U.S. patent application number 11/508756 was filed with the patent office on 2007-03-29 for device and method for microfluidic control of a first fluid in contact with a second fluid, wherein the first and second fluids are immiscible.
This patent application is currently assigned to Applera Corporation. Invention is credited to Steven J. Boege, David M. Cox, Linda G. Lee, Eric S. Nordman, Mark F. Oldham, Richard T. Reel, Dmitry M. Sagatelyan, Willy Wiyatno, Sam L. Woo.
Application Number | 20070068573 11/508756 |
Document ID | / |
Family ID | 37772254 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070068573 |
Kind Code |
A1 |
Cox; David M. ; et
al. |
March 29, 2007 |
Device and method for microfluidic control of a first fluid in
contact with a second fluid, wherein the first and second fluids
are immiscible
Abstract
Various embodiments described in the application relate to an
apparatus, system, and method for fluidically controlling, within a
conduit, discrete volumes of one or more fluids that are immiscible
with a second fluid. The discrete volumes can be used for
biochemical or molecular biology procedures involving small
volumes, for example, microliter-sized volumes, nanoliter-sized
volumes, or smaller. The system can comprise combinations of
detectors, controllers, valves, and fluid supply units to control
the spatial size, location and/or fluidic composition of discrete
volumes separated from one another by a fluid that is immiscible
with the fluid(s) of the discrete volumes, for example, aqueous
immiscible-fluid-discrete volumes separated by an oil.
Inventors: |
Cox; David M.; (Foster City,
CA) ; Woo; Sam L.; (Redwood City, CA) ; Boege;
Steven J.; (San Mateo, CA) ; Oldham; Mark F.;
(Los Gatos, CA) ; Reel; Richard T.; (Hayward,
CA) ; Nordman; Eric S.; (Palo Alto, CA) ; Lee;
Linda G.; (Palo Alto, CA) ; Wiyatno; Willy;
(Union City, CA) ; Sagatelyan; Dmitry M.;
(Alameda, CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.;APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
37772254 |
Appl. No.: |
11/508756 |
Filed: |
August 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60710167 |
Aug 22, 2005 |
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60731133 |
Oct 28, 2005 |
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60818197 |
Jun 30, 2006 |
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Current U.S.
Class: |
137/1 |
Current CPC
Class: |
B01L 3/502715 20130101;
Y10T 137/86863 20150401; B01J 2219/00659 20130101; B01L 2200/0673
20130101; B01L 2300/0867 20130101; C12Q 1/6806 20130101; G01N
27/44769 20130101; B01L 2400/0421 20130101; B01J 2219/00626
20130101; B01J 2219/00353 20130101; B01L 3/0293 20130101; B01L
2300/0864 20130101; G01N 35/08 20130101; B01L 3/502784 20130101;
C12Q 1/6869 20130101; Y10T 137/0318 20150401; Y10T 137/85986
20150401; B01L 7/52 20130101; F16K 99/0011 20130101; C12Q 1/6874
20130101; F16K 99/0001 20130101; F16K 99/0013 20130101; Y10T
436/2575 20150115; B01J 2219/00608 20130101; B01J 2219/00637
20130101; B01J 2219/00657 20130101; B01L 2400/0487 20130101; B01L
2200/10 20130101; B01J 2219/0036 20130101; F16K 2099/0084 20130101;
G01N 27/44743 20130101; B01J 2219/0061 20130101; B01J 2219/00619
20130101; Y10T 137/4259 20150401; Y10T 137/85978 20150401; B01J
2219/00612 20130101; C12Q 2535/101 20130101; F15C 5/00 20130101;
B01J 2219/00364 20130101; B01J 2219/00653 20130101; G01N 1/14
20130101; C12Q 1/6869 20130101; C12Q 2535/101 20130101 |
Class at
Publication: |
137/001 |
International
Class: |
F17D 1/00 20060101
F17D001/00 |
Claims
1. A method of splitting a discrete volume of a liquid contained in
a conduit into two or more smaller discrete volumes of the liquid,
the method comprising: connecting to a first displacement pump a
first conduit for receiving a first, smaller discrete volume of a
first liquid; connecting to a second displacement pump a second
conduit for receiving a second, smaller discrete volume of the
first liquid; supplying to a junction of at least the first and
second conduits a discrete volume of a first liquid in contact with
a second liquid with which the first liquid is immiscible; and
moving the first and second displacement pumps in a first direction
at the same time, thereby splitting the discrete volume of the
first liquid into at least the first and second smaller discrete
volumes of the first liquid; wherein the ratio of the volume of the
first smaller discrete volume of the first liquid to the volume of
the second smaller discrete volume of the first liquid is based on
the ratio of the volume drawn by the first displacement pump to the
volume drawn by the second displacement pump.
2. The method of claim 1, wherein moving the first and second
displacement pumps in a first direction at the same time comprises
moving a linkage attached to at least the first and second
displacement pumps.
3. The method of claim 2, further comprising: connecting the common
port of a first three-way valve to the first displacement pump;
connecting the first of two input/output ports of the first
three-way valve to the first conduit; connecting the second of two
input/output ports of the first three-way valve to a third conduit;
connecting the common port of a second three-way valve to the
output of the second displacement pump; connecting the first of two
input/output ports of the second three-way valve to the second
conduit; connecting the second of two input/output ports of the
second three-way valve to a fourth conduit; after moving the
linkage in a first direction, moving the linkage in a second
direction opposite the first direction, thereby displacing a first
and second volume of the second liquid into the third and fourth
conduits, respectively.
4. The method of claim 2, wherein the supplying to a junction
comprises: detecting the speed of the discrete volume of the first
fluid, which is flowing in a third conduit connected to the
junction of at least the first and second conduits, at a known
distance from the junction; and stopping the flow of the discrete
volume of the first fluid at the junction.
5. An apparatus comprising: a first displacement pump having a
first cross-sectional area; a first conduit for receiving a first,
smaller discrete volume of a first liquid connected to the first
displacement pump; a second displacement pump having a second
cross-sectional area; a second conduit for receiving a second,
smaller discrete volume of the first liquid connected to the second
displacement pump; a third conduit containing a discrete volume of
a first liquid in a second liquid with which the first liquid is
immiscible to, a junction of at least the first and second
conduits; and a linkage attached to at least the first and second
displacement pumps, wherein when the linkage is moved in a first
direction, the discrete volume of the first liquid splits into at
least the first and second smaller discrete volumes of the first
liquid, and the ratio of the volume of the first smaller discrete
volume of the first liquid to the volume of the second smaller
discrete volume of the first liquid is based on the ratio of the
cross-sectional area of the first displacement pump to the
cross-sectional area of the second displacement pump.
6. The apparatus of claim 5, further comprising: a first three-way
valve having a common port, and at least two input/output ports,
wherein the common port of the first three-way valve is connected
to the output of the first displacement pump, the first of two
input/output ports of the first three-way valve is connected to the
first conduit, and the second of two input/output ports of the
first three-way valve is connected to a fourth conduit; a second
three-way valve having a common port and at least two input/output
ports, wherein the common port of a second three-way valve is
connected to the output of the second displacement pump, the first
of two input/output ports of the second three-way valve is
connected to the second conduit, and the second of two input/output
ports of the second three-way valve is connected to a fifth
conduit; wherein when the linkage is moved in a second direction
opposite the first direction, the first displacement pump displaces
a first volume of the second liquid into the fourth conduit and the
second displacement pump displaces a second volume of the second
liquid into the fifth conduit.
7. The apparatus of claim 5, further comprising a detector adjacent
the third conduit and operatively coupled to the motor.
8. The apparatus of claim 5, wherein at least one of the first and
second three-way valves is a rotary three-way valve.
9. A method of adding two or more different miscible liquids in
predetermined volumetric combination to an existing
immiscible-fluid, discrete volume, the method comprising:
connecting to a first displacement pump a first conduit for
supplying a first liquid; connecting to a second displacement pump
a second conduit for supplying a second liquid miscible with the
first liquid; connecting to a junction of the first and second
conduits a third conduit for receiving the predetermined volumetric
combination of the first liquid and second liquid, moving a linkage
attached to the first and second displacement pumps in a first
direction, thereby combining the first and second liquid in the
predetermined volumetric relationship; thereby creating an addition
liquid; supplying the addition liquid to a fourth conduit
containing an immiscible-fluid, discrete volume of a fourth liquid,
miscible with the addition liquid; and adding a volume of the
addition liquid to the immiscible-fluid, discrete volume.
10. The method of claim 9, further comprising: connecting the
common port of a first three-way valve to the first displacement
pump; connecting the first of two input/output ports of the first
three-way valve to the first conduit; connecting the second of two
input/output ports of the first three-way valve to a fourth
conduit; connecting the common port of a second three-way valve to
the second displacement pump; connecting the first of two
input/output ports of the second three-way valve to the second
conduit; connecting the second of two input/output ports of the
second three-way valve to a fifth conduit; before moving the
linkage in a first direction, moving the linkage in a second
direction opposite the first direction, thereby withdrawing a first
and second volume of the second liquid from the fourth and fifth
conduits, respectively.
11. A method of adding a first liquid in predetermined volumetric
relationship to a second liquid with which it is immiscible, the
method comprising: connecting to a first displacement pump a first
conduit for supplying a first liquid; connecting to a second
displacement pump a second conduit for supplying a second liquid
immiscible with the first liquid; connecting to a junction of the
first and second conduits a third conduit for receiving
immiscible-fluid, discrete volumes of at least one of the first
liquid and second liquid, moving a linkage attached to the first
and second displacement pumps in a first direction, thereby
generating immiscible-fluid, discrete volumes of at least the first
liquid in the third conduit; thereby creating a first set of
immiscible-fluid, discrete volumes of the first liquid.
12. A method of adding a first liquid to a third liquid with which
it is miscible, the method comprising connecting to a first
displacement pump a first conduit for supplying a first
immiscible-fluid, discrete volume of a first liquid in contact with
a second fluid with which the first liquid is immiscible;
connecting to a second displacement pump a second conduit for
supplying a second immiscible-fluid, discrete volume of a third
liquid in contact with the second fluid which the third liquid is
immiscible; connecting to a junction of the first and second
conduits a third conduit for receiving a larger, immiscible-fluid,
discrete volume of the first and third liquids in contact with the
second fluid, with which the first and third liquids are each
immiscible, positioning the first immiscible-fluid, discrete volume
in the first conduit and the second immiscible-fluid, discrete
volume in the second conduit; moving a linkage attached to the
first and second displacement pumps in a first direction, thereby
contacting the immiscible-fluid, discrete volume of first liquid
with the immiscible-fluid, discrete volume of the third liquid to
form a larger immiscible-fluid, discrete volume of the first and
third liquids.
13. The method of claim 12, further comprising: connecting the
common port of a first three-way valve to the first displacement
pump; connecting the first of two input/output ports of the first
three-way valve to the first conduit; connecting the second of two
input/output ports of the first three-way valve to a fourth
conduit; connecting the common port of a second three-way valve to
the second displacement pump; connecting the first of two
input/output ports of the second three-way valve to the second
conduit; connecting the second of two input/output ports of the
second three-way valve to a fifth conduit; before moving the
linkage in a first direction, moving the linkage in a second
direction opposite the first direction, thereby withdrawing the
first immiscible-fluid-discrete volume and the second
immiscible-fluid-discrete-volume from the fourth and fifth
conduits, respectively.
14. An apparatus comprising: a conduit containing discrete volumes
of aqueous liquid in contact with an immiscible, non-aqueous
liquid; an expandable and contractible reservoir in fluid
communication with the conduit in at least one location and
containing a volume of the immiscible, non-aqueous liquid; a
diaphragm coupled to the reservoir; an enclosure coupled to the
diaphragm, such that the diaphragm is between the enclosure and the
reservoir; wherein when the space between the enclosure and the
diaphragm is positively pressurized, the fluid in the conduit is
pressurized.
15. The apparatus of claim 14, wherein the conduit is in further
fluid communication with the reservoir at a second location.
16. The apparatus of claim 15, wherein between the first and second
locations, the conduit is adjacent a thermal cycler.
17. The apparatus of claim 15, wherein between the first and second
locations, the conduit is adjacent at least one of the following: a
heating unit, cooling unit, isothermal zone, and a thermal
cycler.
18. An apparatus comprising: a stator having at least three
through-holes from outer diameter to inner diameter; a mating rotor
having at least two through-holes, first, second, and third tubes,
each coupled to one of the three through-holes of the stator,
wherein at least one of the first, second, and third tubes contains
one or more immiscible-fluid, discrete volumes; wherein, when the
stator and mating rotor are in a first relative position, a first
of the at least two through-holes in the mating rotor aligns with
two of the at least three through-holes in the stator, and when the
stator and mating rotor are in a second relative position, a second
of the at least two through-holes in the mating rotor is in fluid
communication with one of the previous two of the at least three
through-holes and the remaining third through-hole in the
stator.
19. The apparatus of claim 18, wherein at least the mating rotor
and the first and second conduits are made from the same type of
material.
20. An apparatus for preventing flow of discrete-volumes of a first
fluid in contact with a second fluid with which the first fluid is
immiscible, the apparatus comprising: a first member connected to a
first tube for supplying one or more discrete volumes of a first
fluid in contact with a second fluid with which the first fluid is
immiscible; a second member having a through-hole for receiving
discrete volumes of the first fluid from the first tube in a first
position, wherein when the second member is in a second position
with respect to the first member, discrete volumes of the first
fluid cannot flow through the through-hole in the second
member.
21. The apparatus of claim 20, wherein the second member is
rotatable between the first and second position.
22. The apparatus of claim 20, wherein the second member is
translatable between the first and second position.
23. An apparatus for preventing flow of discrete-volumes of a first
fluid in contact with a second fluid with which the first fluid is
immiscible, the apparatus comprising: an
immiscible-fluid-discrete-volume tube; a first pinching member
having a radiused surface in at least tangential contact with the
immiscible-fluid-discrete-volume tube; a second pinching member in
a first position with respect to the
immiscible-fluid-discrete-volume tube, wherein a section of the
tube is pinched between the radiused surface of the first pinching
member and the second pinching member, such that discrete volumes
of the first fluid cannot flow past the compressed section.
24. The apparatus of claim 23 further comprising a solenoid in
contact with one of the first and second pinching member.
25. A method of splitting a discrete volume of a first fluid in
contact with a second fluid with which it is immiscible, the method
comprising: positioning a first part of an
immiscible-fluid-discrete-volume in a through-hole of a first
member that is in a first position, wherein a through-hole in the
first member is aligned with an immiscible-fluid-discrete-volume
supply tube; moving the first member to a second position, wherein
the through-hole in the first member is aligned with a
spacing-fluid supply tube, thereby turning the first part of an
immiscible-fluid-discrete-volume into a first-split-part,
immiscible-fluid, discrete volume in the through-hole; moving the
first-split-part, immiscible-fluid, discrete volume into a
first-split-part tube that is aligned with the through-hole in the
second position; moving the first member back to the first
position, wherein the through-hole is aligned with the
immiscible-fluid-discrete-volume supply.
26. The method of claim 25, wherein positioning a first part
comprises: detecting the speed of an immiscible-fluid, discrete
volume in the immiscible-fluid-discrete-volume supply conduit at a
known distance from the first member; stopping the force moving the
immiscible-fluid, discrete volume; and detecting a known point of
the immiscible-fluid, discrete volume in the first member at a
desired distance from an opening of the through-hole in the first
member.
27. A method of removing gas phase bubbles from spacing fluid
between discrete volumes of a first fluid which is immiscible with
the spacing fluid, the method comprising: flowing a stream of fluid
comprising at least two immiscible-fluid-discrete-volumes separated
by spacing fluid and a gas phase bubble therein in a first conduit
in a first direction toward a junction of the first conduit, a
second conduit and at least a third conduit; flowing spacing fluid
in the second conduit away from the junction in a vertical
direction; flowing at least one of the at least two
immiscible-fluid-discrete-volumes separated by spacing fluid into
the third conduit, the longitudinal axis of which forms an angle in
the junction of greater than zero and less than 180 degrees with
the longitudinal axis of the second conduit at the junction;
flowing the gas phase bubble into the junction, wherein the gas
phase bubble transfers, due to a smaller specific gravity than the
spacing fluid, to the spacing fluid flowing away from the junction
in a vertical direction, thereby removing the gas phase bubble from
the spacing fluid between the two of the at least two
immiscible-fluid-discrete-volumes.
28. The method of claim 27, wherein the angle in the junction
between the longitudinal axis of the third conduit and the
longitudinal axis of the second conduit is approximately 90
degrees.
29. The method of claim 27 further comprising: flowing a stream of
spacing fluid in a fourth conduit toward the junction, wherein the
longitudinal axis of the fourth conduit forms an angle in the
junction with the longitudinal axis of the second conduit of
approximately 90 degrees.
30. A method comprising: alternately introducing a first fluid and
a second fluid, that is immiscible with the first fluid, into a
conduit, to form a set of immiscible discrete volumes of the second
fluid, each immiscible discrete volume of the set being separated
from one or more other immiscible discrete volumes of the set by
the first fluid, the set comprising a first end and a second end;
moving the set of immiscible discrete volumes in a first direction
by withdrawing from the conduit, some of the first fluid from the
first end of the set; and moving the set in the first direction by
adding to the conduit, more first fluid at the second end of the
set.
31. The method of claim 30, wherein the first fluid comprises an
oil and the second fluid comprises an aqueous liquid that is
immiscible in the oil.
32. The method of claim 30, further comprising moving the set past
a valve in the conduit and closing the valve before moving the set
in the first direction by adding to the conduit more first fluid at
the second end of the set.
33. The method of claim 32, wherein the closing the valve comprises
rotating a rotary valve.
34. The method of claim 32, wherein the closing the valve
comprising actuating a linear, sliding valve.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority under
35 U.S.C. 119 of earlier filed U.S. Provisional Patent Application
No. 60/710,167, filed Aug. 22, 2005, U.S. Provisional Patent
Application No. 60/731,133, filed Oct. 28, 2005, and U.S.
Provisional Patent Application No. 60/818,197, filed Jun. 30, 2006,
which are incorporated herein in their entireties by reference.
INTRODUCTION
[0002] The section headings used herein are solely for organization
purposes and are not to be construed as limiting the subject matter
described in any way.
[0003] Large scale sequencing projects can involve cloning DNA
fragments in bacteria, picking and amplifying those fragments, and
performing individual sequencing reactions on each clone. Standard
sequencing reactions can often be performed in 5 .mu.l to 20 .mu.l
reaction volumes, even though only a small fraction of the
sequencing product can be analyzed. Such cloning and sequencing
protocols can be time consuming and can use relatively large sample
and reagent volumes. The relatively large volumes can be wasteful
in terms of expensive consumable reagents.
SUMMARY
[0004] Various embodiments of the present teachings relate to
systems, apparatus, and/or methods for sample preparation that can
be used for biochemical or molecular biology procedures involving
different volumes, for example, small volumes such as micro-liter
sized volumes or smaller.
[0005] According to the present teachings, the system can comprise
an apparatus for generating discrete volumes of at least a first
fluid in contact with a second fluid, wherein the first and second
fluids are immiscible with each other, for example, discrete
volumes of an aqueous liquid (herein "aqueous
immiscible-fluid-discrete-volumes"), spaced-apart from one another
by a spacing fluid that is immiscible with the
immiscible-fluid-discrete-volumes. An
immiscible-fluid-discrete-volume can be a partitioned segment in
which molecular biology procedures can be performed. As used
herein, an immiscible-fluid-discrete-volume can be one of many
structures, three of which are: a fluid segment, a slug, and an
emulsified droplet. In some embodiments,
immiscible-fluid-discrete-conduits are formed and/or processed in a
conduit.
[0006] This paragraph defines a conduit as it is used herein. A
conduit can be any device in which an
immiscible-fluid-discrete-volume can be generated, conveyed, and/or
flowed. For example, a conduit as defined herein can comprise any
of a duct, a tube, a pipe, a channel, a capillary, a hole or
another passageway in a solid structure, or a combination of two or
more of these, as long as the spaces defined by the respective
solid structures are in fluid communication with one another. A
conduit can comprise two or more tubes or other passageways
connected together, or an entire system of different passageways
connected together. An exemplary conduit can comprise an
immiscible-fluid-discrete-volume-forming tube, thermal spirals,
valve passageways, a processing conduit, junctions, and the like
components all connected together to form one or more fluid
communications therethrough, which system is also referred to
herein as a main processing conduit. Examples of solid structures
with holes or passageways therein that can function as conduits are
manifolds, T-junctions, Y-junctions, rotary valves, and other
valves. Thus, when connected to conduits, such structures can be
considered part of a conduit as defined herein.
[0007] This paragraph defines a fluid segment, as it is used
herein. A fluid segment is a discrete volume that has significant
contact with one or more conduit wall(s), such that a
cross-sectional area of the fluid segment is the same size and
shape as the cross-sectional area of the conduit it contacts. At
least a portion of a fluid segment fully fills the cross-sectional
area of the conduit, such that the immiscible fluid adjacent it in
the conduit can not flow past the fluid segment. The entire
longitudinal length of the fluid segment may not contact the
conduit walls.
[0008] This paragraph defines a slug as used herein. A slug is a
discrete volume that has at least a portion of which has
approximately the same cross-sectional shape as the conduit in
which it exists, but a smaller size. The smaller size is due to the
insignificant contact, if any, of the slug with the conduit
wall(s). A slug can have a cross-sectional dimension between
approximately 0.5 and approximately 1.0 times the maximum dimension
of a cross sectional area of the conduit. If the conduit has a
circular cross section, the cross-sectional area of a slug can be
concentric with the conduit's cross-sectional area, but it does not
have to be, such as, for example, when the conduit is horizontal
and, due to different specific gravities, one fluid rises toward
the top of the cross-sectional area of the conduit under the
influence of gravity. A slug can be free of contact with the
conduit walls. When not moving relative to the conduit, a slug can
have "feet" that appear as nibs or bumps along an otherwise
smoothly appearing round surface. It is theorized that the feet at
the bottom of the slug may have contact with the conduit wall. In
contrast to a fluid segment, the contact a slug can have with the
conduit wall(s) still permits the immiscible fluid adjacent it in
the conduit to flow past the slug.
[0009] The "slugs" formed by the teachings herein, separated by
spacing fluid, can merge together to form larger slugs of liquid,
if contacted together. The ability of the slugs, for example,
aqueous slugs, described and taught herein, to merge together with
each other, facilitates the downstream addition of aqueous reagents
to the slugs.
[0010] This paragraph defines an emulsified droplet, as used
herein. An emulsified droplet is a discrete volume that has no
contact with the walls of the conduit. The size of an emulsified
droplet is not necessarily constrained by the conduit, and examples
of emulsified droplets described in the prior art range in size
from about 1 femtoliter to about 1 nanoliter. The shape of an
emulsified droplet is not constrained by the conduit, and due to
the difference in surface-energies between it and the continuous
phase liquid in which it is dispersed, it is generally spherical.
It can have a maximum dimension that is not equal to, nor
approximately equal to, but much less than the maximum dimension of
the cross-sectional area of the conduit, for example, 20%, 10%, 5%
or less. An emulsified droplet will not merge upon contact with
another emulsified droplet to form a single, larger discrete
volume, without external control. Put another way, an emulsified
droplet is a stable discontinuous phase in a continuous phase.
[0011] A conduit can contain more than one emulsified droplet, but
not more than one slug or fluid segment, at any cross-sectional
location. Thus, a first emulsified droplet does not necessarily
impede the movement of a second emulsified droplet past it, where
as a fluid segment and a slug necessarily do not permit the passage
of another fluid segment or slug past them, respectively. If two
fluid segments are separated by a fluid with which the first and
second of the two fluids is each immiscible, then the immiscible
fluid also forms a discrete volume. It is likely that it has
significant contact with the conduit walls and thus is another
fluid segment.
[0012] Whether two immiscible fluids, when present in a conduit,
form fluid segments of the first and second of the two immiscible
fluids, slugs of the first immiscible fluid, or emulsified droplets
of the first immiscible fluid depends on at least the method of
introduction of each fluid into the conduit, the relative surface
energies of the first immiscible fluid, the second immiscible
fluid, and the conduit material, the contact angle each forms with
the other two materials, respectively, and the volume of the
discrete volume of the first immiscible fluid. Thus, it is
recognized that these definitions are merely reference points on a
continuum, the continuum of the shape and size of discrete volumes
of a first immiscible fluid in a conduit, and discrete volumes will
exist that, when described, fall between these definitions.
[0013] The molecular biology procedures can, for example, utilize
proteins or nucleic acids. Procedures with nucleic acids can
comprise, for example, a PCR amplification and/or nucleic acid
analysis of an amplification product. The PCR amplification and/or
nucleic acid analysis of an amplification product can comprise an
integrated DNA amplification/DNA sequencing method.
[0014] Using the apparatus, methods, and/or systems provided in
this application, a polymerase chain reaction (PCR) amplification
of single DNA molecules can be performed, for example, to obtain
amplicons. The amplified DNA or amplicons can then be used in a
sequencing reaction and then be sequenced in small volumes. Other
manipulations of nucleic acids or proteins can also be
accomplished, for example, DNA hybridization reactions or
antibody-antigen binding assays.
[0015] The apparatus, system and/or methods described herein can
also be used in conjunction with U.S. Provisional Patent
Application No. 60/710,167 entitled "Sample Preparation for
Sequencing" to Lee et al., filed Aug. 22, 2005 (Attorney Docket No.
5841P), U.S. Provisional Patent Application No. 60/731,133 entitled
"Method and System for Spot Loading a Sample" to Schroeder et al.,
filed Oct. 28, 2005 (Attorney Docket No. 5010-288), and systems
described in U.S. Provisional Patent Application No. 60/818,197
filed June 30, 2006, each of which are incorporated herein in their
entireties by reference.
[0016] An exemplary type of sample preparation can be used for
genotyping, gene-expression, methylation analysis, and/or directed
medical sequencing (VariantSEQr.TM., for example, an Applied
Biosystems product comprising primers for resequencing genes and
detecting variations) that requires multiple liquids to be brought
together in an aqueous discrete volume. For example, in a
gene-expression application, each aqueous discrete volume can
contain individual primer sets. The sample to be analyzed, for
example, complementary DNA (cDNA), can be added to each aqueous
discrete volume. In the VariantSEQrm application, for example, an
aqueous discrete volume can comprise a primer set and genomic DNA
can be added to that discrete volume. According to various
embodiments, a system and method are provided that are able to
generate discrete volumes with unique content. According to various
embodiments of the present teachings, sipping, other aspirating, or
other techniques to generate immiscible-liquid, discrete volumes
can be used. According to various embodiments, an
immiscible-liquid, discrete volume of at least an aqueous sample
fluid can be generated in a tube by alternately drawing into the
tube the aqueous sample fluid and spacing fluid, with which the
aqueous sample fluid is immiscible, from a single container or well
containing both fluids or from different containers or wells each
containing one of the two fluids.
[0017] Using the apparatus, methods, and/or systems provided in
this application, one can control the positioning of
immiscible-fluid, discrete volumes during processing. These
processes can include, for example, polymerase chain reaction (PCR)
amplification of single DNA molecules to obtain, for example,
amplicons. The amplified DNA or amplicons can then be used in a
sequencing reaction and be sequenced using small volumes. Other
manipulations of nucleic acids or proteins can also be
accomplished, for example, DNA hybridization reactions or
antibody-antigen binding assays.
[0018] In some embodiments, flow rates for moving aqueous discrete
volumes can comprise rates of from about 1 picoliter/sec. to about
200 microliters/sec., and can be selected based on the inner
diameter of the conduits through which the liquids are to be
pumped. Within that broad range, in some embodiments, the aqueous
discrete volumes comprising 50% reagents, for example, PCR
reagents, and 50% other reagents, for example, sample fluid, in
contact with oil can flow at 0.5 microliter/sec, or in a range from
about 0.1 microliter/sec. to 2 microliters/sec. In some
embodiments, aqueous volumes comprising reagents, for example, exo
SAP or sequencing mix, can flow at a rate of about 1/3
microliters/sec., or in a range from about 0.1 microliters/sec. to
1 microliters/sec. In some embodiments, when aspirating aqueous
fluid into a conduit as discrete volumes, flow rates of up to about
0.1 microliters/sec. max can be used. In some embodiments, when
outputting an aqueous discrete volume, a flow rate of about 1/3 to
about 1/2 ul/sec can be used, or in a range from about 0.1
microliters/sec. to about 2 microliters/sec. can be used.
[0019] Tubing that can be used with the 1 picoliter/sec. to 200
microliter/sec. flow rate can comprise an inner diameter of from
about 250 microns to about 1000 microns. In other embodiments, the
inner diameter of the inner tube can be from about 10 microns to
about 2000 microns, while the inner diameter of the outer tube can
be from about 20 microns to about 5000 microns, for example, from
about 35 microns to about 500 microns. Other diameters, however,
can be used based on the characteristics of the slug processing
system desired. In some embodiments, a tube having a 10 micron
inner diameter is used with a flow rate of from about 8 to about 10
picoliters/second. In some embodiments, a tube having a 5000 micron
inner diameter is used with a flow rate of from about 25 to about
200 microliters/second. In some embodiments, a tube having a 500
micron inner diameter is used with a flow rate of from about 0.25
to about 2.0 microliters/second.
[0020] The aqueous sample liquid, which in some embodiments can
form discrete volumes in contact with a fluid with which it is
immiscible, can comprise a plurality of target nucleic acid
sequences, wherein at least one of the discrete volumes comprises
at least one target nucleic acid sequence. In some embodiments,
after formation, at least 37% of the plurality of the discrete
volumes in the inner conduit can each comprise a single target
nucleic acid sequence. In various other embodiments, less than
about 37% of the plurality of discrete volumes in the conduit can
each comprise a single target nucleic acid sequence. In other
embodiments, at least 1% or more, 5% or more, 10% or more, or 20%
or more can have a single target nucleic acid sequence, for
example, upon formation of the discrete volumes.
[0021] According to various embodiments, each of the plurality of
discrete volumes in the inner conduit can comprise one or more
respective oligonucleotide primers. Oligonucleotide primers can be
chosen as determined by one of skill in the art to accomplish the
desired objective. For example, universal primers can be used.
[0022] In some embodiments, further downstream processing of the
prepared immiscible-fluid-discrete-volumes can be integrated with
the system, of which embodiments are described herein. Such
downstream processing can include amplifying the at least one
target nucleic acid sequence in the first discrete volume in the
conduit to form an amplicon, and thereafter subjecting the amplicon
to a nucleic acid sequencing reaction. For such purposes, and in
some embodiments, the discrete volumes or
immiscible-fluid-discrete-volumes can comprise reaction components,
for example, oligonucleotide primers. Various embodiments of
downstream processing can include universal PCR, or can comprise
up-front multiplexed PCR followed by decoding, for example, see WO
2004/051218 to Andersen et al., U.S. Pat. No. 6,605,451 to Marmaro
et al., U.S. patent application Ser. No. 11/090,830 to Andersen et
al., and U.S. patent application Ser. No. 11/090,468 to Lao et al.,
all of which are incorporated herein in their entireties by
reference. Details of real time PCR can be found in Higuchi et al.,
U.S. Pat. No. 6,814,934 B1, which is incorporated herein by
reference in its entirety.
[0023] Further devices, systems, and methods that can be used with
or otherwise implement the present teachings include those
described, for example, in U.S. patent application Ser. No. ______,
filed Aug. 22, 2006, entitled "Apparatus, System, and Method Using
Immiscible-Fluid-Discrete-Volumes," to Lee et al. (attorney docket
number 5010-362), in U.S. patent application Ser. No. ______,
entitled "Device and Method for Making Discrete Volumes of a First
Fluid in Contact With a Second Fluid, Which are Immiscible With
Each Other," to Cox et al. (attorney docket number 5010-363), and
in U.S. patent application Ser. No. ______, filed Aug. 22, 2006,
entitled "Apparatus and Method for Depositing Processed
Immiscible-Fluid-Discrete-Volumes," to Schroeder et al. (attorney
docket number 5010-365), which are herein incorporated in their
entireties by reference.
DRAWINGS
[0024] The skilled artisan will understand that the drawings
described below are for illustrative purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way. In the drawings:
[0025] FIGS. 1A and 1B illustrate a system using microfluidic
immiscible-fluid, discrete volumes in which nucleic acid assays can
be performed;
[0026] FIG. 2 illustrates a system using microfluidic
immiscible-fluid, discrete volumes in which nucleic acid assays can
be performed;
[0027] FIG. 3 illustrates a cross-sectional view in the XY plane of
an embodiment of a rotary valve;
[0028] FIG. 4 illustrates a cross-sectional view in the XY plane of
another embodiment of a rotary valve; the rotor of the rotary valve
having a two ducts therethrough, one of which is aligned with a
duct through the stator; and
[0029] FIG. 5 illustrates a cross-sectional view in the XY plane of
the rotary value of FIG. 4, where the relative positions of the
rotor and stator have changed such that the second of the two
parallel ducts is in fluid communication with the first and third
ducts through the stator;
[0030] FIG. 6 illustrates a cross-sectional view in the XY plane of
an embodiment of a linear valve in the open position;
[0031] FIG. 7 illustrates a cross-sectional view in the XY plane of
the linear valve of FIG. 6 in a closed position;
[0032] FIG. 8 illustrates a side view of an embodiment of a pinch
valve in an open position;
[0033] FIG. 9 illustrates a front, cross-sectional view of the
pinch valve of FIG. 8;
[0034] FIG. 10 illustrates a front, cross-sectional view of the
pinch valve of FIG. 8, but in a closed position;
[0035] FIG. 11A illustrates an embodiment of a system that can be
used to (1) combine two miscible fluid streams in preset volumetric
ratios, (2) split an immiscible-fluid, discrete volume into two
smaller, distinct, discrete volumes, (3) generate immiscible-fluid,
discrete volumes of a first fluid in a consistent ratio with a
second fluid with which it is immiscible and (4) combine a first
immiscible-fluid, discrete volume with a second immiscible-fluid,
discrete volume;
[0036] FIG. 11B illustrates the system of FIG. 11A, except the
three-way valves are illustrated with the common port of each
connected to the second of the two input/output ports and the
displacement pumps have been moved in the opposite direction;
[0037] FIG. 12 illustrates an embodiment of a slug splitter with a
slug in position in the supply tube to split;
[0038] FIG. 13 illustrates the slug splitter with a first of the
two portions of the split slug in fluid communication with the
first-split-portion conduit;
[0039] FIG. 14 illustrates the slug splitter after the first of two
portions of the slug has flowed into the first-split-portion tube
and spacing fluid has flowed into the slider through-hole;
[0040] FIG. 15 illustrates the slug splitter with the spacing fluid
in the slider in fluid communication with the second-split-portion
conduit;
[0041] FIG. 16 illustrates advancing the second of two portions of
the split slug through the slider and into the second-split-portion
conduit;
[0042] FIG. 17 illustrates an embodiment of a bubble remover;
[0043] FIG. 18 illustrates another embodiment of a bubble
remover;
[0044] FIG. 19 illustrates an embodiment of an apparatus for
pressurizing an immiscible-fluid-discrete-volume processing
system;
[0045] FIG. 20 illustrates another embodiment of an apparatus for
pressuring an immiscible-fluid-discrete-volume processing
system;
[0046] FIG. 21 illustrates a system of fluidic control using
optical detectors to provide feedback;
[0047] FIG. 22 illustrates an embodiment of an inspection
system;
[0048] FIG. 23 illustrates using a "T" junction to add liquid to a
conduit containing aqueous discrete volumes, with a constant flow
rate of the "addition" liquid matched to add a pre-determined
volume of liquid either before or after an aqueous discrete volume
or to pre-existing aqueous discrete volumes moving past the
junction at a known speed;
[0049] FIG. 24 illustrates using a "T" junction to add discrete
volumes of aqueous fluid in one of the vertical conduits to one or
more discrete volumes of aqueous fluid in a main conduit, the flow
rates and spacing of the immiscible-fluid, discrete volumes in each
conduit being controlled to ensure coalescence;
[0050] FIG. 25 illustrates using a dedicated displacement pump for
each junction of an addition tube and an
immiscible-fluid-discrete-volume supply tube;
[0051] FIG. 26 illustrates a system using a single pressure pump to
positively pressurize liquid supplies in addition tubes to multiple
T-junctions, and a valve for each addition tube to meter a known
volume of liquid to the immiscible-fluid-discrete-volume supply
tube;
[0052] FIG. 27 uses a single pressure pump to positively pressurize
all liquid supply vessels and, as a result, the addition tube to
each individual T-junction, and a valve to meter the amount of
liquid added to the immiscible-fluid-discrete-volume supply
tube;
[0053] FIG. 28 illustrates using a T-junction and valving to merge
two streams of immiscible-fluid, discrete volumes into a stream of
larger-volume, immiscible-fluid discrete volume;
[0054] FIG. 29A-C illustrates using adding microdroplets (e.g.,
stable emulsified nanodroplets) of aqueous liquid using a T to
merge with slugs in main tube after being held by a field and then
expelled from vertical tube into the main conduit;
[0055] FIG. 30 illustrates a system for moving sets of
immiscible-fluid, discrete volumes to a storage tube under vacuum
before introducing them under positive pressure to the main conduit
system of a immiscible-fluid-discrete-volume processing system;
[0056] FIG. 31 illustrates the positions of sets of
immiscible-fluid, discrete volumes in an apparatus during steps of
a method for manipulating them into a processing system; and
[0057] FIG. 32 illustrates the positions of sets of
immiscible-fluid, discrete volumes in an apparatus during steps of
another method for manipulating them into a processing system.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0058] It is to be understood that the following descriptions are
exemplary and explanatory only. The accompanying drawings are
incorporated in and constitute a part of this application and
illustrate several exemplary embodiments with the description.
Reference will now be made to various embodiments, examples of
which are illustrated in the accompanying drawings. Wherever
possible, the same reference numbers are used in the drawings and
the description to refer to the same or like parts.
[0059] Throughout the application, descriptions of various
embodiments use "comprising" language, however, it will be
understood by one of skill in the art, that in some specific
instances, an embodiment can alternatively be described using the
language "consisting essentially of" or "consisting of."
[0060] For purposes of better understanding the present teachings
and in no way limiting the scope of the teachings, it will be clear
to one of skill in the art that the use of the singular includes
the plural unless specifically stated otherwise. Therefore, the
terms "a," "an" and "at least one" are used interchangeably in this
application.
[0061] Unless otherwise indicated, all numbers expressing
quantities, percentages or proportions, and other numerical values
used in the specification and claims, are to be understood as being
modified in all instances by the term "about." Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
the following specification and attached claims are approximations
that may vary depending upon the desired properties sought to be
obtained. In some instances, "about" can be understood to mean a
given value+5%. Therefore, for example, about 100 nl, could mean
95-105 nl. At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques.
[0062] Reference to "nucleotide" should be understood to mean a
phosphate ester of a nucleotide, as a monomer unit or within a
nucleic acid. Nucleotides are sometimes denoted as "NTP", or "dNTP"
and "ddNTP" to particularly point out the structural features of
the ribose sugar. "Nucleotide 5'-triphosphate" can refer to a
nucleotide with a triphosphate ester group at the 5' position. The
triphosphate ester group may include sulfur substitutions for the
various oxygens, for example, .alpha.-thio-nucleotide
5'-triphosphates. Nucleotides can comprise a moiety of substitutes,
for example, see, U.S. Pat. No. 6,525,183 B2 to Vinayak et al.,
incorporated herein by reference in its entirety.
[0063] The terms "polynucleotide" or "oligonucleotide" or "nucleic
acid" can be used interchangeably and include single-stranded or
double-stranded polymers of nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, or internucleotide
analogs, and associated counter ions, for example, H+, NH4+,
trialkylammonium, Mg2+, Na+ and the like. A polynucleotide may be
composed entirely of deoxyribonucleotides, entirely of
ribonucleotides, or chimeric mixtures thereof. Polynucleotides may
be comprised of nucleobase and sugar analogs. Polynucleotides
typically range in size from a few monomeric units, for example,
5-40 when they are frequently referred to in the art as
oligonucleotides, to several thousands of monomeric nucleotide
units. Unless denoted otherwise, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.
A labeled polynucleotide can comprise modification at the
5'terminus, 3'terminus, a nucleobase, an internucleotide linkage, a
sugar, amino, sulfide, hydroxyl, or carboxyl. See, for example,
U.S. Pat. No. 6,316,610 B2 to Lee et al. which is incorporated
herein by reference. Similarly, other modifications can be made at
the indicated sites as deemed appropriate.
[0064] The term "reagent," should be understood to mean any
reaction component that in any way affects how a desired reaction
can proceed or be analyzed. The reagent can comprise a reactive or
non-reactive component. It is not necessary for the reagent to
participate in the reaction. The reagent can be a recoverable
component comprising for example, a solvent and/or a catalyst. The
reagent can comprise a promoter, accelerant, or retardant that is
not necessary for a reaction but affects the reaction, for example,
affects the rate of the reaction. A reagent can comprise, for
example, one member of a binding pair, a buffer, or a DNA that
hybridizes to another DNA. The term "reagent" is used synonymous
with the term "reaction component."
[0065] Methods, apparatuses and systems described herein can use
fluids immiscible in each other. Fluids can be said to be
immiscible in each other when they can be maintained as separate
fluid phases under conditions being used. Immiscible fluids can
also be said to be incapable of mixing with each other or attaining
a solution with each other. An aqueous liquid and a non-aqueous
liquid such as oil can be said to be immiscible with each other.
Throughout the specification, reference is made to aqueous slugs.
This is merely exemplary and does not necessarily preclude the use
or manufacture of non-aqueous liquid slugs in combination with an
immiscible liquid.
[0066] While oil and aqueous liquids are immiscible in each other,
such a combination does not necessarily form aqueous
immiscible-fluid-discrete-volumes in the oil when the two liquids
are mixed or placed together. For example, oil may form the
disperse phase in a continuous aqueous liquid in a larger volume,
as it does in certain salad dressings. For another example, oil and
aqueous liquids may merely form aqueous droplets or microdroplets
in a larger volume of oil, but not necessarily aqueous
immiscible-fluid-discrete-volumes. Aqueous
immiscible-fluid-discrete-volumes can form, however, using an
apparatus such as, for example, those described in U.S. patent
application Ser. No. ______, entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363).
[0067] Aqueous solutions and oil from separate sources can be
combined to form a continuous flowing liquid stream comprising
aqueous immiscible-fluid-discrete-volumes separated from one
another by the oil. Because the aqueous
immiscible-fluid-discrete-volumes entirely or almost entirely fill
the cross-sectional area of the conduit or tube in which they are
formed, the resulting stream of aqueous
immiscible-fluid-discrete-volumes in oil can exhibit a banded
appearance. According to various embodiments, such a pattern can be
exhibited by combining any two immiscible fluids with one another.
The pattern can be formed throughout the length of the conduit. In
various embodiments, a first aqueous
immiscible-fluid-discrete-volume can contain different reagents
than a second aqueous immiscible-fluid-discrete-volume. In other
words, not all aqueous immiscible-fluid-discrete-volumes throughout
the conduit need to contain the same reagents.
[0068] An aqueous immiscible-fluid-discrete-volume can be spaced
apart from an adjacent aqueous immiscible-fluid-discrete-volume by
the oil. In various embodiments, liquids other than oil can act as
a spacing fluid, provided that the spacing fluid and aqueous fluid
are immiscible with respect to each other and provided that they
can form individual aqueous immiscible-fluid-discrete-volumes
spaced apart from one another by the spacing fluid. In various
embodiments, gas can be used as a spacing fluid.
[0069] According to various embodiments, methods are provided that
refer to processes or actions involved in sample preparation and
analysis. It will be understood that in various embodiments a
method can be performed in the order of processes as presented,
however, in related embodiments, the order can be altered as deemed
appropriate by one of skill in the art in order to accomplish a
desired objective.
[0070] According to various embodiments, an apparatus is provided
that can be used as a front-end sample preparation device for
high-throughput sequencing, or other applications requiring
preparation and/or processing of a plurality of small samples. The
sample liquid that can become an immiscible-fluid-discrete-volume
can comprise, for example, nucleic acids, proteins, polypeptides,
carbohydrates, or the like. The apparatus can be part of an
integrated system and/or be adapted to function with other pieces
of equipment adapted for further sample processing of samples, for
example, an ABI 310, ABI 3130, ABI 3130x1, ABI 3700, ABI 3730, or
ABI 3730x1 capillary electrophoresis analyzer (available from
Applied Biosystems, Foster City, Calif.) that can be used for
sequencing. In some embodiments, the apparatus can be part of an
integrated system and/or be adapted to function with other pieces
of equipment adapted for further sample processing of samples, for
example, a PCR detector. Exemplary detectors that can be used
include real-time sequence detection systems and real-time PCR
detectors, for example, the ABI 7900, available from Applied
Biosystems, Foster City, Calif.
[0071] The apparatus, system and/or methods described herein can
also be used in conjunction with downstream processing of
immiscible-fluid-discrete-volumes in conduits as described, for
example, in FIGS. 10 and 11 of U.S. Provisional Patent Application
No. 60/710,167 entitled "Sample Preparation for Sequencing" to Lee
et al., filed Aug. 22, 2005 (Attorney Docket No. 5841P), or U.S.
Provisional Patent Application No. 60/731,133 entitled "Method and
System for Spot Loading a Sample" to Schroeder et al., filed Oct.
28, 2005 (Attorney Docket No. 5010-288) which applications are
incorporated herein in their entireties by reference. If there is
any discrepancy between the description of a slug in an immiscible
fluid in the above provisional applications and this one, this
application is deemed to be correct.
[0072] FIGS. 1A and 1B are the left-side and right-side,
respectively, of a schematic diagram detailing an example of a
fluid processing system 10 for processing immiscible-fluid,
discrete volumes. The six conduits on the right-hand side of FIG.
1A and terminating in arrow heads pointing to the right are
respectively continued as the six conduits shown on the left-hand
side of FIG. 1B and terminating in arrow heads pointing to the
left, such that the top tube of each respective six depicted are
continuations of each other, and so on going down the figures.
[0073] Generally, system 10 can be configured to perform different
types of assays on fluids introduced thereinto. The amounts and
types of fluids introduced into system 10 can be varied depending
on a particular assay to be performed. Exemplary assays can
include, for example, de novo nucleic acid sequencing reactions,
and nucleic acid resequencing reactions, as discussed herein. An
exemplary type of sample preparation can be used for genotyping,
gene-expression, methylation analysis, and/or directed medical
sequencing (VariantSEQr.TM., for example) that requires multiple
liquids to be brought together in an aqueous discrete volume. For
example, in a gene-expression application, each aqueous discrete
volume can contain individual primer sets. The sample to be
analyzed, for example, complementary DNA (cDNA), can be added to
each aqueous discrete volume. In the VariantSEQr.TM. application,
for example, an aqueous discrete volume can comprise a primer set,
and genomic DNA can be added to that discrete volume
[0074] According to various embodiments, one or more samples 22,
24, can be introduced to system 10. Samples 22 and 24, for example,
can comprise a nucleic acid containing fluid. According to some
embodiments, the nucleic acid contained in a sample can be, for
example, a single copy of a genomic DNA sequence of an organism, or
complementary DNA from an organism.
[0075] In some embodiments, a plurality of fluids can be received
by fluid processing system 10 through an
immiscible-fluid-discrete-volume-forming tube 12, which is a part
of main conduit system 50. Other types of conduits may be used
instead of a tube to accept the introduction of fluids into fluid
processing system 10. Immiscible-fluid-discrete-volume-forming tube
12 can be part of a system that can comprise, for example, a pump
or another apparatus adapted to produce controlled intake of fluids
through intake tip 13 into immiscible-fluid-discrete-volume-forming
tube 12. Motive force providers for moving fluids into and along a
tube or other type of conduit include differential pressure,
displacement, electowetting, optoelectrowetting,
magnetohydrodynamic "pumps" among others. The
immiscible-fluid-discrete-volume-forming conduit 12 can be adapted
to control an introduction unit to introduce alternate volumes of
aqueous sample fluid and spacing fluid that together form discrete
volumes of aqueous sample fluid in contact with spacing fluid,
i.e., aqueous sample immiscible-fluid-discrete-volumes, in the at
least one conduit wherein each aqueous sample
immiscible-fluid-discrete-volume can comprise a maximum outer
dimension that is equal to or slightly less than the maximum inner
cross-sectional dimension of
immiscible-fluid-discrete-volume-forming conduit 12. One of skill
in the art will understand that the maximum inner cross-sectional
dimension of a tube is the inner diameter of the tube if the tube
has a circular cross-section.
[0076] According to various embodiments,
immiscible-fluid-discrete-volume-forming tube 12 can comprise a tip
13. Tip 13 can interface with fluids to be drawn into system 10.
Tip 13 can comprise an angled surface or have any suitable geometry
such that the creation of air bubbles in
immiscible-fluid-discrete-volume-forming tube 12 is minimized or
eliminated when tip 13 contacts and draws in a fluid.
Immiscible-fluid-discrete-volume-forming tube 12 can be robotically
controlled, or manually controlled. Robotic configurations can
comprise, for example, stepper motors 14, 16, and 18, which can
move immiscible-fluid-discrete-volume-forming tube 12 in X-axis,
Y-axis, and Z-axis directions, respectively. In some embodiments,
tube 12 can be moved in the Z-axis direction by a stepper motor 18,
and a fluid container can be moved in the X-axis and Y-axis
directions by stepper motors 14 and 16, respectively. In some
embodiments, tube 12 can be stationary and a fluid container can be
moved in the X-axis, Y-axis, and Z-axis directions by stepper
motors 14, 16, and 19, respectively. Motive force providers other
than stepper motors can be used.
[0077] According to various embodiments, a variety of fluids can be
introduced into fluid processing system 10, in a number of
different combinations, depending on the particular type of assay
to be performed. The fluids can reside in or on any suitable fluid
retaining device, for example, in the wells of a multi-well plate
20, an opto-electrowetting plate, a tube of preformed slugs, a tube
of stable emulsified nanodroplets, individual tubes, strips of
tubes, vials, flexible bags or the like.
[0078] According to some embodiments, fluid processing system 10
can comprise a number of different fluid conduits and fluid control
devices. The following description applies to the embodiment as
illustrated in FIGS. 1A and 1B, but one skilled in the art will
understand that alterations to fluid processing system 10 can be
made while the teachings remain within the scope of the present
teachings. As illustrated, fluid processing system 10 can comprise
a main system conduit 50. Main conduit system 50 can comprise a
plurality of tubes connecting together, for example, the following
exemplary components: T-junctions 52, 66, and 84; holding conduits
56, 60, 63, 64 and 65; valves V-1, V-2, V-5, V-6, V-7, V-8, V-9,
V-10, V-11, V-12, and V-13; cross-junctions 68, 70, 76, 86, and 88;
and thermal-spirals 74, 80, 90, and 92. Along conduit 50, thermal
spirals 74, 80, 90, and 92 can be in thermal contact with
respective thermal cyclers 74A, 80A, 90A, and 92A. Each thermal
cycler 74A, 80A, 90A, and 92A can independently comprise a liquid
bath, an oven, a plate, a block comprising fluid passages therein,
a peltier device, or the like thermal cycling device.
[0079] Main conduit system 50 can provide a fluid communication
between T-junction 52 and output tube 54. From T-junction 52,
conduit system 50 comprises two pathways that intersect at
cross-junction 68 and at T 66. A first pathway can take a fluid
sequentially through holding tubes 56, 60 and 64, and T-junction
66, before reaching cross-junction 68. A second pathway can take a
fluid sequentially through holding tubes 63, and 65, and through
either T-junction 66, to cross-junction 68, or directly to
cross-junction 68. Both the first pathway and the second pathways
are configured to hold fluids for later analysis and are configured
to interface with pumps for moving fluids along the conduits as
discussed below.
[0080] From cross-junction 70, fluids can move sequentially to
thermal-spiral 74, cross-junction 76, thermal-spiral 80, and
T-junction 84. At T-junction 84 fluids can sequentially move either
through cross-junction 86, thermal-spiral 90, and output tube 54,
or through cross-junction 88, thermal-spiral 92, and an output tube
54.
[0081] According to some embodiments fluid processing system 10 can
comprise pumps 39 and 40. Pump 40 can be configured to remove or
add oil to main conduit system 50, and thereby move fluids located
therein. Pump 39 can be configured to remove or add oil to main
conduit system 50 to move fluids located therein. All of the pumps
described herein can create positive and/or negative pressures in
the various conduits of system 10.
[0082] According to various embodiments, a T-junction can comprise
any junction having three discrete pathways extending from, for
example, either a Y-junction or a T-junction. In various
embodiments, the junction can comprise a valve-less junction where
a stream of aqueous sample fluid and a stream of non-aqueous
spacing fluid can meet and form at least discrete volumes of the
aqueous sample fluid in contact with the non-aqueous spacing fluid.
For example, microfabrication technology and the application of
electrokinetics or magneto hydrodynamics can achieve fluid pumping
in valve-less, electronically controlled systems. Components
comprising shape-optimized channel turns, optimal injection
methods, micromixers, and/or high flow rate electroosmotic pumps
can be used in such a valve-less system.
[0083] According to some embodiments, system 10 can comprise
discrete volume detectors D-1, D-2, D-3, D-4, D-5, D-6, D-7, D-8,
D-9, D-10, D-11, D-12, D-13, D-14, D-15, D-16, D-17, D-18, D-19,
and D-20, and detector 98. The discrete-volume detectors can
comprise, for example, fluorescent or infra-red, refractive-index
detectors, and possibly capacitive and absorption detectors. In
FIGS. 1A and 1B, all of the detectors depicted are infrared
detectors with the exception of detector 98 which is a fluorescent
signal detector, although other arrangements can be used. The
discrete volume detectors can be configured to distinguish
immiscible-fluid, discrete volumes from spacing fluid or oil as the
discrete volumes travel through the conduits of system 10.
[0084] According to various embodiments, the system can comprise a
thermal-cycling device or thermal cycler, adapted to thermally
cycle an aqueous immiscible-fluid, discrete volume in a conduit
disposed thereon or therein. In some embodiments, the conduit can
contact the thermal cycler in a single straight-line segment, or a
coil around the external perimeter of thermal cycler, or a spiral
of decreasing radius on one surface, or a serpentine pattern across
one or more surfaces of thermal cycler. The thermal-cycling device
can comprise a heat source, for example, a radiant heat source, a
non-radiant heat source, a peltier device, or the like, and a
cooling source, for example, a fan, an air jet, or a
liquid-circulating system in a thermal block. The thermal-cycling
device can comprise one or more temperature sensors and one or more
control units for controlling heating and cooling according to a
desired or programmed thermal cycle.
[0085] The conduits of the present teachings can comprise capillary
tubes having an inner diameter and the inner diameter can be, for
example, about 1000 microns or less, for example, about 800 microns
or less, or about 500 microns or less. In some embodiments, the
tube has a minimum inner dimension, or diameter, of from about 1.0
micron to about 100 microns, or from about 50 microns to about 75
microns. In other embodiments, the tube can have an inner diameter
greater than about 300 microns. In some embodiments, main conduit
system 50 can comprise tubing with a 635 micron inner diameter. In
some embodiments, thermal spirals after T-84 can comprise tubing
with a 480 micron inner diameter. In some embodiments conduit
system 50 can comprise tubing with an inner diameter in a range of
from about 380 microns to about 635 microns, with the smallest
diameters at the ends of the system. Other details about the
thermal-cycling device, capillary channel, and other system
components will become apparent in view of the teachings
herein.
[0086] System 10 can comprise an single molecule amplification
fluid ("SMAF") conduit system 51. SMAF tube system 51 can supply
sample fluid to a T-junction through positive pressure rather than
by aspiration. SMAF conduit system 51 can comprise a supply conduit
connected to and in fluid communication with a supply of single
molecule amplification fluid. The SMAF can comprise a solution or
mixture of target nucleic acids diluted to a degree such that there
is an average of less than about one target nucleic acid per volume
of single molecule amplification fluid that is used to make an
immiscible-fluid-discrete-volume. An exemplary concentration of
target molecules can be 0.4 molecule per volume used to make an
immiscible-fluid-discrete-volume. SMAF conduit system 51 can
comprise conduits connecting a SMAF reservoir 69 sequentially to
valve V-18 and T-junction 67. SAMF conduit system 51 can comprise
conduits that connect T-junction 67 to main conduit system 50 and a
rotary valve 71.
[0087] Fluid processing system 10 can comprise rotary valves 71,
73, 75, 77, and 79. Each rotary valve can function to direct the
flow of metered amounts of different reagents from different
respective reagent reservoirs connected thereto, as described
below, to main conduit system 50. Syringe pumps 58, 66, 78, and 82
can be in fluid communication with rotary valves 73, 75, 77 and 79,
respectively. Pumps 42, 43, 44, and 45 can be in fluid
communication with rotary valves 73, 75, 77 and 79,
respectively.
[0088] Fluid processing system 10 can comprise a first waste tube
system 81. Waste tube system 81 can comprise conduits connecting
the following components: valves V-17, V-20, V-21, V-22, V-23,
V-24, V-25, and a waste reservoir 83. Waste tube system 81 can
provide a fluid communication between and cross-junctions 68, 70,
76, 86, and 88 and waste reservoir 83.
[0089] Fluid processing system 10 can comprise a second waste tube
system 48. Second waste tube system 48 can comprise conduits
connecting a pump 87, a waste reservoir 85, and a valve V-26, that
interface with output tube 54. Second waste tube system 48 can be
used to remove liquids from output tube 54.
[0090] Fluid processing system 10 can comprise reagent reservoirs
89, 91, 93, 95, 97, and 99, which can be in fluid communication
with rotary valves 75, 77, 77, 79, 79, and 73, respectively.
Reagent reservoir 89 can contain, for example, an exo-nuclease and
shrimp alkaline phosphatase. Reagent reservoir 91 can contain, for
example, nucleic acid amplification reaction forward primers.
Reagent reservoir 93 can contain, for example, nucleic acid
amplification reaction chain terminating dyes. Reagent reservoir 95
can contain, for example, nucleic acid amplification reaction
reverse primers. Reagent reservoir 97 can contain, for example,
nucleic acid amplification reaction chain terminating dyes. Reagent
reservoir 99 can contain, for example, a nucleic acid amplification
reaction master mix comprising, for example, reactive single base
nucleotides, buffer, a polymerase, and the like, for example, to
carry out a polymerase chain reaction.
[0091] According to various embodiments, fluid processing system 10
can comprise a rinse system 30. Rinse system 30 can provide a fluid
communication between a rinse fluid reservoir 28, rotary valve 73,
rotary valve 75, rotary valve 77, rotary valve 79, and
immiscible-fluid-discrete-volume-forming tube 12. Rinse fluid
reservoir 28 can contain a rinse fluid 26. Rinse fluid 26 can
comprise microbiologic grade water, for example, distilled,
de-ionized water.
[0092] Rinse fluid 26 can be used to remove residual sample, for
example, from immiscible-fluid-discrete-volume-forming tube 12.
Rinse fluid can be provided to multi-well plate 20, by way of rinse
tube system 30. In some embodiments, rinse fluid 26 can be added to
immiscible-fluid, discrete volumes to adjust the volume or
concentration thereof, in conjunction with an addition station, as
described in FIG. 2.
[0093] According to various embodiments, fluid processing system 10
can comprise a spacing fluid tube system 36. Spacing fluid tube
system 36 can provide a fluid communication between a spacing fluid
reservoir 34, vacuum pump 41, and multi-well plate 20. Spacing
fluid reservoir 34 can contain an oil 32 or other spacing fluid
that is immiscible with an immiscible-fluid-discrete-volume-forming
fluid, for example, an aqueous slug fluid.
[0094] In some embodiments, the spacing fluid can be non-aqueous.
The spacing fluid can comprise an organic phase, for example, a
polydimethylsiloxane oil, a mineral oil (e.g., a light white
mineral oil), a silicon oil, a hydrocarbon oil (e.g., decane), a
fluorinated fluid or a combination thereof.
[0095] Fluorinated compounds such as, for example, perfluoroooctyl
bromide, perfluorodecalin, perfluoro-1,2-dimethylcyclohexane, FC
87, FC 72, FC 84, FC 77, FC 3255, FC 3283, FC 40, FC 43, FC 70, FC
5312 (all "FC" compounds are available from 3M, St. Paul, Minn.),
the Novec.RTM. line of HFE compounds (also available from 3M, St.
Paul, Minn.), such as, for example, HFE-7000, HFE-7100, HFE-7200,
HFE-7500, and perfluorooctylethane can also be used as the spacing
fluid. Combinations, mixtures, and solutions of the above materials
can also be used as the spacing fluid.
[0096] In some embodiments, fluorinated alcohols, such as, for
example, 1H, 1H, 2H, 2H-perfluoro-decan-1-ol, 1H, 1H, 2H,
2H-perfluoro-octan-1-ol, and 1H, 1H-perfluoro-1-nonanol can be
added to a fluorinated compound, such as those listed above, to
improve the stability of aqueous discrete volumes within the
spacing fluid, but still maintain the ability to coalesce upon
contact. In some embodiments, fluorinated alcohols can be added in
a range of approximately 0.1% to approximately 5% by weight. In
some embodiments, the fluorinated alcohol additive can be
approximately 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 3.0%, 4.0% or 5%
by weight of the fluorinated compound. In some embodiments, the
fluorinated alcohol additive can be from approximately 1% to
approximately 10% by volume of the fluorinated compound. In some
embodiments, the fluorinated alcohol additive may comprise
approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by volume
of the spacing fluid. In some embodiments, F-alkyl
dimorpholinophosphates can be added as surfactants to fluorinated
compounds.
[0097] In some embodiments, the organic phase can include non ionic
surfactants such as sorbitan monooleate (Span 80 (no. S-6760,
Sigma)), polyoxyethylenesorbitan monooleate (Tween 80 (no. S-8074,
Sigma)), sorbitan monostearate (Span 60), octylphenoxyethoxyethanol
(Triton X-100 (no. T9284, Sigma)). In some embodiments, Span 80 can
be added in an amount ranging from about 1.0% to about 5.0%, or
about 3.0% to about 4.5%. In some embodiments, adding surfactants
in the quantities of 4.5% Span 80, 0.40% Tween 80, and 0.05% Triton
X-100 to mineral (no. M-3516, Sigma) can result in the creation of
stable emulsified droplets.
[0098] In some embodiments, the organic phase can include ionic
surfactants, such as sodium deoxycholate, sodium cholate, and
sodium taurocholate. In some embodiments, the organic phase can
include chemically inert silicone-based surfactants, such as, for
example, polysiloxane-polycetyl-polyethylene glycol copolymer.
[0099] In some embodiments, the non-aqueous, spacing fluid can have
a viscosity between approximately 0.5 to approximately 0.75
centistokes. In some embodiments, the non-aqueous spacing fluid can
have a viscosity between approximately 0.75 centistokes to about
2.0 centistokes. In some embodiments, the non-aqueous spacing fluid
can have a viscosity greater than 2.0 centistokes. In some
embodiments, the non-aqueous spacing fluid can have a viscosity
between 0.5 to greater than about 2.0 centistokes. In some
embodiments, the non-aqueous spacing fluid can have a viscosity
greater than 2.0 centistokes. In some embodiments, the non-aqueous,
spacing fluid can have a boiling point greater than or equal to 100
C.
[0100] Spacing fluid 32 can function to separate discrete volumes
of an immiscible-fluid-discrete-volume-forming fluid, for example,
an aqueous sample fluid, before, during, or after the
immiscible-fluid-discrete-volume-forming fluid has been introduced
into system 10. Spacing fluid can be provided to multi-well plate
20, from a spacing fluid reservoir 34, by way of a spacing fluid
tube system 36.
[0101] According to some embodiments, a de novo nucleic acid
sequencing method is provided that uses system 10. The de novo
sequencing method can be used to sequence an entire genome or
portions thereof. The de novo sequencing method can be especially
useful when the sequence of the organism is unknown.
[0102] In some embodiments, a de novo sequencing method comprises
pre-processing a sample, separating the sample into a set of
immiscible-fluid, discrete volumes, optionally adding amplification
reagents to each discrete volume of the set, amplifying nucleic
acids in the set of immiscible-fluid, discrete volumes to form a
set of amplified immiscible-fluid, discrete volumes, optionally
detecting, and removing, discrete volumes without amplified sample
molecules therein, adding primer and dNTP deactivation agents to
each discrete volume in the set, or optionally, to only those with
amplified sample molecules, incubating the set of amplified
immiscible-fluid, discrete volumes with primer and dNTP
deactivation agents, subjecting the resulting nucleic acids to
sequencing conditions to form detectable products, and detecting
the detectable products.
[0103] In some embodiments, the method can comprise pre-processing
a sample before it is input into system 10. The pre-processing of a
sample can comprise fragmenting the nucleic acid present in the
sample. The fragmentation can be accomplished by any suitable
method known in the art. For example, the nucleic acid can be
fragmented by enzymatic digestion, or physical disruption methods,
for example, hydro-shearing or sonication. In some embodiments the
nucleic acid can be fragmented to an average size of about 500 B,
750 B, 850 B, 1 KB, 2 KB, or 3 KB, for example.
[0104] According to some embodiments, the pre-processing of sample
can comprise ligating sequences to a sample. In universal sequences
can be used to facilitate universal nucleic acid amplification.
Universal sequences can be artificial sequences that generally have
no homology with the target nucleic acids. Universal sequences can
be designed to resist the formation of dimers between themselves.
Universal sequences can be designed to bind with analogous primers
with a consistent efficiency.
[0105] According to some embodiments, the present teachings can
encompass a de novo sequencing method wherein universal sequences
can be ligated to the 5' and 3' ends of the DNA fragments in a
sample by, for example, T-4 DNA ligase, thereby forming a universal
tail. The universal tail sequences can function as sites of
complementarity for zip code primers. Details of universal tail
procedures can be found in U.S. Pat. App. No. 2004/0185484, to
Costa et al., which is incorporated herein, in its entirety, by
reference.
[0106] According to various embodiments, the amplifying of a
nucleic acid can comprise a thermal cycling nucleic acid sequence
amplification process or an isothermal nucleic acid sequence
amplification process. If a thermal cycling nucleic acid sequence
amplification process is used, the process can comprise, for
example, a polymerase chain reaction (PCR). The nucleic acid
sequence amplification reaction can comprise an exponential
amplification process, for example, PCR, or a linear amplification
process, as can occur during, for example, during Sanger cycle
sequencing. In various embodiments, other nucleic acid
amplification processes can be used, for example, ligase chain
reaction (LCR), nucleic acid sequence based amplification (NASBA),
Q-beta replicase (QB) amplification, or strand displacement
amplification (SDA). These alternatives, as well as others known to
one skilled in the art, can be used either by themselves or in
combination with PCR to amplify nucleic acids.
[0107] According to various embodiments, nucleic acid sequence
processing methods comprising a first type of nucleic acid
amplification reaction followed by one or more of a second
different type of amplification reaction, and/or detection assay
reaction, can be carried out, for example, as described in U.S.
Patent Application No. 60/699,782 to Faulstich et al., filed Jul.
15, 2005, (Attorney Docket No. 5010-297), which is incorporated
herein in its entirety by reference.
[0108] According to some embodiments, the present teaching can
comprise a method of de novo sequencing wherein pre-processing of
sample can comprise adding zip code primers to a sample of nucleic
acid having universal tail sequences ligated therein. Zip code
primers can be complementary to the universal tail sequences. The
use of zip code tails sequences and zip code primers can reduce the
need for target specific primers, resulting in significant cost
savings as well as greater assay flexibility.
[0109] According to various embodiments, pre-processing a sample
can comprise adding to the sample reactants to facilitate a nucleic
acid amplification reaction. For example, the four dNTP's (dATP,
dTTP, dGTP, and dCTP), a polymerase, oligonucleotide primers,
and/or chelating agents can be added to the sample. Oligonucleotide
primers can be chosen as determined by one of skill in the art to
accomplish the desired objective, for example, universal primers
can be used.
[0110] According to various embodiments, pre-processing a sample
can comprise diluting the sample with a miscible solvent, vehicle,
or carrier. The sample can be diluted at a ratio of 1:1, 1:10,
1:100, 1:1000, or 1:10,000, for example. Exemplary ranges of
dilution can be from about 1:1 to about 1:100, or from about 1:10
to about 1:50. For example, the sample can be diluted such that
only a single fragment of nucleic acid is present per 500
nanoliters of diluted sample, or per 200 nanoliters of diluted
sample. In some embodiments, the concentration of target fragments
can be based on the size of the immiscible-fluid-discrete-volumes
generated that carry the target fragments, such that an average of
about 1 target fragment is present per 1.4
immiscible-fluid-discrete-volumes generated. According to various
embodiments, the sample can be diluted such that at least 50%
immiscible-fluid-discrete-volumes produced from a sample in the
process described below can each comprise a single target nucleic
acid sequence. In various other embodiments, less than about 50% of
the immiscible-fluid-discrete-volumes produced can each comprise a
single target nucleic acid sequence. In other embodiments, at least
1% or more, 5% or more, 10% or more, or 20% or more can comprise a
single target nucleic acid sequence, for example, from about 10% to
about 50% or from about 20% to about 40%.
[0111] After optional preprocessing, the sample fluid is introduced
to system 10 to form one or more discrete volumes of the sample
fluid in a spacing fluid with which it is immiscible. A plurality
of immiscible-fluid, discrete volumes can be associated together as
a set of immiscible-fluid, discrete volumes. Each set of
immiscible-fluid, discrete volumes can comprise immiscible-fluid,
discrete volumes separated from one another by a spacing fluid, for
example, an oil. Each immiscible-fluid-discrete-volume of a set can
be equally spaced from one or more adjacent immiscible-fluid,
discrete volumes of the set. Multiple sets of immiscible-fluid,
discrete volumes can be present at the same time in main conduit
50. Each set of immiscible-fluid, discrete volumes can be separated
from one or more other sets of immiscible-fluid, discrete volumes
by larger volumes of spacing fluid, which in a tube of constant
cross-sectional area size and shape is visible by greater length
between the trailing end of the last discrete volume of the
upstream set and the leading end of the first discrete volume in
the downstream set. In some embodiments, two or more sets of
immiscible-fluid, discrete volumes are spaced from one another a
distance that is greater than the average distance between adjacent
immiscible-fluid, discrete volumes with the same set.
[0112] In the embodiment depicted in FIGS. 1A and 1B,
immiscible-fluid, discrete volumes that have been aspirated into
immiscible-fluid-discrete-volume-forming tube 12 can be moved into
holding tube 56 by suction produced by vacuum pump 40. In some
embodiments, pump 40 can be a syringe pump. More details about an
exemplary method of forming sets of immiscible-fluid, discrete
volumes are provided in concurrently filed, U.S. patent application
Ser. No. ______, entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363).
[0113] According to some embodiments, the method can comprise
moving a set of immiscible-fluid, discrete volumes, from T-junction
52, to cross-junction 70, by way of conduit system 50. If a set of
immiscible-fluid, discrete volumes does not contain nucleic acid
amplification reactants, the reactants can be added to each
immiscible-fluid-discrete-volume of the set of immiscible-fluid,
discrete volumes at cross-junction 70. More details about exemplary
methods of adding additional miscible liquid to immiscible-fluid,
discrete volumes is provided herein, at least in FIGS. 2, 21-28,
and 29A-C. As illustrated in FIGS. 1A and 1B, reactant addition to
each immiscible-fluid-discrete-volume can be metered by rotary
valves 71 and 73. Detector D-3 can detect the arrival of the
beginning and/or the end of a set of sample immiscible-fluid,
discrete volumes at cross-junction 70. More details about an
exemplary method of detecting the size and speed of
immiscible-fluid, discrete volumes is provided herein, at least in
FIGS. 21-22. Detector D-21 can detect the arrival of the beginning
and/or the end of immiscible-fluid, discrete volumes at
cross-junction 70. In some embodiments, valve V-7 can control the
movement of a set of immiscible-fluid, discrete volumes out of
cross-junction 70. In some embodiments, valve V7 can isolate a
thermal cycle section during thermal cycling.
[0114] According to some embodiments, the method can comprise
moving a set of immiscible-fluid, discrete volumes from
cross-junction 70, through main conduit system 50, to
thermal-spiral 74. Detector D-8 can be used to detect the arrival
of a set of immiscible-fluid, discrete volumes at thermal-spiral
74. Detector D-8 can be used to detect the end of a set of
immiscible-fluid, discrete volumes, and thereby detect that a set
of immiscible-fluid, discrete volumes is disposed in thermal-spiral
74. A set of immiscible-fluid, discrete volumes can be thermally
cycled, for one or more cycles, for example, for from about 5 to
about 50 temperature cycles or from about 20 to about 30
temperature cycles.
[0115] According to various embodiments, the method can comprise
introducing polymerase chain reaction inactivating reagents into
main tube 50 after amplifying the at least one target nucleic acid
sequence and before subjecting the nucleic acid sequence to a
sequencing reaction. The reagents can be used to inactivate or
remove or eliminate excess primers and/or dNTP's. The inactivating
reagents can be introduced at an junction in the capillary channel,
for example, after an immiscible-fluid-discrete-volume to be
inactivated is aligned with the junction. The junction can
comprise, for example, a T-junction.
[0116] According to some embodiments the method can comprise moving
a set of immiscible-fluid, discrete volumes from thermal-spiral 74,
through cross-junction 76. As the set of immiscible-fluid, discrete
volumes moves through cross-junction 76, the method can comprise
adding exonuclease and shrimp alkaline phosphatase to each
immiscible-fluid-discrete-volume of the set of immiscible-fluid,
discrete volumes. For example, the exonuclease and shrimp alkaline
phosphatase "SAP" can be metered out in discrete volumes which
merge respectively with the immiscible-fluid, discrete volumes of a
set of immiscible-fluid, discrete volumes at an junction in rotary
valve 77. More details about metering "addition" liquid, such as,
for example, exonuclease and SAP is provided herein, at least in
FIGS. 11A-B, 21-24, and 25-27 and their corresponding descriptions.
For example, exonuclease and shrimp alkaline phosphatase can be
added to each immiscible-fluid-discrete-volume of the set of
immiscible-fluid, discrete volumes in cross-junction 76.
[0117] In the exemplary system shown, detector D-6 can detect the
arrival of the beginning and/or the end of a set of sample discrete
volumes at cross-junction 76. Detector D-18 can detect the arrival
of the beginning and/or the end of one or more immiscible-fluid,
discrete volumes of exonuclease and shrimp alkaline phosphatase at
cross-junction 76. Valve V-8 can control the movement of a set of
immiscible-fluid, discrete volumes out of cross-junction 76. In
some embodiments, V8 can be used to isolate one or more thermal
spirals from each other. More details about an exemplary method of
isolating a thermal spiral is provided herein, at least in FIGS.
19-20, and the corresponding description.
[0118] In the exemplary embodiment shown, a set of
immiscible-fluid, discrete volumes containing exonuclease and
shrimp alkaline phosphatase can be moved into thermal-spiral 80,
via main conduit system 50. Detector D-9 can detect the arrival of
the beginning and/or the end of a set of immiscible-fluid, discrete
volumes at thermal-spiral 80. The set of immiscible-fluid, discrete
volumes can be incubated at from about 25.degree. C. to about
35.degree. C. for a time period of from about one minute, to about
60 minutes or from about two minutes to about 10 minutes. The
incubation step can function to facilitate the activities of the
exonuclease and shrimp alkaline phosphatase. A set of
immiscible-fluid, discrete volumes can be further incubated at a
temperature of from about 75.degree. C. to about 85.degree. C., for
a time period of from about 10 seconds to about 10 minutes, or from
about one minute to about five minutes. The incubation at from
about 75.degree. C. to about 85.degree. C. can function to
heat-kill any enzymes that might still be present in the set of
immiscible-fluid, discrete volumes.
[0119] According to some embodiments, the method can comprise
moving a set of immiscible-fluid-discrete-volumes to T-junction 84.
Valve V-9 can control the movement of a set of
immiscible-fluid-discrete-volumes from thermal spiral 80, to
T-junction 84. Detector D-10 can detect the arrival of the
beginning and/or the end of a set of
immiscible-fluid-discrete-volumes at T-junction 84. The method can
comprise dividing one or more immiscible-fluid, discrete volumes of
a set of immiscible-fluid discrete volumes into two or more smaller
immiscible-fluid-discrete volumes to form two newly formed sets of
equal number of immiscible-fluid discrete volumes, but containing
immiscible-fluid discrete volumes of smaller volume. More details
about an exemplary method of splitting immiscible-fluid, discrete
volumes into smaller immiscible-fluid, discrete volumes is provided
herein, at least in FIGS. 11A-B, and 12-16 and the corresponding
description. The method can comprise moving one newly created set
of immiscible-fluid, discrete volumes along main conduit system 50,
to cross-intersection 86. Forward primers and chain terminating
dyes can be moved from reservoirs 91 and 93, to rotary valve 77.
The forward primers and chain terminating dyes can be metered out
by rotary valve 77. The forward primers and chain terminating dyes
can be moved to cross-intersection 86 and be added to each
immiscible-fluid-discrete-volume of the newly-created set of
immiscible-fluid, discrete volumes, thereby creating a forward set
of immiscible-fluid, discrete volumes. According to various
embodiments, the method can comprise moving the second newly
created set of immiscible-fluid, discrete volumes along main
conduit system 50, to cross-intersection 88. Reverse primers and
chain terminating dyes can be moved from reservoirs 95 and 97, to
rotary valve 79. The reverse primers and chain terminating dyes can
be metered out by rotary valve 79. The reverse primers and chain
terminating dyes reagent can be moved to cross-intersection 86 and
be joined with each immiscible-fluid-discrete-volume of the second
newly-created set of immiscible-fluid, discrete volumes, thereby
creating a reverse set of immiscible-fluid, discrete volumes.
[0120] In some embodiments, the method can comprise moving the
forward set of immiscible-fluid-discrete-volumes from
cross-junction 86, along main conduit system 50, to thermal spiral
90. The forward set of immiscible-fluid-discrete-volumes can be
thermally cycled for from about 5 to about 50, temperature cycles,
for example, from about 20 to about 40 thermal cycles.
[0121] In some embodiments, the method can comprise moving the
reverse set of immiscible-fluid-discrete-volumes from
cross-junction 88, along main conduit system 50, to thermal spiral
92. The reverse set of immiscible-fluid-discrete-volumes can be
thermally cycled for from about 5 to about 50 thermal cycles, for
example, from about 20 to about 40 cycles, temperature cycles.
[0122] In some embodiments, the method can comprise thermal cycling
at least four different sets of immiscible-fluid-discrete-volumes,
one in each of thermal spirals 74, 80, 90, and 92. In some
embodiments, valves, such as, for example, can be used to isolate
each thermal spiral from all other thermal spirals. In some
embodiments, the different thermal spirals operate at the same
time, but with different thermal profiles. In some embodiments,
isolating the thermal expansion and contraction of the fluid in the
different spirals can be desirable. In some embodiments, the total
time for more than one temperature cycling can be the same. In
embodiments where different length thermal cycles are desired, an
additional step at non-process inducing temperature can be added
for the remainder of the longest thermal cycles.
[0123] According to various embodiments, the method can comprise
moving the forward and the reverse sets of
immiscible-fluid-discrete-volumes from their respective thermal
spiral to output conduit 54. Movement can be caused by syringe
pumps 82A and 82B that can be controlled independently, or
together, by a motor 88A operatively connected thereto. Syringe
pumps 82A and 82B can push and pull fluids through respective
T-junctions 84A and 84B. This arrangement is useful as syringe
pumps 82A and 82B can initially pull
immiscible-fluid-discrete-volumes into place in the respective
thermal spirals 90 and 92, in conjunction with the positive
pressure from the pumps on the upstream side of tee 84. Valves V-10
and V-11 can be switched so that immiscible-fluid-discrete-volumes
can be pushed out of system 10. In some embodiments, the pushing
can be done with one of pumps 82A and 82B at a time; therefore,
there is no need to merge two separate sets of
immiscible-fluid-discrete-volumes back together into a single set,
but rather the separate sets can be individually dispensed. Output
conduit 54 can deposit both sets of
immiscible-fluid-discrete-volumes on, for example, a multi-well
plate.
[0124] According to some embodiments, a dye can be added to one or
more immiscible-fluid, discrete volumes of a set of
immiscible-fluid, discrete volumes. The dye can comprise a
double-strand (ds), nucleic acid intercalating dye, for example,
SYBR green, SYBR gold, EVA green, LC green, or the like. The dye
can be added to an aqueous immiscible-fluid-discrete-volume-forming
fluid, such as an aqueous sample, before it is added to system 10.
The dye can be added to a set of immiscible-fluid, discrete volumes
at any cross-junction of system 10. The dye can be used to
discriminate between immiscible-fluid, discrete volumes that
contain ds nucleic acids and immiscible-fluid, discrete volumes
that do not contain ds nucleic acids. The immiscible-fluid,
discrete volumes that do not contain ds nucleic acids can be
removed from output tube 54 before the immiscible-fluid, discrete
volumes are deposited on a multi-well plate 47. The
immiscible-fluid, discrete volumes that do not contain ds nucleic
acids can be moved through second waste tube system 48, to waste
reservoir 85. In some embodiments, a dye can be detected by
detector 98 to determine whether a discrete volume should be sent
to second waste reservoir 85 or be collected. Pump 87 can apply a
negative pressure to waste tube system 48, which can cause the
movement of immiscible-fluid, discrete volumes into waste
reservoir.
[0125] Immiscible-fluid, discrete volumes deposited on multi-well
plate 47 can be subjected to a sequencing reaction to form a
detectable product, and the method of the present teachings can
comprise detecting the detectable product. In various embodiments,
the detectable product can be detected using, for example, a flow
cell or a capillary electrophoresis sequencer. In various other
embodiments, an off-capillary detector can be used as deemed
appropriate.
[0126] Shown below is a table showing a state diagram of various
settings that can be implemented for the various valves and
detectors of the system shown in FIGS. 1A and 1B, to achieve
various different functions, for example, to carry out various
different immiscible-fluid-discrete-volume processing useful in the
above described de novo sequencing method. TABLE-US-00001 TABLE 1
V- V- V- 1 2 3 V-4 V-5 V-6 V-7 V-8 V-9 V-10 V-11 V-12 V-13 V-14
V-15 V-16 V-17 Prime SMA Sample 0 1 0 1 0 0 0 0 1 0 1 0 0 0 0 1 0 1
0 0 0 Deliver initial portion 0 1 0 1 0 0 0 of SMAF/MM mixture to
ZT-1 0 0 1 0 1 0 0 0 Deliver initial portion 0 0 0 0 0 0 0 0 1 1 0
1 0 of oil to ZT-1 Form initial SMAF 0 0 1 0 1 0 1 0 0 1 1 0 1 0
Zebra Deliver intermediate 0 1 0 1 0 0 portion(s) of SMAF.MM
mixture toZT-1 0 0 1 0 1 0 1 0 0 1 1 0 1 0 Deliver final portion(s)
0 1 0 1 0 0 of SMAF.MM mixture toZT-1 0 0 1 0 1 0 1 0 0 1 1 0 1 0
Amplify DNA 0 0 Prime ES reagent path 0 0 1 1 0 0 1 1 Add ES
reagents and 0 0 1 1 1 1 1 0 1 1 0 1 0 load clean up thermal cycler
0 0 1 1 1 1 1 0 1 1 0 1 0 Clean up after PCR 0 0 Prime PF + BD
paths 0 0 0 0 0 0 Add FP + BD and 0 0 1 1 1 1 1 0 1 1 0 1 0 RP + BD
& load cycle 0 0 1 1 1 1 1 0 1 1 0 1 0 sequencing sticky bun
Cycle sequence 0 0 0 Dispense sample-laden 0 0 1 1 1 1 0 0 1 1 0 1
0 FP slugs to tray, 0 0 1 1 1 1 0 0 1 1 0 1 0 dispose of other
fluids Rotary Rotary Rotary V-18 V-19 V-20 V-21 V-22 V-23 V-24 V-25
V-26 Valve-ES Valve-FP&BD Valve-MM_SMF Prime SMA Sample 1 0 1 0
0 0 1 Oil 0 0 1 MM Deliver initial portion 1 0 1 MM of SMAF/MM
mixture to ZT-1 1 0 1 MM Deliver initial portion 0 0 0 1 Off of oil
to ZT-1 Form initial SMAF 1 1 0 0 0 1 Zebra Deliver intermediate 1
1 1 MM portion(s) of SMAF.MM mixture toZT-1 1 0 0 0 0 1 Deliver
final portion(s) 1 1 1 MM of SMAF.MM mixture toZT-1 0 0 0 1 Amplify
DNA 0 0 Prime ES reagent path 1 Oil out 1 ES out Add ES reagents
and 0 0 0 0 0 out 0 load clean up thermal cycler 0 0 0 0 0 0 0
Clean up after PCR Prime PF + BD paths 1 1 Oil out FP BD 1 1 Oil
out Add FP + BD and 0 0 0 0 0 0 out RP + BD & load cycle 0 0 0
0 0 0 0 sequencing sticky bun Cycle sequence 0 0 0 Dispense
sample-laden 0 0 0 0 0 1 0 0 FP slugs to tray, 0 0 0 0 0 0 0 0
dispose of other fluids Rotary Valve-MM_VI VICI-1 VICI-2 SP-MM SPES
SP-FP&BD SP-RP&BD Footnote Prime SMA Sample 1 1 0 1 2 0 1 3
Deliver initial portion 0 1 4 of SMAF/MM mixture to ZT-1 0 1 5
Deliver initial portion 0 1 6 of oil to ZT-1 Form initial SMAF 7
Zebra Deliver intermediate 0 1 8 portion(s) of SMAF.MM mixture
toZT-1 1 1 9 Deliver final portion(s) 0 1 10 of SMAF.MM mixture
toZT-1 1 1 11 Amplify DNA Prime ES reagent path 12 13 Add ES
reagents and 0 0 1 0 out 0 0 14 load clean up thermal cycler 0 0 1
0 0 0 0 15 Clean up after PCR Prime PF + BD paths 16 Oil In In 17
out out out In In 18 RF In In 19 BD In In 20 Oil In In 21 out out
out Add FP + BD and out 1 0 0 0 1 1 22 RP + BD & load cycle 0 1
0 0 0 0 0 sequencing sticky bun Cycle sequence 0 Dispense
sample-laden 0 1 23, 24 FP slugs to tray, 0 1 dispose of other
fluids Footnotes: 1 Pull SMAF into T-intersection (67); 2 Pull oil
through T-intersection (67); 3 Pull MM through T-intersection (67);
4 Pull SMAF + MM through D-17; 5 Push SMAF + MM towards
T-intersection (66) until D-5 detects AF; 6 Pull, Push oil towards
T-intersection (66) until D-4 detects oil; 7 Push oil + SMAF + MM
through thermal cycler until D-6 detects zebras or, more likely,
D-2 sees only oil; 8 Pull SMAF + MM through D-17; 9 Push oil + SMAF
+ MM through thermal cycler until D-6 detects zebras or, more
likely, D-2 sees only oil; 10 Pull SMAF + MM towards D-17. After
total volume of SMAF has entered T-intersection (67), close V-18.
After total volume of MM has left Rotarty Valve (71), switch Rotary
Valve (71) to "oil" position. Continue pulling SMAF + MM towards
D-17 until D-2 sees a; 11 Push oil + SMAF + MM through thermal
cycler until D-6 detects zebras or, more likely, D-5 sees only oil;
12 Push oil until D-16 detects oil; 13 Push ES until D-18 detects
ES, then push further distance calculated to advance ES to Zebra
path.; 14 Push until D-6 detects end of batch, then push further
distance calculated to advance batch just past ES adder; 15 Push
until D-9 detects end of batch, then push farther distance
calculate to advance batch completely into cleanup thermal cycler;
16 Push SP (78) until D-19 sees oil. Push SP (82) until D-20 sees
oil.; 17 Pull portion of FP into SP (78). Pull portion of RP into
SP (82); 18 Pull portion of BD into SP (78). Pull portion of BD
into SP (82); 19 Pull alternating sub-portions of primers and big
dyes until complete portion has been loaded; 20 Pull small amount
of oil so all aqueous fluids advance into syringe; 21 Push SP (78)
until D-19 sees FP + BD. Push SP (82) until D-20 sees RP + BD. Push
farther distance calculated to advance FP + BD and RP + BD to Zebra
path; 22 Push with pumps until D-11 and D-7 see oil, then push
further distance calculated to advance batch just past RP + BD and
FP + BD adders; 23 Push with pumps further distance calculated to
advance batch into cycle sequencing thermal cycler; 24 Push until
FSD-1 detects sample-laden FP slug, then push further distance
calculated to move downstream boundary of sample-laden slug just
inside dispense tip; .sup.25Push distance calculated to bead
sample-laden slug on dispense tip. Touch bead to bottom of sample
well.
[0127] According to various embodiments, the present teachings can
encompass a resequencing method using system 10. In general, the
resequencing method is similar to the de-novo method described
herein with modifications as discussed herein.
[0128] In some embodiments, the pre-processing of a sample for
resequencing comprises shearing a robust sample of nucleic acid
having a plurality of copies of one or more nucleic acids of
interest, herein also referred to as target sequences. The nucleic
acids in the sample can be sheared. The method can comprise adding
a set of gene specific primers, for example, at cross-junction 10,
to a set of immiscible-fluid, discrete volumes generated from the
sample. Immiscible-fluid, discrete volumes made from the sample can
contain a single copy of a nucleic acid fragment or can contain a
plurality of copies of one or more different nucleic acid
fragments. Each immiscible-fluid-discrete-volume can contain, for
example, from about 50 to about 150 or more; 384 or a thousand, or
several thousands, or fewer.
[0129] In some embodiments, the method can comprise moving a set of
immiscible-fluid, discrete volumes comprising the concentrations of
primers discussed above, to thermal-spiral 74. The set of
immiscible-fluid, discrete volumes can be thermally cycled and
thereafter processed in any of the many manners disclosed herein
for the de novo sequencing method.
[0130] Various sequencing and re-sequencing methods that can be
carried out according to various embodiments can include, for
example, those depicted in FIGS. 2C-2K of co-pending U.S. patent
application Ser. No. ______, filed Aug. 22, 2006, entitled
"Apparatus, System, and Method Using
Immiscible-Fluid-Discrete-Volumes," to Lee et al. (attorney docket
number 5010-362), which is incorporated herein in its entirety by
reference.
[0131] Shown below are Tables 2A and 2B which are the first and
second halves of another state diagram of various settings that can
be implemented for the various valves and detectors of the system
shown in FIGS. 1A and 1B, to achieve various different functions.
The various functions can include carrying out various different
immiscible-fluid-discrete-volume processing, for example, carrying
out the standard resequencing reactions depicted in FIGS. 2C-2D of
U.S. patent application Ser. No. ______, filed Aug. 22, 2006,
entitled "Apparatus, System, and Method Using
Immiscible-Fluid-Discrete-Volumes," to Lee et al. (attorney docket
number 5010-362). TABLE-US-00002 TABLE 2A V-1 V-2 V-3 V-4 V-5 V-6
V-7 V-8 V-9 V-10 V-11 V-12 V-13 V-14 Prime Primary 0 0 0 0 0 VI
Input Path Form VI Zebra 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 Push Zebra Into Storage 0 0 0 0 1 1 Repeat "Form VI Zebra"
and "Push Zebra Into Storage" until D-4 sees zebras or until the
total required number of slugs is reached. Prime Secondary 1 1 0 1
0 0 0 0 VI Input Path Form secondary 1 1 0 1 0 0 0 0 VI fluid macro
slugs 1 1 0 1 0 0 0 0 1 1 0 1 0 0 0 0 Push Macro-Zebra 0 1 0 0 0
Into Storage Repeat "Form secondary VI fluid macro slugs" and "Push
Macro-Zebra Into Storage" until D-4 sees zebras or until the total
required number of slugs is reached. Add Secondary VI 0 1 0 0 1 0 0
0 1 1 fluid to Zebra slugs Prime MM_VI Add MM to VI Zebra slugs 0 1
0 0 1 1 0 0 1 1 Amplify DNA 0 0 Rotary V-15 V-16 V-17 V-18 V-19
V-20 V-21 V-22 V-23 V-24 V-25 V-26 Valve (75) Prime Primary 1 0 1
VI Input Path Form VI Zebra 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 Push
Zebra Into Storage 0 1 0 1 1 Repeat "Form VI Zebra" and "Push Zebra
Into Storage" until D-4 sees zebras or until the total required
number of slugs is reached. Prime Secondary 0 0 1 VI Input Path
Form secondary 0 0 1 VI fluid macro slugs 0 0 1 0 0 1 Push
Macro-Zebra 1 0 Into Storage Repeat "Form secondary VI fluid macro
slugs" and "Push Macro-Zebra Into Storage" until D-4 sees zebras or
until the total required number of slugs is reached. Add Secondary
VI 0 1 0 1 0 0 1 0 fluid to Zebra slugs Prime MM_VI Add MM to VI
Zebra slugs 0 1 0 1 0 0 0 1 Amplify DNA 0 0 Rotary Rotary Rotary
Rotary Valve (77) Valve (71) Valve (73) Valve (79) Pump (40) Pump
(39) SP (58) SP (66) SP (78) Prime Primary 1 VI Input Path Form VI
Zebra 1 1 1 1 1 Push Zebra Into Storage 1 Repeat "Form VI Zebra"
and "Push Zebra Into Storage" until D-4 sees zebras or until the
total required number of slugs is reached. Prime Secondary 1 VI
Input Path Form secondary 1 VI fluid macro slugs 1 1 Push
Macro-Zebra Into Storage Repeat "Form secondary VI fluid macro
slugs" and "Push Macro-Zebra Into Storage" until D-4 sees zebras or
until the total required number of slugs is reached. Add Secondary
VI 0 1 1 0 fluid to Zebra slugs Prime MM_VI 0 MM 1 Add MM to VI
Zebra slugs Out 1 1 1 Amplify DNA SP (82) Prime Primary Pull oil
from reservoir until it reaches D-15, then pump VI Input Path
distance calculated to advance oil at D-17 just past V-17. Form VI
Zebra Pull 78 nl primary VI fluid into tube through tip. Wash tip.
Pull 800 nl oil into tube through tip. Wash tip. Pull 78 nl primary
VI fluid from next well into tube through tip. Wash tip. Pull 800
nl oil into tube through tip. Wash tip. Continue aspiration steps
until zebras (sequence of immiscible fluid volumes) are detected by
D-15. Push Zebra Into Storage Push oil until D-16 no longer sees
slugs (individual fluid volumes). Repeat "Form VI Zebra" and "Push
Zebra Into Storage" until D-4 sees zebras or until the total
required number of slugs is reached. Prime Secondary Pull oil from
reservoir until it reaches D-17, then pump distance VI Input Path
calculated to advance oil at D-17 just past V-17. Form secondary
Pull m(78 nl) of secondary VI fluid i into tube, where m is the
number of VI fluid macro slugs primary VI fluids that are to be
mixed with the ith secondary fluid. Pull 800 nl oil into tube
through tip. Wash tip. Continue aspiration steps until zebras are
detected by D-17. Push Macro-Zebra Pump oil to push macro-zebra
until D-2 no long sees macro-slugs. Into Storage Repeat "Form
secondary VI fluid macro slugs" and "Push Macro-Zebra Into Storage"
until D-4 sees zebras or until the total required number of slugs
is reached. Add Secondary VI Push micro and macro zebras until D-3
sees slugs fluid to Zebra slugs Prime MM_VI Load Syringe Pump (58)
Add MM to VI Zebra slugs Runs pumps until D-6 sees slugs Amplify
DNA
[0132] TABLE-US-00003 TABLE 2B V- 1 V-2 V-3 V-4 V-5 V-6 V-7 V-8 V-9
V-10 V-11 V-12 V-13 V-14 V-15 V-16 V-17 V-18 Prime ES Reagent path
0 0 1 1 0 0 1 1 Add ES Reagents & 0 0 1 1 1 1 1 0 1 1 0 1 0
load cleanup 0 0 1 1 1 1 1 0 1 1 0 1 0 thermal cycler Clean up
after PCR 0 0 Prime FP + BD 0 0 0 and RP + BD paths 0 0 0 Add FP +
BD and 0 0 1 1 1 1 1 0 1 1 0 1 0 RP + BD & load 0 0 1 1 1 1 1 0
1 1 0 1 0 cycle sequencing thermal cycler Cycle sequence 0 0 0
Dispense sample-laden 0 0 1 1 1 1 0 0 1 1 0 1 0 FP slugs to tray, 0
0 1 1 1 1 0 0 1 1 0 1 0 dispose of other fluids Rotary Rotary
Rotary Rotary Rotary Pump V-19 V-20 V-21 V-22 V-23 V-24 V-25 V-26
Valve (75) Valve (77) Valve (71) Valve (73) Valve (79) (40) Prime
ES Reagent path Oil 1 Out ES 1 Out Add ES Reagents & 0 0 0 0 0
Out 0 0 0 1 load cleanup 0 0 0 0 0 0 0 0 0 1 thermal cycler Clean
up after PCR Prime FP + BD Oil Oil and RP + BD paths 1 1 Out Out FP
RP BD BD Oil Oil 1 1 Out Out Add FP + BD and 0 0 0 0 0 0 Out 0 Out
1 RP + BD & load 0 0 0 0 0 0 0 0 0 1 cycle sequencing thermal
cycler Cycle sequence 0 0 0 0 Dispense sample-laden 0 0 0 0 0 1 0 0
0 0 1 FP slugs to tray, 0 0 0 0 0 0 0 0 0 0 1 dispose of other
fluids SP SP SP Pump (39) (58) (66) (78) SP (82) Prime ES Reagent
path In Out Push oil until D-18 detects oil. In Out Push ES until
D-18 detects ES, then push further distance calculated to advance
ES to zebra path. Add ES Reagents & 0 0 Out 0 0 Push until D-6
detects end of batch, then push further distance load cleanup
calculated to advance batch just past ES adder. thermal cycler 0 0
0 0 0 Push until D-9 detects end of batch, then push further
distance calculate to advance batch completely into cleanup thermal
cycler. Clean up after PCR Prime FP + BD In In and RP + BD paths
Out Out Push SP-FP&BD until D-19 sees oil. Push SP (82) until
D-20 sees oil. In In Pull portion of FP into SP (78). Pull portion
of RP into SP-RP&BD. In In Pull portion of BD into SP (78).
Pull portion of BD into SP (82). Pull alternating sub-portions of
primers and big dyes until complete portion has been loaded. In In
Pull small amount of oil so all aqueous fluids advance into
syringe. Out Out Push SP (78) until D-19 sees FP + BD. Push SP (82)
until D-20 sees RP + BD. Push farther distance calculated to
advance FP + BD and RP + BD to zebra path. Add FP + BD and 0 0 0 1
1 Push with pumps until D-11 and D-7 see oil, then push further
distance calculated RP + BD & load to advance batch just past
RP + BD and FP + BD adders. cycle sequencing 0 0 0 0 0 Push with
pumps further distance calculated to advance batch into thermal
cycler cycle sequecning thermal cycler. Cycle sequence Dispense
sample-laden Push until fluorescent detector (98) detects
sample-laden FP slug, then FP slugs to tray, push further distance
calculated to move downstream boundary of sample-laden dispose of
other fluids slug just inside dispense tip. Push distance
calculated to bead sample-laden slug on dispense tip. Touch bead to
bottom of sample well.
[0133] A simplified system 200 is illustrated in FIG. 2. As
illustrated, box 202 represents a structure that delivers to tube
204 of system 200 discrete volumes 206 of aqueous liquid in a
non-aqueous liquid 208 with which they are immiscible. Examples of
such structures and methods of generating discrete volumes 206 in
contact with spacing fluid 208 are described in concurrently filed
U.S. patent application Ser. No. ______, entitled "Device and
Method for Making Immiscible-Fluid-Discrete-Volumes," to Cox et al.
(attorney docket number 5010-363). In some embodiments, such a
structure could be a tube of preformed discrete volumes 206 of
aqueous fluid. In some embodiments, such a structure could be a
chip or other substrate with a channel therein containing the
discrete volumes 206 of aqueous fluid. As illustrated, tube 204
extends throughout system 200. After entering tube 204, desired
information about aqueous volumes 206 are determined and optionally
manipulated by structures in triangle 210. For example, the length
and speed of a slug and the distance between two adjacent slugs can
be desired information. In that example, a slug detection system
can provide that information. If the distance between adjacent
slugs does not meet preferred values, then additional spacing fluid
can be added between the trailing point of the first slug and the
leading point of the second slug, or one of the slugs could be held
in an electric field, for example, to allow more of the existing
spacing fluid to flow past it in tube 203. If the length, and
therefore the volume, of an aqueous discrete volume does not meet
preferred values, additional non-reactive, miscible liquid can be
added by an apparatus at that area of tube 204. Triangle 210
represents these and other structures of discrete volume
characteristic detection and manipulation. Examples of these
structures and/or component parts of thereof are described
herein.
[0134] System 200, as illustrated in FIG. 2, next incorporates a
processing section 212 of tube 204 (not illustrated, but in the
box), which can include, for example, vibration, heating, cooling,
and electromagnetic radiation exposure. In some embodiments,
processing section 212 can include thermal cycling between one or
more pre-determined temperatures for pre-determined durations as
needed, for example, to perform PCR, or other amplification
methods. In some embodiments, aqueous discrete volumes may continue
to flow at a constant rate through processing section 212 while
undergoing a desired process, or alternatively, they may dwell in a
particular location in processing section 212. System 200, as
illustrated in FIG. 2, includes another aqueous discrete volume
characteristic determination and optional manipulation station 214.
Aqueous discrete volumes 206 then flow through a junction J-1. In
some embodiments, junction J-1 can be a T. As illustrated, fluid
addition station 220 includes pump P-1 and valve V-1 in conjunction
with a supply of different fluid (not shown) and can add that fluid
to tube 204. In some embodiments, a gas phase can be introduced
between aqueous discrete volumes 206. In some embodiments, an
aqueous liquid can be added to aqueous discrete volumes 206 in
junction J-1. In some embodiments, a different aqueous fluid can be
added to a discrete volume between aqueous discrete volumes 206. An
aqueous discrete volume characteristic determination and optional
manipulation station 215, like 214 and 210 described above, follows
liquid addition station 220. In some embodiments, station 215
evaluates the volume of liquid added to aqueous discrete volume
206.
[0135] Next in line, as illustrated in FIG. 2, is junction J-2.
Junction J-2 and junction J-4, further down the line, fluidically
connect back pressure unit 216 to pressurize tube 204 to a desired
pressure. Between junctions J-2 and J-4, system 200 includes a
second processing section 212, a junction J-3, at which point,
fluid adding station 222 can add a volume of liquid to pre-existing
aqueous discrete volumes 206, and an aqueous discrete volume
characteristic determination and optional manipulation station 217
can evaluate the volume of liquid added to aqueous discrete volume
206.
[0136] As illustrated in FIG. 2, system 200 includes a final
processing section 212, and processed aqueous discrete volumes are
delivered from tube 204 to output station 218. Examples of
structures used in output station 218 are described in concurrently
filed U.S. patent application Ser. No. ______, filed Aug. 22, 2006,
entitled "Apparatus and Method for Depositing Processed Discrete
Volumes of a First Fluid in Contact With a Second Fluid, Wherein
the First and Second Fluids are Immiscible," to Schroeder et al.
(attorney docket number 5010-365).
[0137] Reference will now be made to various embodiments of
devices, apparatus, systems, and methods for controlling the fluid
flow to manipulate immiscible-fluid, discrete volumes of a first
fluid separated from one another by an immiscible spacing fluid,
examples of which are illustrated in the accompanying drawings.
Various embodiments of these can be used in the system described
above with reference to FIGS. 1A and 1B. Wherever possible, the
same reference numbers are used in the drawings and the description
to refer to the same or like parts.
[0138] Referring now to FIG. 3, an embodiment 300 of a rotary valve
that can work in any of the locations indicated in the system
diagrams described above includes an annular stator 302 and a
cylindrical rotor 304. Annular stator 302 has a first through-hole
306 and a second through hole 308, both of equal circular
cross-sectional area, with their axes parallel, though they need
not be, to the x-axis, and in alignment with each other. As
illustrated, cylindrical rotor 304 has a through-hole 310 having a
circular cross-sectional area the same shape and size of that both
through-hole 306 or through-hole 308 and having its axis at an
angle with the x-axis. As illustrated, valve 300 is in the closed
position: through-hole 310 is not aligned with through-holes 306
and 308. In some embodiments, a larger cross-section counter bore
316 from the outer diameter of annular stator 302, and a larger
cross-section counter bore 318 from the outer diameter of annular
stator 302 are co-axial with through-hole 308 and through-hole 306,
respectively, and tube 312, which can be, for example, PTFE
capillary tubing, is disposed in counter bore 316 and tube 314,
which can also be, for example, PTFE capillary tubing, is disposed
in counter bore 318. In some embodiments, the inner cross-sectional
shape and size of tube 312 matches the cross-sectional shape and
size of through-hole 306. In some embodiments, the inner
cross-sectional shape and size of tube 312 matches the
cross-sectional shape and size of through-hole 310. In some
embodiments, the inner cross-sectional shape and size of tube 314
matches the cross-sectional shape and size of through-hole 310. In
this position, rotary valve 300 prevents the flow of fluid between
tube 312 and tube 314 through through-hole 310. If cylindrical
rotor 304 is rotated such that the axis of through-hole 310 is
parallel with the x-axis, rotary valve 300 will permit the flow of
fluid between tube 312 and tube 314 through through-hole 310. Any
known mechanism providing rotary motion can be coupled to rotor 302
to actuate valve 300.
[0139] In some embodiments, annular stator 302 and cylindrical
rotor 304 can be formed from PTFE. In some embodiments, annular
stator 302 or cylindrical rotor 304 can be formed poly
oxy-methylene, Nylon 66, Tefzel (a modified ETFE
(ethylene-tetrafluoroethylene) fluoropolymer (manufactured by Du
Pont)), Polytetrafluoroethylene (PTFE), Noryl classico family of
modified PPE resins consists of amorphous blends of PPO*
polyphenylene ether resin and polystyrene. (manufactured by General
Electric), ultra high density polyethylene, polyphenol sulfide, and
red Turcite TX or Turcite X ((Ethylene-chlorotrifluoroethlyene
[ECTFE]) (manufactured by Busak+Shamban). In some embodiments,
annular stator 302, cylindrical rotor 304, tube 312 and tube 314
comprise the same material. In some embodiments, at least one of
annular stator 302, cylindrical rotor 304, tube 312, and tube 314
comprises a different material than at least the remaining listed
components.
[0140] In some embodiments, conduits 312 and 314 are connected to
annular stator 302 via fittings. In some embodiments, conduits 312
and 314 do not touch the cylindrical surface of cylindrical rotor
304, but fit in bores coaxially aligned with through-hole 306 or
308.
[0141] Use of rotary valve 300 does not displace a volume of fluid
relative to its longitudinal location in tube 312 or 314. If a slug
is located partially in one of conduits 312 and 314 and partially
in through-hole 310, rotation of annular rotor 304 will split the
slug into at least two parts. However, upon rotating annular rotor
304 back such that through-hole 310 aligns with conduits 312 and
314, the two or more volumes of the "split" slug will coalesce and
the original slug can be flowed out of rotary valve 300.
[0142] Rotary valves as depicted in FIGS. 1A and 1B have multiple
input tubes connected thereto. Rotary valve 300 described above can
be modified to accomplish such a use. For example, multiple sets of
aligned through-holes in annular stator 302 can accommodate
additional tubes and the fluids contained therein, and which of the
different fluids is passed through the rotary valve depends on the
angular orientation of cylindrical rotor 304 relative to annular
stator 302. As another possible modification, multiple
through-holes could exist in rotor 304 at different angular
orientations and different "Z" planes within rotor 304 than
through-hole 310 illustrated in FIG. 3. A set of through-holes in
stator 304 would align with each additional through-hole in rotor
304, and, accordingly, be at the appropriate "Z" value
corresponding to the appropriate Z plane in rotor 304. The position
of the holes in rotor 304 and stator 302 can be either aligned,
with respect to the rotation of the rotor, with another set of
holes in the rotor and stator such that both align at the same
time, or they can align at different through-holes at a different
angle. This permits the valves to be either opened and closed
together, or separately.
[0143] FIG. 4 illustrates a cross-sectional view in the XY plane of
embodiment 400 of a rotary valve. As illustrated, and in some
embodiments, rotary valve 400 can be a three-way valve. In other
words, it can connect a first passageway with one of a second and
third passageways. Rotary valve 400 is the same as rotary valve 300
except that cylindrical rotor 304 has a second through-hole 402 and
annular stator 302 has a third through-hole 404 and third
counter-bore 406 forming from the outer diameter of annular stator
302. A tube 408 is disposed in counter-bore 404. Second
through-hole 402 is parallel to through-hole 310 and, as
illustrated, when its axis is parallel to the x-axis, through-hole
402 is not aligned with any of the through-holes in annular stator
302. It is understood that second through-hole 402 does not have to
be parallel, nor even entirely in the same plane as through-hole
310
[0144] FIG. 5 illustrates a cross-sectional view in the XY plane of
the rotary value of FIG. 4, where the relative angular positions of
cylindrical rotor 304 and annular stator 302 have changed such that
through-hole 404 is in fluid communication with through-hole 314
and through-hole 404 of annular stator 302. In this position,
rotary valve 400 permits flow of fluid between tube 314 and tube
408 through through-hole 402. As illustrated, and in some
embodiments, through-hole 310 is not aligned, and therefore is not
in fluid communication with either of tubes 312 or 314.
[0145] FIG. 6 illustrates a cross-sectional view in the XY plane of
an embodiment 600 of a linear valve in an open position. In some
embodiments, tube 602 is disposed in a through-hole 606 in member
604, which as illustrated in FIG. 6 is a block of rectangular
shape. In some embodiments, a tube 608 is disposed in a
through-hole 612 in member 610, which as illustrated in FIG. 6, can
be a block of rectangular shape. As illustrated, tube 602 and tube
608 are axially aligned, and fluid can flow from one to the other.
Member 610 is slidably mounted with respect to member 604. In some
embodiments, a solenoid 614 can contact member 610.
[0146] FIG. 7 illustrates a cross-sectional view in the XY plane of
the linear valve of FIG. 6 in a closed position. As illustrated,
and in some embodiments, solenoid 614, or other motive force
element, can apply a force to member 610 and slide it in the X
direction with respect to member 604. Tube 608 and tube 602 no
longer align, and, therefore, no fluid can flow between them, thus
"closing" linear valve 600.
[0147] In some embodiments of a system such as that depicted in
FIGS. 1A and 1B or FIG. 2, a pinch valve can be used to stop flow
in conduits. FIG. 8 illustrates a side view of an embodiment 1000
of a pinch valve in an open position. Pinch valve 1000 provides a
structure to collapse the inner cross-section of conduit 1002 until
flow therein stops. In some embodiments, tube 1002 is between a
radiused valve body 1004 and lever 1006. In some embodiments,
radiused valve body 1004 is mounted to a base 1008. Springs 1010
and 1012 support lever 1006 a fixed distance above radiused valve
body 1004. In some embodiments, spring 1010 is compressed between
lever 1006 and base 1008 by a force applied to lever 1006 by
fastener 1014. Fastener 1014, then, limits the distance between
lever 1006 and base 1008. Lever 1006 is cantilevered beyond spring
1012. The minimum length that lever 1006 extends is determined by
the required torque to rotate lever 1006 and the force supplied by
the solenoid or other motive force element. In some embodiments, a
solenoid can be mounted above lever 1006 to contact lever 1006 with
its actuator in the un-actuated position.
[0148] FIG. 9 illustrates a front, cross-sectional view of the
pinch valve of FIG. 8, more clearly illustrating the curvature of
radiused valve body 1004 and the uncompressed tube 1002.
[0149] When solenoid 1020 actuates, actuator 1022 extends under
force, rotating lever 1006 a pre-determined number of degrees,
thereby collapsing or "pinching" tube 1002 between lever 1006 and
radiused valve body 1004. FIG. 10 illustrates a front,
cross-sectional view of the pinch valve of FIG. 8 in a closed
position when tube 1002 is collapsed.
[0150] The ratio of volumes of the two smaller discrete volumes
formed when a slug is split in a T-junction depends on the
differences in the flow rate, diameter, and length of each of the
two conduits flowing away from the T-junction. If the diameter and
length are the same, then the ratio of the flow rates determines
the ratio of the volumes of the two smaller, immiscible-fluid,
discrete volumes.
[0151] FIGS. 11A and 11B illustrate an embodiment 1500 of a system
that can be used to split an immiscible-fluid slug into two,
smaller, immiscible-fluid discrete volumes. In some embodiments,
and as illustrated in FIGS. 11A and 11B, system 1500 includes a
T-junction 1502, out of which fluid flows through tube 1504 in the
positive X direction and through tube 1506 in the negative X
direction. The other end of tube 1504 is connected to and is in
fluid communication with port 1508 of a three-(or more) way valve
1510. Port 1512 of three-way valve 1510 is in fluid communication
with tube 1513. Port 1514 of three-way valve 1510 is in fluid
communication with tube 1516. As illustrated, three-way valve is
positioned such that the internal pathway between port 1508 and
port 1514 is open, and the internal pathway between port 1512 and
port 1514 is closed. As illustrated, three-way valve 1510 is a
linear three-way valve operated, for example, by a solenoid. Other
three-way valve structures can be used. For example, the rotary
three-way valve described in FIGS. 4-5 can be used. One end of tube
1516 is connected to and is in fluid communication with port 1514
of three-way valve 1510. The other end of tube 1516 is connected to
and is in fluid communication with an outlet of a displacement pump
1518.
[0152] The other side of T-junction 1502 can be plumbed in a
separate, but similar way. Thus, one end of tube 1506 connects to
and is in fluid communication with T-junction 1502 and the other
end is connected to and is in fluid communication with port 1520 of
three-way valve 1522. One end of tube 1523 is connected to and is
in fluid communication with port 1524 of three-way valve 1522. Port
1526 of three-way valve 1522 is connected to and is in fluid
communication with one end of tube 1528. The other end of tube 1528
is connected to and is in fluid communication with an outlet of
displacement pump 1530.
[0153] Displacement mechanism 1531 and 1519 can be moved at the
desired speed, in the same direction, and starting at the same time
to create a desired ratio of flow rates through the fluid pathway
between an outlet of T-junction 1502. If the movement does not
start at the same time, in the same direction, or at the desired
speed, the ratio of flow rates in each divergent pathway may not be
as desired, resulting in an undesired volumetric split of a slug in
T-junction 1502.
[0154] To reduce the likelihood of an undesired volumetric split,
the displacement mechanisms of displacement pumps 1530 and 1518 can
both be moved by a single motion. As illustrated in FIGS. 11A and
11B, one structure for moving displacement mechanism 1531 at the
same time, and at the same speed is a link 1532 rigidly coupled to
each of displacement mechanisms 1531 and 1519 and to the linear
output of a motor 1534. In some embodiments, the ratio of flow
rates can be changed by changing the ratio of the cross-sectional
areas of displacement pumps 1530 and 1514. Thus, for even splitting
of slugs, in some embodiments, the cross-sectional area of
displacement pump 1530 equals the displacement area of displacement
pump 1518. In some embodiments, to prevent bubbles, the movement of
pumps 1530 and 1518 can permit flow from additional positive
pressure pump feeding into tee 1502 from tube at "FIG. 11A" (not
shown); thus negative pressures are never created.
[0155] After motor 1534 has moved link 1532 the desired distance in
the positive Y direction, it stops. Three-way valves 1510 and 1522
change position to close internal pathways between ports 1526 and
1520 and 1514 and 1508, respectively, and open internal pathways
between ports 1526 and 1524 and 1514 and 1512, respectively. FIG.
11B illustrates three-way valves 1522 and 1510 in this position.
Motor 1534 moves link 1532 in the negative Y direction and fluid in
conduits 1528 and 1516 flow through three-way valve 1522 and 1510,
respectively, into conduits 1523 and 1513, respectively. In some
embodiments, tube 1523 and tube 1513 can be in fluid communication
with a waste reservoir.
[0156] The configuration of displacement pumps, three-way valves
and connected conduits described above can also be used to control
the volumes of miscible fluid combined in a T, if the flow
directions are reversed, along with the steps. In other words, the
three-way valves start in the position shown in FIG. 11B and the
motor then moves in the positive Y direction, pulling a volume of
fluid through conduits 1523 and 1513, respectively, through the
respective three-way valve and into tube 1528 and 1516,
respectively. The three-way valve then changes position from that
illustrated in FIG. 1B to that illustrated in FIG. 1A, and the
motor then moves in the negative Y direction, pushing a desired
volume of fluid through each of conduits 1528 and 1516, through the
respective three-way valve and into tube 1506 and 1504,
respectively. The two streams of fluid then combine in preset
volumetric ratio and exit T-junction 1502. One use for such a
configuration and method of operation is for combining two liquid
reagents prior to forming discrete volumes to coalesce with
existing discrete volumes. Referring to FIG. 1B, such a
configuration and method of operation could be used in at least at
cross-junction 86 and cross-junction 88 to add dye terminators and
forward or reverse primers to existing immiscible-fluid, discrete
volumes. In some embodiments, the intake to the pump can be between
90 and V10 and 92 and V11.
[0157] The method of operation of the configuration described
immediately above can also generate immiscible-fluid discrete
volumes of a first fluid in a consistent ratio with a second fluid
with which it is immiscible. Thus, referring to FIGS. 1A and 2,
such a configuration could be used in at least at T-junction 66 and
in supply unit 202.
[0158] Another method of use can combine slugs as in the zipper
approach to merging samples and primer sets. After creation of
slugs sets of primers in tubes 1523 and 1513 they can be pulled
into 1528 and 1516, and then pushed so that they merge in tube
1502. A pair of detectors can monitor the position of the slugs,
oil adding devices can be used to add oil into tubes 1506 1504 so
that the slugs will meet precisely.
[0159] Another system for splitting aqueous immiscible-fluid,
discrete volumes spaced apart by spacing fluid, in a conduit, is
depicted in FIGS. 12-16. As shown, the system 1200 comprises an
immiscible-fluid-discrete-volume supply tube 1202 and a spacing
fluid supply tube 1204 which deliver immiscible-fluid, discrete
volumes in spacing fluid and spacing fluid, respectively, to a
housing 1206. System 1200 can be used to form immiscible-fluid,
discrete volumes of any first fluid separated from one another by
an immiscible second fluid. Each of supply lines 1202 and 1204 can
be operatively connected through a respective delivery unit (not
shown) that can comprise, for example, a pump and a fluid
reservoir. Housing 1206 houses a slider 1212 therein, which is
configured for sliding movement in housing 1206. Slider 1212 is
provided with a through hole 1214 that, in the position shown in
FIG. 12, is aligned with immiscible-fluid-discrete-volume supply
tube 1202. Any suitable drive unit can be provided for effecting
sliding movement of slider 1212 in housing 1206, for example, a
programmable drive unit. Slider 1212 is snugly seated in housing
1206 and causes a sealing action to seal off spacing supply tube
1204, when positioned as shown in FIG. 12A.
[0160] Housing 1206 is provided with an upper wall 1210 and a lower
wall 1208. Lower wall 1208 is provided with through holes 1226 and
1216 to accommodate and/or provide a fluid communication with
aqueous immiscible-fluid-discrete-volume supply tube 1202 and
spacing-fluid supply tube 1204, respectively. Upper wall 1210 is
provided with through holes 1224 and 1218 to accommodate and
provide fluid communication with a right-split-slug tube 1222 and a
left-split-slug tube 1220, respectively. In the position shown in
FIG. 12, a slug 1226 has been flowed upwardly through
immiscible-fluid-discrete-volume supply tube 1202 to fill
through-hole 1214 of slider 1212 with half of its volume. The
forward most point of slug 1226 is lower than upper wall 1210. For
even splitting of slug 1226, as illustrated, the midpoint of slug
1226 is positioned at the interface of lower wall 1208 and slider
1212. In some embodiments, slug 1226 can be held in position by an
electric field, regardless of whether oil flows past it.
[0161] In the next step of a method using system 1200, slider 1212
is shifted to the right-side position shown in FIG. 13. As shown in
FIG. 13, in the right-side position, slider 1212 seals off
immiscible-fluid-discrete-volume supply tube 1202 and
left-split-slug tube 1220. Furthermore, when in the right-side
position shown, through-hole 1214 of slider 1212 lines up with
spacing fluid tube 1204 and right-split-slug tube 1222, as well as
with respective through-holes 1224 and 1216. As illustrated,
because spacing-fluid supply tube 1204 contains spacing fluid, the
volume of liquid that used to be half of slug 1226 forms as a new
right split slug 1230.
[0162] Next, spacing fluid is flowed upwardly through the
spacing-fluid supply tube 1204 to move a slug 1230 out of
through-hole 1214 and into right-split-slug tube 1222, as shown in
FIG. 14. In some embodiments, enough spacing fluid is flowed
through through-hole 1214 to completely flow slug 1230 from
through-hole 1214 and past upper wall 1210 of housing 1206. As
illustrated, through-hole 1214 is filled with spacing fluid in FIG.
14.
[0163] Subsequently, slider 1212 is moved back to the left-side
position as shown in FIG. 15 so that through-hole 1214 of slider
1212 can then be again aligned with
immiscible-fluid-discrete-volume supply tube 1202 and
left-split-slug tube 1220. Because through-hole 1214 is filled with
spacing fluid, the volume of fluid that used to be the remaining
half of slug 1226 forms a smaller slug 1232.
[0164] Thereafter, as shown in FIG. 16, fluid in
immiscible-fluid-discrete-volume supply tube 1202 is flowed
upwardly to completely flow slug 1232 through through-hole 1214 and
into left-split-slug tube 1220 and, in some embodiments, above
upper wall 1210. In some embodiments, through-hole 1214 becomes
filled with spacing fluid, depending on the spacing between slugs
in immiscible-fluid-discrete-volume supply tube 1202. As
illustrated, through-hole 1214 is filled with spacing fluid.
[0165] Thereafter the series of steps described in conjunction with
FIGS. 12-16 can be repeated. The ratio of post-split slug volumes
1230 and 1232 is determined by the positioning of slug 1226 with
respect to the interface between lower wall 1208 and slider 1212
and the height of slider 1212.
[0166] In some embodiments, the initial
immiscible-fluid-discrete-volume can be flowed into through hole
1214 such that part of it remains in through-hole 1214, and part of
it extends into left-split-part conduit 1220. Then, when slider
1212 is moved back to align with conduit 1220 after "splitting"
slug 1228, slug 1232 can be moved further into conduit 1220 with
less, if any, spacing fluid flowing through through-hole 1214.
[0167] Another embodiment of a method of splitting slugs using a
two part slider mechanism described in U.S. patent application Ser.
No. ______, entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363) comprises splitting a slug into at least two parts
and moving the first and second sliders to align with distinct sets
of through-holes and conduits to deliver their respective split
portion 1230 or 1232 of initial slug 1228, wherein each of those
conduits is not in alignment with the
immiscible-fluid-discrete-volume supply conduit. An advantage of
this embodiment is it requires less time to flow both smaller split
slugs in their respective conduits.
[0168] FIG. 17 illustrates an embodiment 1700 of a bubble remover,
which takes advantage of the buoyancy of gas in a spacing fluid to
remove undesired bubbles from spacing fluid between
immiscible-fluid, discrete volumes. As illustrated in FIG. 17,
conduits 1702, 1704, and 1706 form a junction in the shape of T. As
illustrated, tube 1702 contains two slugs 1708 and a gas-phase
discrete volume (herein after referred to as a bubble) 1710 in a
spacing fluid 1712 flowing at a rate, Q combined. As illustrated,
tube 1704 contains two whole slugs and a part of a third slug 1708
in spacing fluid 1712 flowing at a rate, Q discrete volumes. Note
that spacing fluid 1712 in tube 1704 is free of bubbles 1710. As
illustrated, tube 1706 contains two bubbles 1710 in spacing fluid
1712 flowing at a rate, Q bubbles. Bubbles 1710 in tube 1706 may
have a speed greater than the speed of spacing fluid 1712 due to
the differences in specific gravity (buoyancy) until they reach the
top of the bend in tube 1706. As the diameter of each tube 1702,
1704, and 1706 is the same in FIG. 17, slugs 1702 flow into tube
1704 after the junction due to the difference in flow rates between
Q discrete volumes and Q bubbles. In some embodiments, the three
flow rates can have the following relationship: Q combined>Q
discrete volumes>Q bubbles. In some embodiments, flow rate Q
combined can be 10 times as great as Q bubbles and can be up to 100
times as great As illustrated, slugs 1708 in tube 1704 are separate
by less distance than in tube 1708 due to the movement of spacing
fluid into tube 1706.
[0169] In some embodiments, conduit 1702 and 1704 can form an angle
of about 90 degrees and conduit 1702 and conduit 1706 can form an
angle of about 180 degrees. In this embodiment, slugs bend
approximately ninety degrees as the move from the junction of
conduits 1702 and 1704. Gas bubbles 1710 flow straight up from
conduit 1702 into conduit 1706.
[0170] FIG. 18 illustrates another embodiment 1800 of a bubble
remover. Bubble remover 1800 includes a "cross" junction connected
to conduits 1802, 1804, 1806, and 1808. As illustrated, tube 1802
contains an immiscible-fluid, discrete volume, depicted as a slug
1812, and a gas-phase discrete volume, depicted as a bubble 1814 in
spacing fluid 1816 flowing at a rate, Q oil+DVs+bubbles, in the
positive Z direction. As illustrated, tube 1804 contains two slugs
1812 in spacing fluid 1816 flowing at a rate, Q oil+DVs, in the
positive X direction. Tube 1804 is free of bubbles 1814. As
illustrated, tube 1806 contains spacing fluid 1816 flowing at a
rate, Q oil, in the positive X direction. As illustrated, tube 1808
contains three bubbles 1814 in spacing fluid 1816 flowing at rate,
Q oil+bubbles, flowing in the positive Z direction. As a bubble
1814 reaches the junction volume, it transfers from the stream of
spacing fluid 1816 from tube 1802 to the stream of spacing fluid
1816 in tube 1808 due to buoyancy. In some embodiments, Q
oil+DVs+bubbles is equal to Q oil+DVs. In some embodiments, Q
oil+DVs+bubbles is greater than Q oil+DVs.
[0171] Application of additional pressure to the system can cause
the gas phase to be dissolved into solution, which is in effect,
another embodiment of a bubble remover. Such pressure is sometimes
called "back pressure." The use of back pressure can be useful
during batch thermal cycling of the fluids can apply to all thermal
cycling processing sections, whether in thermal spirals, for
example, in a system as illustrated in FIG. 1, or in a flow through
system, to prevent movement of discrete volumes that may coalesce
if in contact. If no back pressure is applied, pre-existing bubbles
between two slugs may expand and cause one or more of those slugs
to contact, and, therefore, merge with, another slug, reducing the
desired separation of reactions within each slug. During thermal
cycling, without back pressure, bubbles may form in previously
bubble-free spacing fluid, which may then expand and cause merging
of slugs.
[0172] FIG. 19 illustrates an immiscible-fluid-discrete-volume
processing apparatus 1900 including an embodiment of a
back-pressure device. Apparatus 1900 includes a tube 1902
containing two or more discrete volumes of aqueous liquid 1904,
each of which is in contact with an immiscible, non-aqueous liquid
1906. As illustrated, tube 1902 is blocked at one end by a valve in
a closed position or other pressure retaining structure,
illustrated here as a box 1908. Tube 1902 is connected to, as
illustrated, and in fluid communication with, a reservoir 1910
containing a volume of the immiscible, non-aqueous liquid 1906. In
some embodiments, reservoir 1910 can be expandable and
contractible. In some embodiments, and as illustrated here, the
immiscible, non-aqueous liquid 1906 is in contact with a diaphragm
1912, which is between reservoir 1910 and an enclosure 1914. In
some embodiments, enclosure 1914 is filled with pressurized fluid
1916. In some embodiments, pressurized fluid 1916 is an inert gas.
In some embodiments, pressurized fluid 1916 is air.
[0173] In some embodiments, the pressure of pressurized fluid 1916
is supplied by a pressure pump 1922. In some embodiments, the
pressure of pressurized fluid 1916 is regulated to a set pressure
by regulator 1920 connected between pressure pump 1922 and
enclosure 1914. In some embodiments, and as illustrated in FIG. 19,
a three-way valve 1918 may be connected between enclosure 1914 and
regulator 1920 or pressure pump 1922. In some embodiments, and as
illustrated, the common port of three way valve 1918 is connected
to reservoir 1914 and is in fluid communication with the space
between enclosure 1914 and diaphragm 1912. The first of the two
input/output ports can be in fluid communication with regulator
1920, if present in the apparatus, or with pressurized pump 1922.
The second of the two input/output ports can be in fluid
communication with the external atmosphere, for releasing the
pressure in the space between enclosure 1914 and diaphragm when
back pressure is not intended.
[0174] FIG. 20 illustrates another embodiment of an
immiscible-fluid-discrete-volume processing apparatus 2000
including an embodiment of a back-pressure device. Apparatus 2000
includes a tube 1902 containing two or more discrete volumes of
aqueous liquid 1904, each of which is in contact with an
immiscible, non-aqueous liquid 1906. As illustrated, tube 1902 is
blocked at least one end by a valve in a closed position or other
pressure retaining structure, illustrated here as a box 1908, or
box 2002. As illustrated, tube 1902 is positively pressurized by
being in fluid communication with a back pressure device as
illustrated in FIG. 19 (for convenience, not all structures
discussed in conjunction with FIG. 19 are illustrated in FIG. 20)
at least at a first location in tube 1902. One location on tube
1902 in fluid communication with reservoir 1910 is at the junction
2004 with tube 2006. A second location on tube 1902 in fluid
communication with reservoir 1920 is at junction 2008 with tube
2010. Between junctions 2004 and 2008, tube 1902 is heated or
cooled or maintained at one or more temperatures by
temperature-control unit 2012. Temperature-control unit 2012 can
comprise, for example, a heating unit, a cooling unit, an
isothermal unit, a thermal-cycler. While illustrated as contacting
temperature-control unit 2012 in a single straight-line segment,
tube 1902 may form, for example, a coil around the external
perimeter of temperature-control unit 2012, a spiral of decreasing
radius on one surface, or a serpentine pattern across one or more
surfaces of temperature-control unit 2012.
[0175] FIG. 21 illustrates a discrete volume characterization
determination and optional manipulation control system 2100. System
2100, as illustrated, includes sources 2102 and 2104. Sources 2102
and 2104 can be optical sources, such as a laser, an LED, a high
intensity white light source, such as for example, a tungsten or a
halogen lamp, with or without a band pass filter, positioned near
conduit 2106. In some embodiments, sources 2102 and 2104 can be a
single source, configured to illuminate two or more different
areas. Conduit 2106 contains a spacing fluid 2108 which is
immiscible with the fluid in immiscible-fluid, discrete volumes
2114 and 2112, which are depicted as slugs. System 2100, as
illustrated, includes detectors 2116 and 2118, which receive
signals from sources 2102 and 2104, respectively, as modified by
slugs 2114 and 2112, respectively. Detectors 2116 and 2118 can be,
for example, fluorescence detectors or infra red detectors. In some
embodiments, detectors 2116 and 2118 can each be a photo multiplier
tube (PMT) or an infra red, refraction index detector. In some
embodiments, only one source and detector are needed to gather
desired characteristics of aqueous discrete volumes.
[0176] Detectors 2116 and 2118 are coupled to sense circuitry 2120
and 2122, respectively, which senses the detected signal and
provides it in a useable format to a controller 2124. Controller
2124 can include for example, a set of instructions, memory, and a
processing unit. Controller 2124 can communicate with one or more
valves 2126, which permit or prevent flow upstream or downstream of
a detector, one or more pumps 2128 and 2130, which pressurize
(negatively or positively), displace, or otherwise move fluid
upstream and/or downstream of a detector, motors 2132 to move the
relative locations of, for example, containers of liquids, and
conduits for removing liquid from or adding liquid to those
containers, and one or more monitors for viewing and further
interface with the presented information. In communicating,
controller may direct a component to maintain or change its state
based on the signal received from sense circuitry 2120 and/or
2122.
[0177] FIG. 22 illustrates an imaging station data flow of system
2200. In some embodiments, a detector can comprise a camera to
gather characteristics of aqueous discrete volumes in an immiscible
spacing fluid within a conduit. As illustrated, conduit 2202
contains aqueous discrete volumes of interest (not shown), which is
imaged by camera 2204. Camera 2204 can transmit the image data to a
computer 2206 via Firewire. Computer 2206 transmits the image
recorded by camera 2204 as optionally modified by software, to
monitor 2208. Computer 2206 can also transmit data to an external
memory device, such as, for example, a hard disk drive. As
illustrated in the image displayed on monitor 2208, conduit 2202 is
shaped in a spiral. In some embodiments, this image data can be use
in monitoring PCR to provide real-time quantization of expression
within each discrete volume of aqueous sample. In some embodiments,
this image data can be used to estimate what percentage of discrete
volumes contained DNA before and/or during to amplification.
[0178] In some embodiments, a method is provided that can comprise
using the system described herein to process an aqueous
immiscible-fluid-discrete-volume. Additional compounds may be
needed in the aqueous discrete volume during the processing and, as
discussed above, the spacing of adjacent aqueous discrete volumes
may need to change to accommodate the addition of liquids
comprising the desired compounds involved in the reaction or
processing within an aqueous discrete volume.
[0179] According to various embodiments of the present teachings
illustrated, for example, in FIG. 23, a plurality of tubes 710,
712, 714, 716, and 718, can be connected to, or otherwise be in
fluid communication with, a manifold 720 at a plurality of
respective openings 722, 724, 726, 728, and 730. While tubes 710,
712, 714, 716, and 718 are shown terminating at manifold 720 and in
fluid communication with respective passageways in manifold 720, it
is to be understood that the tubes can, in some embodiments, extend
into manifold 720. For example, instead of the arrangement shown in
FIG. 23, tubes 712, 714, and 716 can be inserted into bores formed
in manifold 720 and which extend all the way to respective
junctions with immiscible-fluid-discrete-volume channel 756, or
closely adjacent to immiscible-fluid-discrete-volume channel 756.
Manifold 720 is shown in cross-section and tubes 710, 712, 714,
716, and 718 are depicted as transparent, although they do not have
to be, so that the oil and reagents therein can be seen in the
figure. Tube 718 can be transparent in some embodiments such that a
reaction involving an aqueous immiscible-fluid-discrete-volume, or
the position of an immiscible-fluid-discrete-volume can be
monitored inside tube 718 can be detected from outside tube 718. In
the embodiment shown in FIG. 7, tube 710 is shown as a supply tube
that supplies pre-formed immiscible-fluid, discrete volume 732 in
spacing fluid 766 to manifold 720. Immiscible-fluid, discrete
volume 732 can be moved from a supply unit 734. In some
embodiments, supply unit 734 can include a temporary storage tube
of pre-formed discrete volumes of a first type of fluid in contact
with a second type of fluid with which it is immiscible. In some
embodiments, supply unit 734 can include an
immiscible-fluid-discrete-volume-forming apparatus as described in
concurrently filed, U.S. patent application Ser. No. ______,
entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363). Supply unit 734 can also include a
discrete-volume characterization determination and optional
manipulation apparatus (not shown) as described in conjunction with
FIGS. 21 and 22.
[0180] According to the various embodiments, manifold 720 can
comprise a fluorocarbon material, for example, a perfluorocarbon
material such as polytetrafluoroethylene. In some embodiments,
manifold 720 can comprise the same material as is used for the
tubes 710, 712, 714, 716, and/or 718, or other materials known to
those skilled in the art. Materials can be selected that are
non-reactive or minimally reactive with the liquids passing through
manifold 720.
[0181] According to various embodiments, tube 710 can be connected
to manifold 720 by any appropriate connection, for example, using a
fitting or connector that extends from manifold 720, by
frictionally fitting tube 710 into a bore formed in manifold 720
wherein the bore has an inner diameter that is about equal to the
outer diameter of tube 710, or by using an adhesive, or the like.
Similarly, tubes 712, 714, 716, and 718, can be connected to
manifold 720.
[0182] In the embodiment shown, tube 712 is connected at a first
end to manifold 720 and at an opposite, second end to a first
liquid supply unit 736. First liquid supply unit 736 can comprise,
for example, a supply of a first liquid 738 and a pump for moving
first liquid 738 into and through tube 712. First liquid supply
unit 736 can comprise a pump of the same type, or of a different
type, as the type used for supply unit 734. Controller 2124 (shown
in FIG. 21) can control the flow rate of first liquid 738, whether
a result of differential pressure or displacement or other motive
force. In some embodiments, controller 2124 can control the
pressure independently of the other supply units in the system.
[0183] A tube 714 can be connected at a first end to manifold 720
and at a second, opposite end, to a second liquid supply unit 740.
Second liquid supply unit 740 can comprise, for example, a supply
of a second liquid and a pump for moving the second liquid 742 into
and through tube 714. Second liquid supply unit 740 can comprise a
pump that can be the same type as, or different than, the type of
pump used in supply unit 734. Controller 2124 (shown in FIG. 21)
can control the flow rate of second liquid 742, whether a result of
differential pressure or displacement.
[0184] A tube 716 can be connected at a first end to manifold 720
and at a second, opposite end to a third liquid supply unit 744.
Third liquid supply unit 744 can comprise, for example, a supply of
a third liquid 746 and a pump for moving third liquid 746 into and
through tube 716. The pump can be the same type as, or different
than, the type of pump used for supply unit 734. Controller 2124
can control the flow rate of third liquid 746 in tube 716, whether
a result of differential pressure or displacement/time or other
motive force.
[0185] According to various embodiments, first liquid 738, second
liquid 742, and third liquid 746, can each comprise an aqueous
medium, for example, an aqueous solution, and each can be miscible
with the other two reagents. In some embodiments, each of first
liquid 738, second liquid 742, and third liquid 746, can be
immiscible with spacing fluid 766 in contact with aqueous discrete
volume 732. In some embodiments, the first liquid 738, second
liquid 742, or third liquid 746 can comprise spacing fluid 766 to
increase or decrease the spacing between immiscible-fluid discrete
volumes prior to receiving an additional volume of aqueous liquid
at a subsequent junction. Controller 2124 (see FIG. 21) can control
the speed and direction of pumps in liquid supply units 736, 740,
and 744 according to a set program and as optionally modified with
data from detectors upstream and/or down stream of additional
liquid addition and/or removal. As such, as the first liquid 738,
second liquid 742, and third liquid 746 pass through passageways
750, 752, and 754, respectively, and flow into
immiscible-fluid-discrete-volume channel 756 in the body of
manifold 720.
[0186] In an exemplary embodiment, a first aqueous
immiscible-fluid-discrete-volume 732 flowing through
immiscible-fluid-discrete-volume channel 756 can be supplemented
with second liquid 742 flowed out at a constant rate or injected by
second liquid supply unit 740 as aqueous
immiscible-fluid-discrete-volume 732 lines-up with the junction of
passageway 752 and immiscible-fluid-discrete-volume-forming channel
756. As a result, a supplemented immiscible-fluid-discrete-volume
760 can be formed that comprises a mixture of the aqueous liquid of
aqueous immiscible-fluid, discrete volume 732 and second liquid
742. As supplemented immiscible-fluid-discrete-volume 760 proceeds
through immiscible-fluid-discrete-volume-forming channel 756 and
reaches the junction of immiscible-fluid-discrete-volume-forming
channel 756 with passageway 754, third liquid 746 can be flowed out
at a constant rate or injected from third liquid supply unit 744,
when properly timed, to form a further supplemented
immiscible-fluid-discrete-volume 762 comprising a mixture of the
aqueous liquid of aqueous immiscible-fluid, discrete volume 732,
second liquid 742, and third liquid 746. It is to be understood
that additional tubes can be connected to additional respective
passageways (not shown) if it is desired to provide such additional
features in a system. It is to be understood that system 2300 can
also comprise a separate manifold for each junction of a liquid
adding or removal conduit and an immiscible-fluid-discrete-volume
supply conduit, adjacent manifold being connected by tubes. While
the manifold is not illustrated, such a concept as just mentioned
is illustrated in FIG. 25, which illustrates junctions of four
liquid-adding and/or liquid removal conduits with an
immiscible-fluid-discrete-volume supply conduit.
[0187] Such an embodiment that permits the addition of one or more
different miscible liquids to be added to an
immiscible-fluid-discrete-volume, can assist in adding a first
individual primer to a selected slug at a first junction, and a
second individual primer to a selected slug at a second junction.
After receiving both primers, the selected slug will contain a
desired primer set for amplification in a later section. In this
way, the number of different primer liquids needed to provide a
predetermined number of primer pairs for requencing methods, such
as those described in conjunction with SYSTEM APPLICATION, can be
reduced. Table 3 illustrates the combination of eight different
primer liquids to provide 16 different primer pairs for use with a
set of slugs, or other discrete volumes. TABLE-US-00004 TABLE 3 M
.times. N Zip code Primers 3' primer A B C D A AA AB AC AD B BA BB
BC BD 5' primer C CA CB CC CD D DA DB DC DD
[0188] Referring again to FIGS. 23 and 24, an outlet from manifold
720 can be provided at opening 730 and can be connected to tube
718, for example, using a connection as described above. In an
exemplary embodiment, tube 718 is connected by a first end to
manifold 720, and at a second, opposite end, to a collection unit
764 where the further supplemented
immiscible-fluid-discrete-volumes 762 can be processed, detected,
or otherwise manipulated, analyzed, and/or transferred to another
device or system.
[0189] According to various embodiments, system 2300 shown in FIG.
23 can comprise a single pump and a valving scheme that replaces
the four individual pumps described above in connection with units
734, 736, 740, and 744. Embodiments of two such single pump and
valving schemes are illustrated in FIGS. 26 and 27 and will be
described below in conjunction with those figures
[0190] System 2400 illustrated in FIG. 24 includes the same solid
structures as FIG. 23, but the liquids in tubes 712 and 716 are
configured differently. For example, tube 712 contains two distinct
types of aqueous liquids, each in a discrete volume, namely, first
liquid 2402 and second liquid 2404, spaced apart by spacing fluid
766. The volume of first liquid is sufficient, as illustrated, to
add a pre-determined amount to several immiscible-fluid, discrete
volumes flowing past the junction with
immiscible-fluid-discrete-volume supply channel 756. When all of
first liquid 2402 has been flowed out of 736, spacing fluid 766
will advance into immiscible-fluid-discrete-volume supply channel
756, increasing the amount of spacing fluid between discrete
volumes. Then, if the flow through tube 712 continues, second
liquid 2404 can be added to immiscible-fluid, discrete volumes
flowing past the junction of passageway 750 and channel 756. If the
discrete volumes are slugs, then tube 712 and passageway 750 are
not contacted by the first or second liquids, reducing the chance
that second liquid 2404 will be contaminated by first liquid
2402.
[0191] As illustrated, tube 716 and passageway 754 contain a third
liquid 746 (which number is not in the figure) in three separate
discrete volumes 2406, 2408, and 2410 separated by spacing fluid
766. As illustrated, the volumes of third liquid 746 in volumes
2406, 2408, and 2410 is roughly equal to the volume of the passing
immiscible-fluid, discrete volumes 732 in through-hole 756. By
controlling the flow rates in each conduit, discrete volumes, 2406,
2408, and 2410 can each coalesce with a discrete volume 732 to form
a supplemented (not illustrated) discrete volume of combined
liquids 732 and 746. In some embodiments, a particular liquid is
desired to be added to only certain slugs, and having preformed
volumes of the addition liquid can accomplish this task with
minimal contamination of the slugs to which no addition liquid is
to be added with that addition liquid, as may be the case when the
addition liquid is present up to the junction of the addition
conduit and the main conduit, even if its flow rate is zero.
[0192] While the methods described in conjunction with FIG. 23
involve adding more than one of the first, second, and third
reagents, it is to be understood that separate control units, or a
single controller 2124 (See FIG. 21), described herein can be used
to control the addition of just one, two, or all three, of those
reagents. For example, if it is desired to add only the second
liquid to an aqueous immiscible-fluid-discrete-volume, the control
units for supply units 736 and 744, or controller 2124 (see FIG.
21) can control those units not to add the first and third reagents
to that aqueous immiscible-fluid-discrete-volume.
[0193] The various reagents, mixtures, samples, oils, and other
fluids and liquids that can be used with or moved through the
systems described herein include those fluids and liquids described
in detail in U.S. Provisional Patent Application No. 60/710,167
entitled "Sample Preparation for Sequencing," to Lee et al., filed
Aug. 22, 2005 (Attorney Docket No. 5841P), and in U.S. Provisional
Patent Application No. 60/731,133 entitled "Method and System for
Spot Loading a Sample," to Schroeder et al., filed Oct. 28, 2005
(Attorney Docket No. 5010-288), each of which is incorporated
herein in its entirety by reference.
[0194] According to various embodiments, different approaches or
mechanisms can be used to pump or drive liquid into a manifold.
According to various embodiments, and as illustrated in FIG. 25,
each different liquid supply conduit (2502, 2504, 2506, 2508) is in
fluid communication with either a syringe pump (2510, 2512, 2514,
2516) or a liquid supply vessel (2518, 2520, 2522, 2524) via a flow
path of a three-way valve (2526, 2528, 2530, 2532, and illustrated
as a circle around the junction of the conduit from the liquid
supply vessel and the conduit between the
immiscible-fluid-discrete-volume supply conduit 2534 and the
respective, dedicated syringe pump). In some embodiments, when
liquid is to be added to immiscible-fluid-discrete-volume supply
conduit, a valve 2526 connects supply vessel 2518 and syringe pump
2510, and syringe pump 2510 withdraws liquid into through valve
2526. Valve 2526 is then switched to allow flow from syringe pump
2510 to immiscible-fluid-discrete-volume supply conduit 2534
through liquid supply conduit 2502 and syringe pump 2510 advances
the liquid to the junction at the desired flow rate to add a
desired volume of liquid to either the spacing fluid or an
immiscible-fluid-discrete-volume. Conduit 2502 between valve 2526
and immiscible-fluid-discrete-volume conduit 2534 may be primed
with spacing fluid drawn from the immiscible-fluid-discrete-volume
conduit 2534 by syringe pump 2510 before withdrawing the liquid to
be added from supply vessel 2518. This volume of spacing fluid is
then added back to immiscible-fluid-discrete-volume supply conduit
2534 before the desired liquid can enter
immiscible-fluid-discrete-volume supply conduit 2534.
[0195] A consideration in this regard can be the number of liquids
to be introduced, in the case when a large number of samples are to
be introduced into a tube. According to various embodiments, and as
illustrated in FIGS. 26 and 27, one pump, which communicates with
one or more valves that regulate the opening, closing, and/or
diversion of channels or liquids in channels, can deliver a
different liquid through each liquid-addition conduit to which it
is connected. FIG. 26 illustrates a system the same as that
illustrated in FIG. 25, except that instead of a dedicated syringe
pump displacing a liquid, one pressure pump 2610 can flow the
liquid in each conduit to add that liquid to the
immiscible-fluid-discrete-volume supply conduit. In FIG. 26, a
single pump 2610 can negatively or positively pressurize the
conduits to which it is connected. As illustrated, pump 2610 is
connected, via conduits and a three-way valve, to a liquid supply
vessel (2518, 2520, 2522, and 2524) and to an
immiscible-fluid-discrete-volume supply conduit 2534. Between the
three-way valve and the single pump, a conduit contains a structure
past which the liquid does not flow (2602, 2604, 2606, and 2608).
Liquid-stopping structures 2602, 2604, 2606, and 2608 can be a
liquid-impermeable membrane (porous or gas permeable), a diaphragm,
an orifice, or a set of orifices which are large enough for air to
flow through, but too small for the aqueous slug to wet and flow
through. In some embodiments, an orifice could be configured to
also allow the passage of oil, to remove any priming oil.
Liquid-stopping structures 2602, 2604, 2606, and 2608 permit
different usage rates for the different fluids, with a common
refill action, rather than requiring a fluid monitor for each line
as would be required if the tubes were to be controlled
individually.
[0196] One method of operating the system in FIG. 26 follows. With
three-way valve 2526 connecting immiscible-fluid-discrete-volume
supply conduit 2534 to pressure pump 2610, pressure pump 2610
pressurizes supply conduit 2502 to withdraw spacing fluid from
immiscible-fluid-discrete-volume supply conduit 2534 up to a
predetermined point as determined and controlled by, for example, a
system as described in FIG. 21. A controller (not shown) switches
three-way valve 2526 to connect pump 2610 and liquid supply vessel
2518. The controller causes pump 2610 to pressurize the conduits
between it and supply vessel 2518 to flow liquid from supply vessel
2518 through the conduits toward pump 2610 up to liquid-stop
structure 2602. The controller switches valve 2526 to connect
immiscible-fluid-discrete-volume supply conduit 2534 and pump 2610
and causes pump 2610 to pressurize the conduits between it and
immiscible-fluid-discrete-volume supply conduit 2534 to flow liquid
from liquid-stop structure 2602 toward
immiscible-fluid-discrete-volume supply conduit 2534. Three-way
valve 2526 meters the amount of liquid added to
immiscible-fluid-discrete-volume supply conduit 2534. In some
embodiments, valve 2526 may have at least an additional position in
which it can prevent fluid communication between all three tubes
connected to it.
[0197] Another method of operating the system illustrated in FIG.
26 is to prime the line between the pump and the
immiscible-fluid-discrete-volume supply conduit with the desired
"add" liquid, prior to the flow of
Immiscible-fluid-discrete-volumes in the
immiscible-fluid-discrete-volume supply conduit. In contrast to the
previously described method, this method allows the immediate
introduction of the "add" liquid to the
immiscible-fluid-discrete-volume supply conduit, rather than having
to first flow the spacing fluid in the "add" conduit to the
immiscible-fluid-discrete-volume supply conduit.
[0198] According to various embodiments, and as illustrated in FIG.
27, single pressure pump 2610 described in conjunction with FIG. 26
can directly pressurize liquid supply vessels 2518, 2520, 2522, and
2524. Thus, valve 2702, which no longer needs to be at least a
three way valve, meters the amount of liquid added to
immiscible-fluid-discrete-volume supply conduit 2524.
[0199] Another method to pump or transport liquids through the
manifold can involve displacement of liquids localized near the
junction of the addition conduit and the
immiscible-fluid-discrete-volume supply conduit. Compression of the
tube with a solenoid, roller, or other mechanism, may more dispense
a more consistent volume of addition liquid each time in comparison
to metered flow of pressurized liquid. Displacing the liquid in the
addition conduit by compressing or pinching the tubes with rollers
to press the tubes down is one example. As a roller is moved along
the tube the pinching action of the tube can be used to push liquid
into or out of the manifold. In some embodiments, a roller can be
positioned in a range from about 2 inches to 10 inches away from
the junction. Another is actuating a solenoid against the tube. In
some embodiments, a solenoid can be positioned in a range from
about 1 inch to about 1 foot away from the junction. Positioning
may depend on conduit thickness and the actuation force of a
selected solenoid.
[0200] Pumping and routing techniques, according to various
embodiments of the present teachings, can eliminate the need for
re-dipping a tube tip into different supply or other wells. This in
one regard can minimize contamination. In various embodiments, the
disclosed techniques can be performed in a totally enclosed,
vacuum-sealed or otherwise isolated system, such that the
introduction of air bubbles can be avoided.
[0201] In FIG. 28, an apparatus for and method of joining two
streams of immiscible-fluid-discrete-volumes are illustrated In
some embodiments, additional spacing fluid can be added and slug
spacing can be monitors to control alignment of a slug from each
stream with a slug from the other stream. In some embodiments, the
two slug streams can be a set of samples and a set of primers which
are loaded from two XYZ mechanisms from trays, permitting complete
flexibility in the sorting and matching of primers and samples.
[0202] FIGS. 29A, B, and C illustrate a progression of directing a
microdroplet 2902 of aqueous liquid into a slug 2904 flowing in a
main conduit 2906, as controlled with feed back from a detector
2908 of the position of the slug 2904. Knowing the distance of
detector 2908 from a junction of a conduit 2903 containing
microdroplet 2902, and the speed of slug 2904, the movement of
microdroplet 2902 toward slug 2904 can be timed such that while
slug 2904 is in the junction of conduits 2903 and 2906,
microdroplet 2902 will contact and coalesce forming a larger
discrete volume 2910, which as it moves past the junction will form
a supplemented slug 2910.
[0203] FIG. 30 depicts a system 3000 for generating a set of
immiscible-fluid-discrete-volumes 3002 and subsequently pushing the
set of immiscible-fluid-discrete-volumes 3002 into a downstream
processing conduit 3004. System 3000 can be used to carry out a
method wherein an immiscible-fluid-discrete-volume-forming conduit
3006, comprising an injection tip 3008, is manipulated in a two
fluid-containing vessel 3010 to form spaced apart aqueous
immiscible-fluid-discrete-volumes having spacing fluid disposed
between adjacent immiscible-fluid-discrete-volumes in the set.
Immiscible-fluid-discrete-volume generation can involve, for
example, the methods as generally described in connection with the
embodiments of FIGS. 8, 9A, 9B, and 10A-10D of U.S. patent
application Ser. No. ______, entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363). A set of immiscible-fluid-discrete-volumes
generated in immiscible-fluid-discrete-volume-forming conduit 3006
can be pulled through a Y-junction body 3012 and into a temporary
holding conduit 3014 by negative pressure created in temporary
holding conduit 3014 via a syringe pump 3016, although other
suitable types of pumps can be used. By reversing the action of
syringe pump 3016, a set of immiscible-fluid-discrete-volumes 3002
that have been pulled into temporary holding conduit 3014 can then
be pushed through and out of the temporary holding conduit 3014,
through Y-junction body 3012, and downstream into processing
conduit 3004.
[0204] As shown in FIG. 30, each of conduits 3004, 3006, and 3014
can be connected to Y-junction body 3012 through a ferrule, such as
ferrule 3018 as shown. In some embodiments, Y-junction body 3012
can comprise a valve-free junction 3020, as shown. In other
embodiments, Y-junction body 3012 can be provided with a valve, for
example, a multi-channel diverter valve such as valve 400 shown in
FIGS. 4 and 5.
[0205] Using the system shown in FIG. 30, many different sets or
batches of aqueous immiscible-fluid-discrete-volumes can be
generated, temporarily held, and pushed into a downstream
processing tube. For example, if the holding tube accommodates 100
aqueous immiscible-fluid-discrete-volumes spaced apart therein by
spacing fluid, and 1000 immiscible-fluid-discrete-volumes are
desired, 10 processes can be carried out wherein, for example, the
temporary holding conduit 3014 is filled with a set of 100
immiscible-fluid-discrete-volumes, a diverter and/or valve in the
Y-junction body 3012 is switched to cause a fluid communication
with a downstream processing conduit, and each set of 100
immiscible-fluid-discrete-volumes are pushed through the Y-junction
body 3012 into fluid processing conduit 3004, one set at a time.
Automated control of valving, if provided, within the Y-junction
body 3012 can facilitate the synchronization of valve actuation so
that when syringe pump 3016 applies positive pressure to push
fluid, the set of immiscible-fluid-discrete-volumes in temporary
holding tube 3014 can only exit the Y-junction body 3012 to the
downstream fluid processing conduit 3004.
[0206] In order to have great control over very small fluid
volumes, a conventional syringe pump can be used as syringe pump
3016, and in some embodiments, gearing can be implemented to gear
down the otherwise conventional syringe pump to accommodate small
movements of finite volumes of fluid. In some embodiments, a
reciprocating pump can be used with appropriate gearing to provide
both negative pressure and positive pressure, alternately.
[0207] According to various embodiments, an
immiscible-fluid-discrete-volume can be generated in an
immiscible-fluid-discrete-volume-forming conduit, and spaced apart
by spacing fluid, according to any of the various methods described
herein. To minimize and/or eliminate the formation of air bubbles
in an immiscible-fluid-discrete-volume-forming conduit, and to
minimize or eliminate merging of adjacent spaced-apart
immiscible-fluid-discrete-volumes, methods of pushing a pattern of
immiscible-fluid-discrete-volumes and spacing fluid through a
conduit can be used after the immiscible-fluid-discrete-volumes are
generated. In so doing, a pattern of
immiscible-fluid-discrete-volumes can be moved through a processing
conduit without the use of negative pressure. An exemplary system
for pushing a pattern of immiscible-fluid-discrete-volumes through
a conduit, after the pattern is formed, is depicted in FIGS. 31 and
32.
[0208] As shown in FIG. 31 a method is provided that can involve
the generation of a relatively small number of
immiscible-fluid-discrete-volumes in a pattern, spaced apart from
one another by an average distance by a spacing fluid, which set of
immiscible-fluid-discrete-volumes can then be separated from a
subsequent set of immiscible-fluid-discrete-volumes to achieve a
separation distance between sets that is greater than the average
distance between immiscible-fluid-discrete-volumes in a single set.
Once each set of immiscible-fluid-discrete-volumes is generated,
the set can be pushed, rather than pulled, into a main flow path or
main processing conduit, using positive pressure, thereby reducing
and/or eliminating the creation of air bubbles or merging of
adjacent imrniscible-fluid-discrete-volumes in the processing
conduit. A system 3100, as depicted in FIG. 31, can be used to
carry out such a method.
[0209] As shown in FIG. 31, system 3100 can include a pump 3102
operatively connected to a selector valve 3104 that can be
manipulated to perform a number of actions in an
immiscible-fluid-discrete-volume-forming conduit 3101. As shown in
FIG. 31, selector valve 3104 is operatively connected to ports 3106
and 3108 which can each be used as an inlet port and an outlet
port, depending upon a desired action selected.
Immiscible-fluid-discrete-volume-forming conduit 3101 is provided
with valves 3116 and 3118 adjacent ports 3106 and 3108,
respectively.
[0210] FIG. 31 shows a total of 11 method steps that can be used to
carry out an immiscible-fluid-discrete-volume-forming operation as
described above, and depicts the various states of valves 3116 and
3118, and the direction of flow through ports 3106 and 3108, in
each step. In the first step shown at the top of FIG. 31, both
valves 3116 and 3118 are closed and an intake tip 3103 of
immiscible-fluid-discrete-volume-forming conduit 3101 is positioned
within a spacing fluid vessel 3112. Next, valve 3116 is opened
while valve 3118 remains closed, and pump 3102 is actuated to draw
fluid into port 3108. The drawing action is timed with an
alternating disposition of intake tip 3103 back-and-forth between
spacing fluid vessel 3112 and an aqueous
immiscible-fluid-discrete-volume fluid vessel 3114. The alternating
submersion of intake tip 3103 into the spacing fluid in vessel 3112
and the aqueous immiscible-fluid-discrete-volume fluid in vessel
3114, as described elsewhere herein, generates a pattern of aqueous
immiscible-fluid-discrete-volumes in the
immiscible-fluid-discrete-volume-forming conduit 3101, separated
from one another by spacing fluid. As an example of such an uptake
technique, reference is made to FIGS. 8, 9A, 9B, and 10A-10D of
U.S. patent application Ser. No. ______, entitled "Device and
Method for Making Immiscible-Fluid-Discrete-Volumes," to Cox et al.
(attorney docket number 5010-363) and the accompanying descriptions
thereof. The pattern of spaced aqueous
immiscible-fluid-discrete-volumes in
immiscible-fluid-discrete-volume-forming conduit 3101 is referred
to herein as a zebra pattern.
[0211] After a first set of aqueous
immiscible-fluid-discrete-volumes is formed in conduit 3101, for
example as illustrated, 15 spaced-apart aqueous
immiscible-fluid-discrete-volumes, tip 3103 is then held in spacing
fluid vessel 3112 for a period of time sufficient to enable the
uptake of a large spacing fluid spacer following the first set of
15 aqueous immiscible-fluid-discrete-volumes. The large spacer can
be used to separate the first set of aqueous
immiscible-fluid-discrete-volumes from a subsequent set of aqueous
immiscible-fluid-discrete-volumes, as shown in the third and fourth
steps depicted in FIG. 31.
[0212] After two complete sets of aqueous
immiscible-fluid-discrete-volumes are generated in
immiscible-fluid-discrete-volume-forming conduit 3101, intake tip
3103 is held in spacing fluid 3112 and valve 3116 is closed such
that the first set of aqueous immiscible-fluid-discrete-volumes,
but not the second set of aqueous
immiscible-fluid-discrete-volumes, is located along conduit 3101
between port 3106 and 3108, as shown in the fifth step of the
process identified in FIG. 31 as the first "Loaded" step. Once the
first set of aqueous immiscible-fluid-discrete-volumes is loaded as
shown in the fifth step, valve 3118 is opened and pump 3102 is
configured along with selector valve 3104 to push spacing fluid
through port 3106 into conduit 3101, and through and past valve
3118, as shown in the sixth step identified as the "Push a Batch"
step. Once the first set of immiscible-fluid-discrete-volumes
passes valve 3118, valve 3118 is closed and the first set of
immiscible-fluid-discrete-volumes is ready for down-stream
processing as shown in the seventh step identified as the "ready"
step. Next, as depicted in steps 8-11, the second set of
immiscible-fluid-discrete-volumes is pulled through open valve 3116
until it is positioned between ports 3106 and 3108, while at the
same time a third set of immiscible-fluid-discrete-volumes is
generated by the alternating disposition of intake tip 3103 in
vessels 3112 and 3114. As shown in the ninth step identified as the
"Loaded" step, once the second set of
immiscible-fluid-discrete-volumes is positioned between ports 3106
and 3108, valve 3116 is closed and the second set of
immiscible-fluid-discrete-volumes is pushed through valve 3118
(step 10 "Push a Batch") in the same manner that the first set of
immiscible-fluid-discrete-volumes was pushed in the sixth step
("Push a Batch") described above. The method described in
connection with FIG. 31 can be repeated so that multiple sets of
immiscible-fluid-discrete-volumes can be pushed into a down-stream
processing conduit under positive pressure, with each set being
spaced apart from a subsequent set by a relatively large spacing
fluid spacer.
[0213] An alternative method to that shown in FIG. 31 is depicted
in FIG. 32 wherein a pump 3202, selector valve 3204, ports 3206 and
3208, and valves 3216 and 3218, are shown. In the method shown in
connection with FIG. 32, the intake tip of the
immiscible-fluid-discrete-volume-forming conduit can be disposed
initially in a rinse fluid retained in vessel 3220 prior to being
disposed alternately in a spacing fluid vessel 3212 and an aqueous
immiscible-fluid-discrete-volume fluid vessel 3214. Such a rinse
vessel can be used similarly in FIG. 31. As shown in FIG. 32, the
negative pressure used to initially uptake the spacing fluid and
aqueous immiscible-fluid-discrete-volumes is drawn through upstream
port 3206 as opposed to being drawn through downstream port 3208.
Another difference between the system and method shown in FIG. 32
relative to the system and method shown in FIG. 31 is that a first
set of aqueous immiscible-fluid-discrete-volumes is moved all the
way to a ready position, identified as the sixth step shown
("Ready"), before a second set of immiscible-fluid-discrete-volumes
is generated as shown in the seventh step identified as ("Suck a
Batch").
[0214] As can be understood with reference to FIGS. 30, 31, and 32,
the present teachings provide, in some embodiments, a method
comprising: applying a negative pressure to a conduit system
comprising an intake tip; contacting the intake tip with a first
fluid and a second fluid that is immiscible with the first fluid,
while applying the negative pressure, to draw the first fluid and
the second fluid into the conduit system and form a set of discrete
volumes of the first fluid spaced apart from one another by the
second fluid, the set moving in a first direction in the conduit
system; and thereafter, applying a positive pressure to the conduit
system to push the set of discrete volumes in the conduit system.
In some embodiments, the method can comprise applying a positive
pressure that causes the set to move in the first direction. In
some embodiments, the method can comprise applying a positive
pressure that causes the set to move in the conduit system in a
second direction that is opposite the first direction. In some
embodiments the method can comprise applying the negative pressure
to the conduit system until the set moves past a first diverter and
the method can further comprise then changing a position of the
diverter before applying the positive pressure, for example, to
change the pathway of the set. In some embodiments, the method can
comprise applying the negative pressure to the conduit system until
the set moves past a valve and a port, and the method can further
comprise then closing the valve, and furthermore, the applying of
positive pressure can comprise applying a positive pressure through
the port. In some embodiments, the method can comprise applying the
negative pressure with a reversible pump, and reversing the action
of the reversible pump to apply the positive pressure. In some
embodiments, the contacting further comprises applying an
electro-wetting force to move at least one of the first fluid and
the second fluid to a location adjacent the intake tip. As shown in
FIGS. 18 and 30-48 of U.S. patent application Ser. No. ______,
entitled "Device and Method for Making
Immiscible-Fluid-Discrete-Volumes," to Cox et al. (attorney docket
number 5010-363).
[0215] According to various embodiments of the present teachings, a
method is provided that comprises: alternately introducing a first
fluid and a second fluid, that is immiscible with the first fluid,
into a conduit, to form a set of immiscible discrete volumes of the
second fluid, each immiscible discrete volume of the set being
separated from one or more other immiscible discrete volumes of the
set by the first fluid, the set comprising a first end and a second
end; moving the set of immiscible discrete volumes in a first
direction by withdrawing from the conduit, some of the first fluid
from the first end of the set; and moving the set in the first
direction by adding to the conduit, more first fluid at the second
end of the set. In some embodiments, the method can involve
processing a first fluid that comprises an oil and a second fluid
that comprises an aqueous liquid, for example, an aqueous sample
that is immiscible in the oil. In some embodiments, the method can
further comprise moving the set past a valve in the conduit and
closing the valve before moving the set in the first direction by
adding to the conduit more first fluid at the second end of the
set. In some embodiments, closing a valve can comprise rotating a
rotary valve as described herein, for example, in connection with
FIGS. 3-5.
[0216] Other embodiments of the present teachings will be apparent
to those skilled in the art from consideration of the present
specification and practice of the present teachings disclosed
herein. It is intended that the specification and examples be
considered as exemplary only and not be limiting. All cited
references, patents, and patent applications are incorporated in
their entireties herein by reference.
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