operation of the MINDS system 3acgcgcagcg tgaccgctac acttgcca
28
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