U.S. patent application number 10/125045 was filed with the patent office on 2002-08-15 for low volume chemical and biochemical reaction system.
Invention is credited to Jovanovich, Stevan B., Roach, David J..
Application Number | 20020110900 10/125045 |
Document ID | / |
Family ID | 26844248 |
Filed Date | 2002-08-15 |
United States Patent
Application |
20020110900 |
Kind Code |
A1 |
Jovanovich, Stevan B. ; et
al. |
August 15, 2002 |
Low volume chemical and biochemical reaction system
Abstract
A method and device for preparing nanoscale reactions. An
automated system utilizes an array of reaction chambers. The ends
of the chambers are temporarily sealed with deformable membranes
and reactions effected by incubation of temperature cycling.
Reaction mixtures may be assembled by using the reaction containers
to meter reaction components. After the reaction is finished, the
reaction containers may be dispensed onto a substrate and the
reaction products analyzed. An automated transfer device may be
used for automated transport of the reaction container array or
other transportable elements.
Inventors: |
Jovanovich, Stevan B.;
(Livermore, CA) ; Roach, David J.; (Los Gatos,
CA) |
Correspondence
Address: |
Amersham Biosciences Corp.
800 Centennial Avenue
Piscataway
NJ
08855
US
|
Family ID: |
26844248 |
Appl. No.: |
10/125045 |
Filed: |
April 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10125045 |
Apr 18, 2002 |
|
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09577199 |
May 23, 2000 |
|
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60146732 |
Aug 2, 1999 |
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Current U.S.
Class: |
435/286.4 ;
422/400; 435/286.5; 435/287.2; 435/288.1; 435/309.1; 436/180 |
Current CPC
Class: |
C12Q 1/6869 20130101;
B01J 2219/00702 20130101; Y10S 435/809 20130101; C12Q 1/6869
20130101; B01L 7/52 20130101; C12Q 1/6806 20130101; Y10T 436/113332
20150115; B01J 2219/00722 20130101; B01J 2219/00585 20130101; B01L
2300/0838 20130101; C12Q 1/6806 20130101; Y10T 436/2575 20150115;
B01L 2400/0409 20130101; C12Q 2527/125 20130101; C12Q 2565/518
20130101; C12Q 2565/518 20130101; C12Q 2535/101 20130101; C12Q
2535/101 20130101; C12Q 2527/125 20130101; B01L 3/5025 20130101;
C12Q 1/6834 20130101; Y10T 436/143333 20150115; B01L 2400/0487
20130101; B01L 2300/1844 20130101; B01J 2219/00691 20130101; B01J
2219/00511 20130101; C40B 40/06 20130101; B01J 2219/00369 20130101;
C40B 60/14 20130101; B01J 2219/00286 20130101 |
Class at
Publication: |
435/286.4 ;
435/286.5; 435/287.2; 435/288.1; 435/309.1; 422/100; 436/180 |
International
Class: |
C12M 001/36 |
Claims
What is claimed is:
1. A device for dispensing small quantities of liquid, the device
comprising: a capillary cassette, comprised of a plurality of
capillary tube sections disposed in an array through a substrate;
and a capillary cassette dispensing means, said dispensing means
receiving said capillary cassette and dispensing an array of
capillaries of said capillary cassette to an array of
locations.
2. The device of claim 1, wherein said dispensing means is a
pressure driven dispenser.
3. The device of claim 2, wherein said pressure driven dispenser
includes an enclosed area, wherein one end of said capillary tube
sections in said capillary cassette may be sealed in said enclosed
area, and wherein the enclosed area enclosing one end of
capillaries in said capillary cassette may be pressurized.
4. The device of claim 2, wherein said pressure driven dispenser
includes a centrifuge.
5. The device of claim 1, wherein said capillary cassette is an
array of 96 capillaries.
6. The device of claim 1, further including a wash station and a
means for transferring said capillary cassette between said
dispensing means and said wash station.
7. The device of claim 6, wherein said means for transferring said
capillary cassette is an automated transfer device.
8. The device of claim 7, wherein said automated transfer device is
a robotic arm.
9. The device of claim 6, wherein said wash station includes a
housing for receiving a capillary cassette and a liquid
introduction and removal system that pumps liquid through said
capillary tube sections and evacuates said liquid from said
capillary tube sections.
10. The device of claim 1, wherein each capillary tube section of
said capillary cassette has an interior volume of less than one
microliter.
11. A device for transfer of an amount of liquid from a plurality
of locations in a highly parallel manner, the device comprising: a
capillary cassette comprised of a substrate and a two dimensional
array of capillary tube sections extending through said substrate,
each of said capillary tube sections having a first and a second
opposing open ends, said first open ends being coplanar and said
second open ends being coplanar; a capillary cassette dispenser;
and an automated capillary cassette transfer device, wherein said
capillary cassette transfer device may move said capillary cassette
between said a filling location and said capillary cassette
dispenser.
12. The system of claim 11, wherein said substrate is bendable.
13. The system of claim 11, wherein said substrate is curved.
14. The device of claim 11, wherein said capillary cassette
dispenser employs a pressure differential to dispense said
capillary tube sections in said capillary cassette.
15. The device of claim 12, wherein said dispenser is a centrifuge
dispenser.
16. The device of claim 12, wherein said dispenser includes an
enclosed area, wherein one end of said capillary tube sections in
said capillary cassette may be sealed in said enclosed area, and
wherein the enclosed area enclosing one end of capillaries in said
capillary cassette may be pressurized.
17. The device of claim 11, wherein said capillary cassette is
comprised of a 8 by 12 array of capillary tube sections of equal
length extending through a substrate.
18. The device of claim 11, further including a wash station,
wherein said automated capillary cassette transfer device is
disposed to transfer said capillary cassette between the capillary
cassette dispenser and said wash station.
19. The device of claim 16, wherein said wash station has an
enclosure that receives said capillary cassette and a fluid
distribution manifold for introducing fluid through capillary tube
sections of said capillary cassette.
20. The device of claim 11, wherein each capillary tube section in
said capillary cassette has an interior volume of less than one
microliter.
21. A method to dispense small quantities of fluid in a highly
parallel manner, comprising: filling an array of capillary tube
sections in a capillary cassette with fluid at a filling location,
moving said capillary cassette to a dispensing location; and
simultaneous dispensing said capillary tube sections at said
dispensing location.
22. The method of claim 19, further including a step of washing
capillary tube sections of said capillary cassette.
23. The method of claim 20, wherein all steps are repeated a
plurality of times.
24. The method of claim 19, wherein said filling occurs by
capillary action without mechanical force.
25. The method of claim 19, wherein said dispensing is effected by
establishing a pressure differential.
26. The method of claim 23, wherein said dispensing is effected by
centrifugal force.
27. The method of claim 19, wherein moving said capillary cassette
is effected by gripping said capillary cassette by an automated
transfer device and moving said capillary cassette to a programmed
location.
28. The method of claim 19, where the step of filling said array of
capillary tube sections includes filling said capillary tube
sections with a volume of fluid less than one microliter.
29. The method of claim 19, wherein all steps are controlled by a
central electronic control.
30. A device for transfer of an amount of liquid from a plurality
of locations in a highly parallel manner, the device comprising: a
capillary cassette comprised of a substrate and a linear array of
capillary tube sections extending through said substrate, each of
said capillary tube sections having a first and a second opposing
open ends, said first open ends being coplanar and said second open
ends being coplanar; a capillary cassette dispenser; and an
automated capillary cassette transfer device, wherein said
capillary cassette transfer device may move said capillary cassette
between said a filling location and said capillary cassette
dispenser.
31. The system of claim 30, wherein said substrate is bendable.
32. The system of claim 30, wherein said substrate is curved.
33. The device of claim 30, wherein said capillary cassette
dispenser employs a pressure differential to dispense said
capillary tube sections in said capillary cassette.
34. The device of claim 33, wherein said dispenser is a centrifuge
dispenser.
35. The device of claim 33, wherein said dispenser includes an
enclosed area, wherein one end of said capillary tube sections in
said capillary cassette may be sealed in said enclosed area, and
wherein the enclosed area enclosing one end of capillaries in said
capillary cassette may be pressurized.
36. The device of claim 30, further including a wash station,
wherein said automated capillary cassette transfer device is
disposed to transfer said capillary cassette between the capillary
cassette dispenser and said wash station.
37. The device of claim 36, wherein said wash station has an
enclosure that receives said capillary cassette and a fluid
distribution manifold for introducing fluid through capillary tube
sections of said capillary cassette.
38. The device of claim 30, wherein each capillary tube section in
said capillary cassette has an interior volume of less than one
microliter.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/577,199 filed May 23, 2000, and claims
priority from U.S. provisional application No. 60/146,732 filed
Aug. 2, 1999.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates to a method and apparatus for
performing small scale reactions. In particular, the instant
disclosure pertains to small scale cycling reactions and devices
for assembly of sub-microliter reaction mixtures.
BACKGROUND OF THE INVENTION
[0003] The Human Genome Program is a scientific endeavor which is a
national priority of the United States. The original goal of the
federally funded U.S. effort had been to complete the sequence at
ten-fold coverage by the year 2005. A draft, five-fold deep version
of the human genome will now be produced by the year 2001. To
accomplish this goal, the effort has accelerated to improve
sequencing throughput rates and reduce DNA sequencing costs.
[0004] In the late 1970s, Sanger et al. developed an enzymatic
chain termination method for DNA sequence analysis that produces a
nested set of DNA fragments with a common starting point and random
terminations at every nucleotide throughout the sequence. Lloyd
Smith, Lee Hood, and others modified the Sanger method to use four
fluorescent labels in sequencing reactions enabling single lane
separations. This resulted in the creation of the first automated
DNA sequencers. More recently, fluorescent energy-transfer dyes
have been used to make dye sets that enhance signals by 2- to
10-fold and simplify the optical configuration.
[0005] Automated fluorescent capillary array electrophoresis (CAE)
DNA sequencers appear to be the consensus technology to replace
slab gels. Capillary gel electrophoresis speeds up the separation
of sequencing products and has the potential to dramatically
decrease sample volume requirements. The 96-channel CAE instrument,
MegaBACE.TM., which is commercially available from Molecular
Dynamics (Sunnyvale, Calif.), uses a laser induced fluorescence
(LIF) confocal fluorescence scanner to detect up to an average of
about 625 bases per capillary (Phred 20 window) in 90 minute runs
with cycle times of two hours. Confocal spatial filtering results
in a higher signal-to-noise ratio because superfluous reflections
and fluorescence from surrounding materials are eliminated before
signal detection at the photomultiplier tube (PMT). Accordingly,
sensitivity at the level of subattomoles per sequencing band is
attainable. Confocal imaging is also particularly important in
capillary electrophoresis in microchip analysis systems where the
background fluorescence of a glass or plastic microchip may be much
higher than that of fused silica capillaries. Capillary array
electrophoresis systems will solve many of the initial throughput
needs of the genomic community for DNA analysis. However, low
volume sample preparation still presents a significant opportunity
to increase throughput and reduce cost.
[0006] While fluorescent DNA sequencers are improving the
throughput of DNA sequence acquisition, they have also moved the
throughput bottleneck from sequence acquisition back towards sample
preparation. In response, rapid methods for preparing sequencing
templates and for transposon-facilitated DNA sequencing have been
developed as have magnetic bead capture methods that eliminate
centrifugation. Thermophilic Archae DNA polymerases have been
screened and genetically engineered to improve fidelity, ensure
stability at high temperatures, extend lengths, and alter
affinities for dideoxynucleotides and fluorescent analogs. These
improvements have resulted in lower reagent costs, simpler sample
preparation, higher data accuracy, and increased read lengths.
[0007] The sequencing community has also developed
higher-throughput methods for preparing DNA templates, PCR
reactions, and DNA sequencing reactions. Sample preparation has
been increasingly multiplexed and automated using 96- and 384-well
microtiter plates, multi-channel pipettors, and laboratory robotic
workstations. In general, these workstations mimic the
manipulations that a technician would perform and have minimum
working volumes of about a microliter, although stand-alone
multi-channel pipettors are being used to manipulate smaller
volumes.
[0008] A typical full-scale sample preparation method for DNA
shotgun sequencing on capillary systems begins by lysing phage
plaques or bacterial colonies to isolate subcloned DNA. Because
capillary electrophoresis is more sensitive to impurities in
sequencing reactions than slab gels, the subcloned DNA insert is
PCR amplified to exponentially increase its concentration in the
sample. Next, exonuclease I (ExoI) and arctic shrimp alkaline
phosphatase (SAP) are added to perform an enzymatic cleanup
reaction to remove primer and excess dNTPs that interfere with
cycle sequencing. ExoI is used to degrade the single-stranded
primers to dNMPs without digesting double-stranded products. SAP
converts dNTPs to dNMPs and reduces the dNTP concentration from 200
:M, as used for the PCR reaction, to less than 0.1 :M for use with
fluorescent sequencing. The reaction is performed at 37EC and then
heated to 65EC to irreversibly denature the ExoI and SAP.
[0009] Because the PCR amplification produces excess template DNA
for cycle sequencing, the ExoI/SAP treated PCR sample can be
diluted five-fold before cycle sequencing. This reduces the
concentration of contaminants into a range that causes less
interference with CAE analysis. Cycle sequencing reagents are
added, typically with fluorescently labeled dye primers or
terminators and the reaction is thermal cycled to drive linear
amplification of labeled fragments. Finally, after cycling, the
samples are ethanol precipitated, formamide or another denaturant
is added, and the sample is electrokinetically injected into the
CAE system.
[0010] This workflow has resulted in a dramatic improvement in the
performance of the MegaBACE system and currently appears to be the
method of choice for other CAE systems as well. Using actual
samples from single plaques and colonies of human genomic random
subclones or Expressed Sequence Tags (ESTs), this workflow with
linear polyacrylamide as a separation matrix has improved the
success rate of samples over 200 base pairs from about 60% to
85-90%, and has improved the average readlength from about 350 to
greater than 500 bases. Furthermore, this method has proven to be
quite robust.
[0011] While the above sample preparation methods have greatly
increased throughput, the cost of reagents remains a major
component of the cost of sequencing. CAE requires only subattomoles
of sample. Reducing the reaction volume will therefore reduce the
cost of DNA sequencing. However, substantial reductions in reaction
volume can only be achieved if satisfactory methods can be
developed for manipulating and reacting samples and reagents.
Ideally, such a method would be automated and configured in order
that multiple samples could be produced at one time. Moreover, it
would be desirable to integrate such a method as a module capable
of interfacing with additional components, such as CAE and a
detector for separation and analysis.
[0012] Several devices have been designed to aid in the automation
of sample preparation. For example, U.S. Pat. No. 5,720,923
describes a system in which small scale cycling reactions take
place in tubes with diameters as small as 1 mm. The tube are
subsequently exposed to thermal cycles produced by thermal blocks
to effect a desired reaction. Multiple samples may be processed in
a single tube by drawing in small amounts of sample, each of which
are separated in the tube by a liquid which will not combine with
the sample. Fluid moves through the tubes by means of a pump. These
features are incorporated into a system which automatically cleans
the tubes, moves sample trays having sample containing wells, and
brings the tubes into contact with the wells in the sample
trays.
[0013] U.S. Pat. No. 5,785,926 discloses a system for transporting
small volumes of sample. In this system, at least one capillary
tube is used to transport small amounts of sample. A precision
linear actuator connected to a computer controlled motor acts as a
pneumatic piston to aliquot and dispense liquid using the tube. The
sample amount is monitored by an optical sensor that detects the
presence of liquid within the capillary segment. The system
includes a fluid station containing liquids to be deposited and a
positioning device for positioning the transport capillary.
[0014] U.S. Pat. No. 5,897,842 discloses a system for the automated
sample preparation using thermal cycling. In this system a reaction
mixture is pumped into a capillary tube. One end of the tube is
sealed using pressure from an associated pump while the other end
is sealed by pressing the tube against a barrier. The pump also
serves to move fluid within the tube. Once the ends are sealed, the
tube is exposed to thermal cycles. In this system a robotic
transfer device moves the tubes between the sample preparation
station where the pump loads the components of the reaction mixture
into the tubes and the thermal cycling station.
[0015] There is an additional need for an automated system that is
able to perform small scale thermal cycling reactions in a highly
parallel manner. The system should allow for rapid preparation of
cycling reactions with minimal reagents. The combination of
reducing the amount of reagents required for a reaction and
reducing the time required for a reaction will greatly reduce the
overall cost of preparation of cycling reactions.
[0016] Capillary array electrophoresis systems and capillary
electrophoresis microchip analytical systems can detect
subattomoles of reaction products. It is one object of the
invention to disclose a method and system for cycling reactions
that operate on a submicroliter scale that takes advantage of the
high sensitivity of these analytical systems. This reduction of
reaction volume will lower the reagent requirements and cost of
each reaction. It is a further object to provide an automated
system that is able to reduce the time required for cycling
reaction preparation. It is an additional object of the invention
to provide a system that may be integrated with current analytical
instruments including capillary array electrophoresis systems and
electrophoresis chips.
[0017] It is a further object of the invention to provide an
automated system for preparing reactions and filling a reaction
container using capillary action. This allows metering a quantity
of liquid into a capillary tube length of fixed volume without
using external force to pump liquids. It is a further object to
disclose a reagent metering device which also may act as the
reaction container. It is also an object of the invention to
provide a system which allow the nanoscale reaction containers to
be cleaned and reused, saving material costs.
[0018] It is a further object of the invention to provide a system
with highly parallel processing, allowing greater throughput.
Preferably the system would match the density of microwell plates.
It is also an object of the invention to have an automated system
in which a number of different cycling reactions could be performed
in parallel using a single temperature regulation source, allowing
more efficient use of the thermal cycling apparatus. It is a
further object to perform isothermal reactions in a highly parallel
manner in submicroliter volumes. It is also an object of the
invention to provide an automated reaction preparation system that
is able to utilize available automation tools by being compatible
with standard plate size formats.
SUMMARY OF THE INVENTION
[0019] The above objects have been achieved through a system and
method for preparing cycling reaction mixtures. The system uses a
capillary cassette comprised of a number of capillary tube segments
arranged in parallel alignment. The tube segments extend through a
substrate and are generally positioned with uniform spacing. The
capillary cassette may be used both to meter reagents and as a
reaction chamber in which the reaction is conducted.
[0020] A reaction mixture containing a nucleic acid sample and
reaction reagents for performing a thermal cycling reaction (such
as the polymerase chain reaction, ligase chain reaction, or
preparing a chain termination sequencing reaction) is introduced
into the capillaries of a capillary cassette. In one embodiment
each capillary contains a unique nucleic acid sample but the same
reaction reagents.
[0021] The reaction mixture may be generated in various manners. In
one sample preparation method, sample DNA adheres to the interior
of the capillary tubes of the capillary cassette or onto a
substrate. The liquid in which the DNA was suspended may be
eliminated from the capillary or substrate while the nucleic acid
is retained, bound to the capillary or substrate. The reaction
reagents may then be introduced into the capillary or substrate,
combining the sample and reaction reagents to form an assay
mixture. In another sample preparation method, the capillaries in a
capillary cassette or the wells in a multiwell plate are coated
with dehydrated reaction reagents. The nucleic acid sample is
introduced into the capillaries of the capillary cassette or the
wells of a multiwell plate and the reaction reagents are dehydrated
to form a reaction mixture. If the multiwell plate is used, the
reaction mixture is subsequently transferred into the capillaries
of a capillary cassette. In another sample preparation method, both
the reaction reagents and the nucleic acid sample are metered by
the capillaries of a capillary cassette. The capillaries are dipped
into the wells of a sample plate and a fixed amount of fluid
(defined by the interior volume of the capillary) is drawn into the
capillary. The volume of liquid metered by the capillary tubes is
dispensed by positive displacement, centrifugal force, or other
displacement method into the wells of a microplate. A capillary
cassette is used to meter both the reaction reagents in a similar
manner and dispense the metered liquids onto a location on a
substrate combining the sample and reaction reagents to form a
reaction mixture. In any of these reaction mixture preparation
methods reaction reagents, nucleic acid sample and assembled
reaction mixture are introduced into the capillary tubes of a
capillary cassette or drawn into the capillary cassette by
capillary action. Liquids may also be introduced into the
capillaries by active filling, such as by pressure or vacuum. For
example one end of the capillaries may be sealed with a liquid
impermeable (hydrophobic), gas permeable membrane. By applying a
vacuum force to one side of the membrane, the capillary will fill
with liquid to the level of the membrane where hydrophobic forces
will prevent further filling of the capillary.
[0022] The capillary cassette filled with the reaction mixture is
next sealed by pressing the two ends of the capillary tube segments
against deformable membranes. The capillary cassette with ends
sealed against deformable membranes is contained within an interior
chamber of a temperature cycling device. The temperature cycling
device exposes the contents of the capillaries to thermal cycles,
causing the thermal cycling reaction to occur. In one embodiment
the thermal cycling apparatus is an air thermal cycling device.
This device receives the capillary cassette into an interior
chamber where the ends of the capillaries are sealed. The
temperature changes occur using rapidly flowing air. The
temperature of the cycling air may be rapidly lowered by venting
air to outside the interior cycling chamber. A thermocouple sensor
in the air path of the capillary cassette allows for precise
monitoring of the temperature of the reaction mixture. Given the
rapid transfer of heat through the capillary and precise
temperature sensing allowed by the thermal couple, rapid reaction
times are possible. The complete thermal cycling times needed for
30 cycles of denaturing heating followed by a period of lower
temperature for extension of a 600-700 base DNA strand are
performed in 30 minutes or less and could theoretically be effected
in as little as 8 minutes. Following a programmed number of thermal
cycles, the capillary cassette is removed from the temperature
cycling chamber.
[0023] The reaction mixture is next dispensed from the capillary
cassette and transferred onto a substrate. In one embodiment the
substrate onto which the completed reaction mixture is dispensed is
an analytical chip. Following transfer from the capillary cassette
the reaction mixture may be separated and analyzed. Alternatively,
the sample may be dispensed into a microplate or other substrate.
The substrate may then be placed, manually or by an automated
system, in a location where it may be analyzed by capillary array
electrophoresis. In addition to electrophoresis, the instant
reaction preparation system may also be adapted for use in
preparing nucleic acid, protein or other biomolecules for
microarray analysis, mass spectrometry analysis or other analysis
methods. The capillary cassette may also be used for conducting
ELISA or other assays requiring binding to a substrate.
[0024] The use of the present system allows a simplified transition
between nanoscale and larger scale preparation steps. For example
the PCR step may be performed on a nanoscale in the capillary
cassette of the present invention. The resulting products could be
dispensed into a microplate well for enzymatic clean-up on a larger
scale. Following clean-up, the amplified nucleic acid may be again
metered into a nanoscale capillary cassette for subsequent reaction
mixture preparation (e.g. cycle sequencing). This achieves a simple
transition method from nanoscales to larger scales.
[0025] Depositing the reaction mixtures from the capillary cassette
into the wells of a 96 well plate allows subsequent processing by
capillary array electrophoresis systems. Post reaction processing
is also possible. This could include depositing the reaction
mixture into ethanol to precipitate the DNA fragments produced in
the reaction or dispensing the reaction mixture into formamide to
denature double stranded DNA reaction products.
[0026] Following each use, the capillary cassette may be placed
into a capillary cassette washer and washed. Following washing, the
capillary cassette may be reused.
[0027] The system can be designed with magazines for holding the
sample plates, the multiwell mixing plates, and the plates
containing the finished reactions. This would allow the system to
continuously operate and prepare reaction mixtures. In addition, an
integrated system with a central electronic control would allow for
a system which may simultaneously assemble reaction mixtures,
perform thermal cycling, and wash capillary cassettes.
[0028] The system is useful in the preparation of sequencing
reactions, but may also be used in highly parallel preparation of
cell lysing and plasmid extraction, polymerase chain reactions,
ligase chain reactions, rolling circle amplification reactions,
screening compound libraries for drug discovery or compound
activity, protein digestion/sequencing, ELISA, radioimmunoassays
and other chemical or biochemical reactions or assays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic of an integrated system for the
preparation of cycling reaction products.
[0030] FIG. 2 is a flow chart illustrating the steps in reaction
production using the present system.
[0031] FIG. 3A is a perspective view of the capillary cassette of
the present invention.
[0032] FIG. 3B is a perspective view of the capillary cassette
inserted into a capillary cassette holder.
[0033] FIG. 3C is a flexible capillary cassette.
[0034] FIG. 3D illustrates the capillary cassette of FIG. 3C bent
to a frame and mating with the wells of an analytical chip.
[0035] FIG. 3E shows a two layer substrate with microchannels
contained within.
[0036] FIG. 4A illustrates the dispense head for dispensing liquid
from the capillary cassette.
[0037] FIG. 4B shows an internal cross section of an air
displacement dispense head of FIG. 4A.
[0038] FIG. 4C shows the dispense head of FIG. 4A with the dispense
head closed.
[0039] FIG. 5A illustrates a top view of a centrifuge used to move
fluid from the capillary cassette of FIG. 3A.
[0040] FIG. 5B illustrates a cross-section of a rotor arm of FIG.
5A holding a swinging microplate bucket.
[0041] FIG. 6 shows a schematic of an air based thermal cycling
device with the capillary cassette and holder shown in FIG. 3B
inserted into the temperature cycling device.
[0042] FIG. 7A shows an internal cross section of an air based
thermal cycler with integrated capillary cassette sealing
membranes.
[0043] FIG. 7B shows a detail of the air based thermocycler of FIG.
7A, with the lid raised to illustrate the chamber into which the
capillary cassette is inserted.
[0044] FIG. 7C shows a cross section of the cassette compartment
with the capillary cassette inserted into the internal chamber of
the thermal cycler of FIG. 7A.
[0045] FIG. 8A is a front view of the capillary cassette wash
station.
[0046] FIG. 8B is a side view of the view of FIG. 8A with the wash
manifold lowered and the wash rank raised.
[0047] FIG. 8C is the view of FIG. 8B with the wash manifold raised
and the wash tank lowered.
[0048] FIG. 8D is an interior cross-section of the wash
manifold.
[0049] FIG. 8E is a schematic plumbing diagram of the wash
station.
[0050] FIG. 8F is a top perspective view of the wash tank.
[0051] FIG. 9 shows a histogram of the percent success versus
readlength window for the sequencing analysis of example 2.
[0052] FIG. 10 is an electropherogram of the reaction products of
example 2.
[0053] FIG. 11 shows a histogram of the percent success versus
readlength window for the sequencing analysis of example 3.
[0054] FIG. 12A shows a scanned gel image of electrophoretically
separated PCR products prepared at full volume.
[0055] FIG. 12B show a scanned gel image of electrophoretically
separated PCR products prepared at nanoscale (500 nL).
[0056] FIG. 13 is an electropherogram of analysis of prepared
sequencing mixtures.
[0057] FIG. 14 is a graph comparing signal strength of reaction
products prepared in tubes, capillaries, and capillaries using
surface binding.
DETAILED DESCRIPTION OF THE INVENTION
[0058] In the present invention, it was realized that a capillary
segment could be used both to meter reagents and as a reaction
container for preforming temperature cycling reactions. The length
of the capillary and the internal diameter (ID) of the bore of the
capillary tube define the volume of the interior of the capillary
tube segment. Capillaries with a 50-150 um ID are commonly
available. The small internal diameter of the capillary tubes
allows creation of a reaction container having an interior volume
less than one microliter. With the present invention, capillaries
with interior volumes from 10-500 nanoliters are adaptable to the
preparation of DNA cycle sequencing reactions or any other
reaction.
[0059] The process carried out by the present automated system is
shown in the flow chart of FIG. 2. The process begins by the
assembly of the reaction mixture, box 52, by combination of
reagents and a sample nucleic acid. The combined reagents are then
introduced into the capillaries of a capillary cassette, box 54.
The ends of the capillaries are next sealed, box 56. The sealed
capillary segments are exposed to thermal cycles, box 58, which
effect the cycling reaction. The capillaries of the capillary
cassette are then dispensed onto a substrate, box 60. The substrate
is then transferred to an analytical system for analysis of the
reaction mixture, box 62. Details of this process and the structure
of the apparatus for carrying out this process are detailed
herein.
[0060] In reference to FIG. 1, an automated system is shown for
assembly of reaction mixtures, temperature cycling to effect the
chemical reaction, and dispensing the volume of the completed
reaction mixture onto a substrate for subsequent analysis. In the
system an automated robot 102 may move the length of stage 114 and
may rotate such that automated robot 102 may be moved in relation
to other components of the automated system. The automated robot
102 may be rotated to allow the transfer head 104 on automated
robot 102 to access objects on all sides adjacent to stage 114. The
assembly of a reaction mixture would begin by the transfer head 104
picking up a capillary cassette from cassette hotel 106.
[0061] Capillary cassette 15 is shown in FIG. 3A. The capillary
cassette is comprised of a number of capillary tubes 12 extending
through a substrate 10. It is preferred that the capillary cassette
have at least one row of eight capillary tubes and that the
capillary tubes have equal spacing. The capillary cassette shown
has substrate 10 with 96 capillary tubes arranged in an 8 by 12
array, with spacing of the tubes matching the spacing of the wells
of a 96 well microplate. The length of capillary tubes 12 extending
from either side of substrate 10 is unequal. It is preferred that
the shorter end of capillary tube segments 12 be shorter than the
depth of a microplate well. This allows the short end of capillary
tubes 12 to be inserted into the wells of a microplate while
substrate 10 rests on the top of the microplate.
[0062] The capillary tubes may be made of any material compatible
with the assay and preparation to be performed, but preferred
capillary materials include, but are not limited to, glass and
silica capillaries, although plastic, metals and other materials
may also be used. Capillary tubes of various dimensions may be
used, such as 75 um ID capillary tubes or 150 um ID/360 um O.D.
capillary tubes.
[0063] The capillary tubes 12 extend through a substrate 10 and
preferably are arranged in a uniform pattern. The capillary tubes
are of equal length and extend through the substrate in a
substantially parallel orientation such that each of the two
opposing ends of the capillary tubes 12 are coplanar and the planes
defined by the ends of the capillary tubes 12 are substantially
parallel to the substrate 10. The spacing of the capillary tubes
may be uniform and selected to match the center to center spacing
of wells on a microplate. For example on a standard 96 well
microplate the capillary tubes would be arranged with a 9 mm center
to center spacing, on a 384 well microplate the capillary tubes 12
would be arranged with a 4.5 mm center to center spacing. Higher
density capillary formats, compatible with 1536 well microplates or
plates with even higher well density, should also be possible. The
capillary tubes 12 are preferably secured within the substrate such
that the length of capillary tubes 12 extending from one side of
the substrate 10 are shorter than the length of the capillary tube
on the opposite side of substrate 10. The length of the capillary
tubes 12 on the shorter side of the substrate may be matched to the
depth of wells in a microplate, such that the length of the shorter
side is a shorter length than the depth of a well in a microplate.
This feature enables the capillary cassette to be inserted into a
microplate such that the substrate 10 rests against the top lip of
the multiwell plate and the capillaries on one side of the
substrate may extend into the multiwell plate without touching the
bottom. For example, in a 96 well microplate the capillary tubes
may be disposed on a substrate such that the shorter side of the
capillary tube extending from the substrate may be inserted into
wells in a microplate without the capillary touching the bottom of
the well. This ensures that liquid dispensed into a well is clear
of the capillary to prevent re-entering the capillary.
[0064] The capillary cassette substrate 10 may be made of a
fiberglass board or other rigid or semi-flexible material. The
capillary tubes 12 may be inserted through evenly spaced holes in
the substrate and secured with adhesive. In one embodiment, the
length and width of the substrate are similar to the length and
width of a standard 96 well microplate. This simplifies adapting
automated systems designed for manipulation of microplates to
handle the capillary cassette.
[0065] In some embodiments it may be advantageous to coat the
interior of the capillary with various surface coatings such as
ionic and non-ionic surfactants. Coatings which may be used include
bovine serum albumin (BSA), glycerol, polyvinyl alcohol and Tween
20. The coatings are introduced into the capillary and dried onto
the interior surface of the capillary tube. Alternative-ly,
covalent modification of the interior surface with silanization or
Griganard reaction may be desired. For example, covalent
modification of capillary tubes interior surfaces which reduce
electroendoosmosis may also be useful in reducing charge surface
effects between a capillary interior surface and components of a
reaction mixture. U.S. patent application Ser. No. 09/324,892,
hereby expressly incorporated by reference for all purposes herein,
discloses the use of acryloyldiethanolamine as a covalent capillary
coating with advantageous alkaline stability. In addition to this
coating, acrylimide or other known coatings may also be used to
covalently modify capillary interior surfaces.
[0066] A. Assembly of Reaction Mixture
[0067] Returning to FIG. 1, the automated system allows for the
combination of reaction reagents and sample DNA using the capillary
cassette. The capillary cassette would be taken by transfer head
104 from the cassette hotel 106 and brought into contact with the
samples contained in a sample plate at location a. The sample plate
is dispensed from sample plate hotel 108. The sample would be drawn
into the capillary tubes of the capillary cassette by capillary
action. The internal volume of the capillary tube is defined by the
length of the capillary tube and its internal diameter. The
capillary cassette of FIG. 3A acts as a fixed volume parallel
pipettor, allowing a number of capillary tubes to be filled in
parallel. Each capillary tube segment will meter a discrete amount
of liquid which may be subsequently dispensed.
[0068] Once one end of each capillary is inserted into the sample
containing well, a liquid will be drawn into the capillary. This
small amount of sample may be combined with other liquids to form a
reaction mixture. The sensitivity of analytical instruments such as
a capillary array electrophoresis system and the exponential
amplification of reaction mixture products enabled by cycling
reactions allow for nanoscale reactions and analysis. Very small
scale reactions are able to reliably produce reaction mixture
products of sufficient quantity for analysis on a capillary array
electrophoresis system or a capillary electrophoresis chip.
Significantly less reaction reagents are required if a nanoscale
reaction mixture is enabled.
[0069] The automated system may be used in various ways to prepare
reaction mixtures. A few of the many such methods for use of the
system in production of reaction mixtures follow.
[0070] Reaction Mixture Preparation Example 1:
[0071] Metering Reagents with Capillary Cassette and Mixing on a
Substrate
[0072] One method to prepare the reaction mixture is to use the
pipettor to separately meter the components of a reaction mixture.
For example for a PCR mixture, the nucleic acid sample and PCR
reagents would be separately metered and dispensed into a single
container combining the liquids. In using the automated system of
FIG. 1, the automated robot 102 moves transfer head 104 containing
a capillary cassette to location a where a sample plate is located.
The ends of the capillary tubes of the capillary cassette are
dipped into the wells. The capillary tubes fill by capillary
action, metering a precise amount of sample. The wells of sample
plate contain the nucleic acid sample. The DNA sample should be
sufficiently dilute such that 5-20 ng of DNA is contained in the
10-10,000 nL volume metered by each capillary tube segment in the
capillary cassette.
[0073] FIG. 4A shows the capillary cassette transferring fluid
samples from a multiwell plate 36 into a capillary cassette 15. The
capillary tube segments 12 on capillary cassette 15 are extended
into the wells of multiwell plate 36. The wells of multiwell plate
36 are conical and liquid in the well will flow to the bottom
central area of each well. This allows a small amount of liquid
within the well to be positioned such that a capillary inserted
into the center of the well and above the bottom of the well will
contact the liquid. The capillary tube segments of the capillary
cassette then fill by capillary action with the liquid in the
wells. It is preferred that the capillary cassette have capillary
tube segments which have the same center to center spacing as the
wells of the multiwell plate containing the DNA samples. In one
embodiment the capillary cassette has the same number of capillary
tube segments as the number of wells in a multiwell plate holding
samples.
[0074] After the capillary cassette is dipped into the nucleic acid
sample containing wells, the filled capillary cassette may be moved
by transfer head 104 to a dispensing device location 122. At the
dispensing device location 122, the sample is dispensed onto a
substrate. A clean capillary cassette is then retrieved and dipped
into a sample plate containing the PCR reagents. As seen earlier,
the capillary cassette meters a precise amount of liquid defined by
the interior volume of the capillary tubes held in the capillary
cassette. The metered amount of reaction reagents may be the same
volume as the volume of sample dispensed. The reaction reagents are
dispensed from each capillary tube segment onto locations on the
mixing substrate containing the nucleic acid sample.
[0075] The present reaction mixture assembly may be used for
assembly of numerous types of reactions. The same basic method used
to assemble the PCR reaction mixture may be adapted to assembly of
a cycle sequencing mixture, rolling circle amplification reaction
mixture, or other reaction mixtures.
[0076] When dispensing the contents into a microplate some care
must be taken to mix the sample and reaction reagents in a manner
which avoids splattering. A number of different methods have been
envisioned to dispense liquid from the capillary cassette.
[0077] Capillary Cassette Dispensing Example 1:
[0078] Centrifugal force
[0079] The first method to dispense the contents of the capillary
cassette while avoiding splattering uses a centrifuge to dispense
the fluid by centrifugal force. The centrifugal force is applied
evenly to all of the capillaries in the capillary cassette such
that capillaries independently dispense into microplate wells. The
dispensed liquid is drawn by centrifugal force to the bottom of
wells in the multiwell plate.
[0080] In FIG. 5A, the centrifuge 42 is shown having a swinging
microplate bucket 43 which may contain a multiwell plate with an
inserted capillary cassette. The swinging microplate buckets are
held on rotor 41.
[0081] FIG. 5B shows a cross-section of swinging microplate bucket
43. The capillary tubes 12 of the capillary cassette are inserted
into wells 36a of multiwell plate 36. The cassette is inserted such
that the portion of the capillary tubes 12 extending from the
substrate 10 are shorter than the depth of the wells 36a. As shown
in FIG. 5B, the capillary tube 12 extending from substrate 10 do
not reach the bottom of the wells 36a of multiwell plate 36.
Microplate swinging bucket 43 is comprised of an arm 45 and a
platform 44. An upper end of arm 45 fits onto latch head 42 on
rotor 41. Microplate 36 is positioned on platform 44 of microplate
swinging bucket 43. When the centrifuge is in motion, platform 144
rotates on latch head 42 such that the multiwell plate faces the
side wall of the centrifuge and the centrifugal force on the liquid
in the capillary tubes dispenses the liquid into the bottom of the
wells 36a of the multiwell plate 36. When conical shaped wells are
used, the centrifugal force will draw the liquids within the well
to the well center, causing the sample to locate at a more precise
location The liquid will be displaced from the capillary at fairly
low centrifuge speeds.
[0082] In FIG. 1, a low speed centrifuge may optionally be included
in the automated system at the dispensing device location 122.
Automated robot 102 uses transfer head 104 to pick up a nanotiter
plate dispensed onto location b by nanotiter plate hotel 110. The
nanotiter plate is transferred by transfer head 104 to the stage of
the low speed centrifuge. A capillary cassette is filled with
samples or reaction reagents as described and is transferred onto
the nanotiter plate on the stage of the low speed centrifuge. The
plate and cassette are then spun in the centrifuge, dispensing the
liquid from the capillaries into the wells of the nanotiter plate.
Once the liquid has been dispensed and the centrifuge has stopped
rotating, the capillary cassette may by removed by the transfer
head and transferred to the cassette washer 118. The transfer head
104 can then pick up a clean capillary cassette from the capillary
cassette hotel 106. The clean capillary cassette can be used to
meter a second liquid reaction component which is similarly
dispensed using the centrifuge. In the automated system the
centrifuge includes a sensor associated with the rotor used in
conjunction with a rotor braking system to stop the rotor in a
position which transfer head 104 can access such a sensor could be
magnetic, optical, mechanical, or use other known means of sensing
rotor position.
[0083] Capillary Cassette Dispensing Example 2:
[0084] Air Displacement
[0085] A second method of dispensing the liquid contained in the
capillary tube segments of a capillary cassette is through the use
of an air displacement device. With reference to FIG. 1, a
nanotiter plate dispensed from nanotiter plate hotel is transferred
by transfer head 104 to the dispensing device location 122. At this
location an air dispenser, such as the one pictured in FIG. 4A-C is
located. Subsequently a capillary cassette is retrieved by transfer
head 104, filled with either sample from a sample multiwell plate
or reaction reagents. The capillary cassette is then moved to the
dispensing device location 122 and brought into contact with air
displacement head. The substrate of the capillary cassette is
placed on a receiving platform on the air displacement head.
Alternatively, the air displacement head may be joinable to
automated transfer robot 102.
[0086] With reference to FIG. 4A, the air displacement head 301 is
shown with a capillary cassette 15 held on bottom plate 302. The
bottom plate 302 is attached to a manifold assembly by hinge 318.
Capillary cassette substrate 10 is held on foam rubber pad 304
which is secured onto bottom plate 302. An array of holes 325
extend through foam rubber pad 304 and bottom plate 302 which are
spaced to allow the capillary tubes 12 to extend through foam
rubber pad 304 and bottom plate 302 when the capillary cassette is
positioned on bottom plate 302. The manifold assembly of the air
displacement head is comprised of an upper housing 306, chamber
unit 310 and a set of clamps 314. Clamps 314 secure membrane 312 to
the lower surface of the chamber unit 310. Membrane 312 forms a
seal to the top surface of the capillary cassette 15 when the
manifold assembly is closed over the cassette. Membrane 312 has
holes 316 corresponding to capillary 12 positions in the cassette
when the capillary 12 positions in the cassette when the capillary
cassette 15 is placed on bottom plate 302. When the top manifold of
air displacement head 301 is closed against bottom plate 302,
capillary tubes 12 are positioned in capillary tube receiving holes
316 on membrane 312. When the air displacement head 301 is closed
it may be secured by latch 322 which mates with hole 324 to clamp
the capillary cassette between the foam rubber pad 304 and membrane
312 resulting in a seal between the top surface of cassette 15 and
the membrane 312.
[0087] FIG. 4B illustrates a cross sectional view of displacement
head 301. Upper housing 306 is constructed of metal, acrylic or
other rigid material. Gas input coupler 303 is disposed on upper
housing 301. When a pressurized gas or vacuum line 305 is attached
to gas input coupler 303, a vacuum or pressure force may be
introduced into upper chamber 307. Held between upper housing 306
and chamber unit 310 is a gas impervious elastic membrane 308. The
area between elastic membrane 308 and upper housing 306 defines
upper chamber 307. Secured onto clamps 314 is membrane 312.
Membrane 312 is pressed against substrate 10 of a capillary
cassette inserted into displacement head 301. Substrate 10 is
secured within displacement head 301 by bottom plate 302. Rubber
pad 304 provides a deformable surface which exerts uniform force
pressing substrate 10 against membrane 312. Membrane 312 has an
array of holes 316 which allow the capillaries 12 of the capillary
cassette to extend through membrane 312. When a capillary cassette
is inserted into air displacement head 301, the substrate seals
holes 316 enclosing lower chamber 313. When pressurized gas is
introduced into chamber 307 by gas line 305, elastic membrane 308
will be pressed into lower chamber 313. Membrane 308 is located
between upper chamber 307 and lower chambers 313. Membrane 308
serves both as seal for the upper end of chambers 313 and the
chamber displacement actuator when pressure is applied to the upper
chamber 307 through coupler 303. The degree of displacement is
dependent on the pressure applied. The resulting air displacement
will act to dispense liquid from capillary tubes 12 which extend
through the capillary and into the lower chamber 313. By regulating
the amount of pressure applied through line 305, a consistent
displacement force will be applied to each capillary tube. Given
the submicroliter volume of the capillary tube segments,
fluctuations in the amount of dispensing pressure should not
adversely affect displacement from the tubes.
[0088] FIG. 4C illustrates the closed air displacement head 301.
Upper housing 306 is pulled toward bottom plate 302 by latch 322 in
order to compress membrane 312 against the top of the capillary
cassette substrate thereby forming a seal. Clamps 314 secure
membrane 312 onto chamber unit 310. Air displacement head 301 is
mounted on arm 320. Arm 320 may extend from automated transfer
robot 102 shown in FIG. 1 or be positioned at dispense location
122. Pressurized gas may be introduced into upper housing 306
through gas input couple 303.
[0089] This displacement head provides an individual displacement
chamber for each of the capillaries dispensed. Although a 16
capillary cassette is depicted, the displacement head may be
constructed to dispense capillary cassettes having an array of 96
capillaries or greater capillary densities. The dispensing force
applied to each capillary is sufficiently small to allow dispensing
directly onto a substrate with the sample dispensed at a discrete
location.
[0090] Air displacement or centrifugal displacement may be used to
dispense liquid from the capillary tube segments in a capillary
cassette. It may also be possible to dispense liquid from the
capillary tubes using a bank of syringe pumps, applying pressure
through a gas permeable/liquid impermeable (hydrophobic) membrane,
using electrokinetic dispensing, or other known dispensing means.
The air displacement head may also be used to dispense finished
reaction mixtures onto a substrate for subsequent analysis.
[0091] Reaction Mixture Assembly Example 2:
[0092] Dehydrated Reagents
[0093] A second method to assemble the reaction mixture is to have
the regents required for the reaction stored as a dehydrated
coating either on the interior of a capillary or on a substrate,
such as within a well of a multiwell plate. If the reaction
reagents are dehydrated onto the interior of capillary tube
segments in a capillary cassette, introducing a sample into the
capillary would cause rehydration, mixing and formation of the
reaction mixture. In a similar manner, if the wells of a microplate
are coated with the dehydrated reaction reagents, adding a nucleic
acid sample into the wells would bring the reaction reagents into
solution forming an assay mixture. The sample could be metered with
a capillary cassette and dispensed from the capillary cassette by
one of the methods set out above. The sample would bring the
dehydrated reaction reagents into solution and mix with the sample
containing nucleic acid by diffusion. This provides a method to
assemble the reaction mixture in a very simple manner, potentially
without the need to dispense the capillary tubes with a centrifuge
or air displacement device. This could both simplify the reaction
processing system and shorten the reaction assembly time.
[0094] For PCR, a dehydrated reagent mixture is commercially
available, sold as Ready-to-Go.RTM. (Amersham Pharmacia
Biotechnology, Piscataway, N.J.). The stabilized, dehydrated
reagents may be coated onto the interior surface of capillary
segments or the interior of the wells of a multiwell plate. The
Ready-to-Go.RTM. product uses a carbohydrate matrix to stabilize
the reaction reagents (DNA polymerase, buffer reagents, dNTPs) in a
desiccated state. Bringing the reagents in the Ready-to-Go.RTM.
mixture into solution with the liquid nucleic acid sample and
primers in solution produces the final reaction mixture required
for the reaction. The combination of the stabilized
Ready-to-Go.RTM. compounds, the template DNA, primers, and
sufficient water produces a final reaction product. It is
contemplated that reagents for chain termination sequencing
reactions could also be stored in a desiccated state.
[0095] The coating could be applied to surfaces by a number of
different methods including vapor phase coating, filling a
capillary (by capillary action, pressure filling, etc.) with the
Ready-to-Go.RTM. mixture and emptying the bulk phase (under vacuum,
pressure or other forces), or dipping a substrate (such as a bead)
into the reagents and subsequently drying the bead.
[0096] Reaction Mixture Assembly Example 3:
[0097] Nucleic Acid Capture
[0098] A third method of assembly of the reaction mixture is to
capture the sample nucleic acid on the surface of a substrate, such
as the interior of a capillary tube segment. The sample nucleic
acid may be attached onto the surface by a number of methods. These
include covalent attachment, DNA hybridization, hydrophobic
interactions, electric field, magnetic field, or other chemical or
physical forces. Once the sample has been attached, the remaining
liquid in which the sample was suspended may evacuated from the
capillary or microchip by chemical reaction or physical force. Air
displacement or centrifugal dispensing method may be used to empty
the capillary, as can a vacuum. The sample nucleic acid would
remain on the surface of the substrate. In this single step, the
sample nucleic acid may be substantially purified. The reaction
reagents may then be combined with the sample nucleic acid,
producing the reaction mixture.
[0099] One method to immobilize the nucleic acid sample is to
attach the nucleic acid directly to a surface. This may be done by
non-covalent modification (such as surface treatment with NaSCN,
DMSO, etc.) or covalent linkage. There are a number of different
covalent attachment methods for DNA known in the art. For example,
an amino group can be attached to the deoxyribose base of DNA and
incorporated during a synthetic reaction, such as during PCR
amplification of a DNA plasmid insert. The glass or silica of a
capillary interior could be silanized and the amino group on the
modified DNA would covalently bond to the silanized interior of the
capillary. Alternatively, other chemistries are available to
covalently immobilize DNA onto a surface. Once the DNA is bound to
the surface of a capillary or other substrate, the liquid in which
the DNA was suspended may be eliminated from the capillary and the
capillary may be filled with reaction reagents.
[0100] An alternative method of attaching a nucleic acid to the
interior of the capillaries of a capillary cassette is through
affinity chemistry. One common affinity chemistry procedure labels
a biomolecule with biotin and then binds the biotinylated
biomolecules to avidin or streptavidin. The avidin/streptavidin may
be used to link the biotinylated molecules. Nucleic acid labeled
with biotin may be subsequently attached to a surface, such as the
interior of a capillary tube. This may be accomplished by binding
streptavidin to the interior of the capillary.
[0101] One example of the use of affinity chemistry for the binding
of DNA to the interior of a capillary is disclosed in U.S. Pat. No.
5,846,727, hereby expressly incorporated herein for all purposes.
This reference describes the binding of DNA to the interior surface
of the capillary tubes. The technique requires primers labeled with
biotin which are combined with dNTPs, a DNA polymerase, and a
reaction buffer. This is combined with template DNA, such as
plasmids from a DNA library with sub-cloned DNA inserts, to form
the reaction mixture. In the present invention a microplate may
contain 96 or more reaction mixtures, each with a unique plasmid
with a subcloned DNA sequence. This reaction mixture could be
assembled by the method stated in reaction mixture assembly example
1: namely the reaction reagents and the plasmid sample could be
separately metered and dispensed into a 384 well microtiter plate.
In a microplate well the liquids are combined to form a reaction
mixture. The reaction mixture is metered into the capillary tube
segments of a capillary cassette. The PCR reaction may be effected
by temporarily sealing the ends of the capillary tube segments and
exposing the capillary cassette to thermal cycles, as described
below. The results of the PCR reaction are exponentially amplified
copies of the subcloned plasmid DNA insert containing the biotin
labeled primer.
[0102] The template DNA containing the biotin labeled primer may
then be immobilized on the walls of the capillary tubes of a
capillary cassette. The immobilization capillary cassette would
have capillary tubes with avidin or streptavidin coated onto the
interior surface of each capillary tube. The chemistry for
attachment of avidin/streptavidin may be that disclosed in, for
example, L. Amankwa et al., "On-Line Peptide Mapping by Capillary
Zone Electrophoresis," Anal. Chem., vol. 65, pp. 2693-2697 (1993).
The capillary is filled with (3-aniopropyl)trimethoxysilane
(3-ATPS), incubated for 30 minutes, and air dried. The dried
capillaries in the capillary cassette are next filled with
sulfosuccinimidyl-6-(bioti- namido)hexonate (NHS-LC biotin) which
is again incubated followed by air drying. Avidin or streptavidin
in phosphate buffer at 7.4 pH is added to each capillary tube. The
avidin binds to the biotin immobilized on the interior of each
capillary. The double stranded amplified biotinylated PCR products
suspended in a buffer (e.g. Tris-HCl, or EDTA with either NaCl or
LiCl at 1-3M added for efficaceous binding) are added to the
capillary tube and incubated for 5-10 min. This results in a
capillary wall modified as follows: capillary
wall-Si--C.sub.3H.sub.6--NH--CO-bioti- navidin or
streptavidin-amplified oligonucleotide with associated biotin
primer.
[0103] Once the DNA is immobilized on the interior surface of the
capillary, the contents of the capillary tube may be dispensed in
one of the methods described and the DNA would remain bound to the
surface of the capillary. This removes debris and other impurities
from the DNA presenting a rapid and effective method of DNA
purification. The capillary may be rinsed with a buffer for
additional purification. The defined area of the interior surface
of the capillary provides a known amount of binding sites for the
DNA attachment. This provides a simple method for normalization of
DNA concentration. The capillary cassette may then be dipped into
wells or a reagent reservoir containing the reagents for cycle
sequencing. The cycle sequencing reaction can be performed by
temporarily sealing the ends of the capillary tubes by pressing
each end of the capillary tubes against a deformable membrane. The
capillary cassette may then be exposed to thermal cycles which
effect the cycle sequencing reaction.
[0104] In this embodiment biotin, rather than avidin or
streptavidin, is covalently attached first to the capillary wall.
This aids in the regeneration of the capillary cassette for
subsequent binding reactions. After completing the cycle sequencing
reaction, it would be difficult to remove the amplified
biotinylated DNA without also denaturing the avidin protein. By
having biotin bound to the interior surface of the capillary the
amplified DNA may be easily removed by filling the capillary with
phenol or formamide solution at 65-90 degrees C. This denatures the
avidin protein without removal of the biotin bound to the interior
surface of the capillary. This mixture is then dispensed. The
capillary cassette may then again be filled with the avidin
containing solution and reused for binding subsequent biotinylated
amplified template DNA.
[0105] Prior to filling, the capillary tube segments of the
capillary cassette may be coated with a variety of compounds.
Coating the interior surface of the capillary tube segments with
bovine serum albumin (BSA) or polyvinyl alcohol has been shown to
improve performance of some reactions, such as preparation of chain
termination sequencing reactions.
[0106] B. Thermal Cycling
[0107] Once the reaction mixture is introduced into the capillary
tube segments of the capillary cassette, the ends of the
capillaries of the capillary cassette are sealed and the capillary
cassette is exposed to temperature cycles. The ends of the
capillary cassette capillaries are sealed by pressing each of the
ends of the capillary tubes against a deformable membrane.
Returning to FIG. 1, once the capillary cassette has been filled
with the reaction mixture, the ends of the capillaries are sealed
and the capillaries are exposed to thermal cycles in thermal
cycling device 116.
[0108] In one thermal cycling device, shown in FIGS. 7A-7C, the
thermal cycling device has integrated membranes that seal the ends
of the capillaries and exposes the capillary cassette to thermal
cycles. In this apparatus the means for sealing the ends of the
capillaries in the capillary cassette is incorporated into the
thermal cycling device.
[0109] With reference to FIG. 7A, the capillary cassette 15 is held
on lip 280 within internal passageway 256 between deformable
membranes 264a and 264b. As seen in FIG. 7B, deformable membrane
264a is mounted on platform 261. Lid 262 is secured on platform
261. Platform 261 is attached by pivot 286 to base 265. Pneumatics
284a, 284b are attached at an upper end to platform 261 at pivot
263. Screw 282 acts as a stop for platform 261 when platform 261 is
lowered onto housing 270, enclosing passageway 256. Diffuser 258
promotes temperature uniformly of air circulating in internal
passageway 256. Thermocouple 260 measures temperature of the
circulating air. The function of pivot 277 and bottom membrane
platform 200 is described in conjunction with FIG. 7C.
[0110] FIG. 7C shows a cross section of the capillary cassette
holding chamber with capillary cassette 15 inserted into the
internal passageway 256. The capillary cassette could be inserted
into this area by automated robot 102 of FIG. 1 after the capillary
tube segments have been filled with the reaction mixture. Capillary
cassette 15 is positioned such that substrate 10 rests on ledge
280. Capillary cassette is positioned such that the ends of
capillary tube segments 12 are depressed against top deformable
membrane 264a and bottom deformable membrane 264b when upper
platform 261 is lowered over the capillary cassette and lower
platform 271 is raised. Notches 262a, 262b seal along the side
lengths of housing 270 when upper platform 261 is lowered to
provide a host retaining seal. Screw 282 acts as a stop for upper
platform 261 to prevent the platform from lowering so far that
capillary tube segments are bowed or damaged. Base platform 266 is
secured to post 273 and secured to housing 270. The lower end of
pneumatics, 284b is secured at a lower pivot 271a to low platform
271. Extending through lower platform 271 are shoulder screws 268
which extend through housing 270 and stationary platform 266 and
are secured to lower platform 200. When upper platform 261 is
lowered by pneumatic 284b lower platform 271 is also raised toward
housing 270. When pneumatic cylinders 284b, 284a are retracted, the
pneumatic cylinders move to a vertical orientation. Upper platform
261 is lowered and lower platform 271 is raised slightly in an arc.
Lower platform 271 will arc upward on pivot 277 to move to a
position substantially parallel to platform 261 when pneumatic
cylinder 284b is fully retracted. When a capillary cassette 15 is
inserted into internal chamber 258 the ability of platform 200 to
"float" on springs 275 prevents excess pressure from damaging
capillary tubes 12 or membranes 264a, 264b. Platforms 261 and 200
exert 400 pounds per square inch force on capillary tubes 12
providing sufficient sealing pressure. With upper platform 262
lowered, the capillary tube segments 12 are sealed at each end by
deformable membranes 264a, 264b. Deformable membranes 264a, 264b
may be made of silicon rubber or other deformable material.
[0111] Returning to FIG. 7A, a motor 250 turns shaft 251 which
rotates squirrel cage blower 253. Blower 253 produces air movement
through diffuser 254 to flow into internal passageway 256. The
blower generates sufficient circulation flow that the air flowing
through internal passageway 256 circulates at 2000 feet per minute.
Diffuser 254 ensures that the heat of the air blown by blower 253
is uniform throughout passageway 256. Cone 255 on diffuser 254 aids
in mixing the flowing air, promotion temperature uniformity through
passageway 256. Diffuser 254 acts to ensure an even flow of air
through passageway 256 in the region of the capillary cassette and
reduces non-uniformity from wall loss effects in internal
passageway 256.
[0112] The internal passageway 256 is defined by outer housing 270.
Outer housing 270 is preferably of rectangular cross section and
comprised of sheet metal, plastic or other durable material. The
internal surface of outer housing 270 at all locations except for
inlet 278 is lined with thermal foam insulation 272. Insulation 272
prevents excess heating of outer housing 270 and helps retain heat
and aids temperature uniformity of the air circulating through
internal passageway 256. After flowing through first diffuser 254
the air flows through second diffuser 258. Diffusers 254 and 258
promote uniform air flow and temperature uniformity through
internal passageway 256. Past first diffuser 254 internal
passageway 256 transitions, to match the dimensions of passageway
256 to accommodate. The heated air flows past thermocouple 260
which is vertically disposed at the center of internal passageway
256 just beyond second diffuser 258. Thermocouple 260 acts to
monitor the temperature within internal passageway 256.
Thermocouple 260 may be a temperature monitoring device inserted
into a capillary tube section which extends through outer housing
270 and through foam insulation 272. Alternatively thermocouple 260
may be selected such that it accurately reflects the internal
temperature of a capillary tube.
[0113] The air circulating through internal passageway 256 passes
thermocouple 260 and flows past the capillary tube segments 12 of
capillary cassette 15. The ends of the capillary tube segments are
sealed at their upper end by deformable membrane 264a mounted on
upper platform 261 which has been lowered to form an air tight seal
with housing 270. The lower end of capillary tube segments 12 are
sealed by deformable membrane 264b. Deformable membrane 264b is
mounted on platform 200 which is secured on a bottom surface by
shoulder screws 268. Shoulder screws 268 extend through housing 270
and retained by platform 271. Springs 275 located between platform
200 and platform 271 provide a biasing force while allowing for
platform 200 to float such that deformable membrane is biased
against the ends of capillaries 12. The function of double acting
pneumatics act to seal lid 262 and apply force to position platform
271 is described in conjunction with FIG. 7C. Lid 262 fits onto
housing 270 such that the sheet metal or other material comprising
the edge of lid 262 fits on top of housing 270. Membrane 264a is
mounted on upper platform 261 such that membrane 264a extends into
internal passageway 256 at least far enough that membrane 264a is
even with insulation 272. As the air travels past capillary tube
segments 12, the length of the capillary tube segments 12 below
substrate 10 are rapidly heated and cooled to the temperature of
the air rapidly moving through internal passageway 256.
[0114] Door 274 controlled by motor 276 is used in conjunction with
thermocouple 260 and heating element 252 to control the temperature
within internal passageway 256. When door 274 is closed, the air
circulating within internal passageway will not be exchanged with
outside air. As the air continuously passes over heating element
252 the air is rapidly heated until the air comes to the selected
temperature. Once thermocouple 260 senses that the temperature is
at a selected temperature, heating element 252 may be kept at a
lower heat output such that the internal temperature is maintained.
If the temperature needs to be rapidly dropped, as in during a
thermal cycling reaction, door 274 may be moved to orientation 274a
by motor 276 with the door 274 moved into internal passageway 256,
allowing all air which has passed capillary cassette 15 to be
exhausted to outside internal passageway 256. It is envisioned that
a filter or exhaust duct could be mounted about door 274 to prevent
compounds in the circulating air from being exhausted to the
environment. The rapidly circulating air will be quickly exhausted
to outside of the thermal cycler while ambient air is drawn in
through air intake 278. Air drawn into internal passageway 256
through intake 278 flows through heater 252. The area through which
the air moves is restricted by block 259 positioned above heater
252 within internal chamber 256. Again the temperature of the air
within internal passageway 256 is monitored by thermocouple 260 and
when the desired temperature drop has occurred, door 274 will be
brought toward housing 270, reducing air volume drawn through air
intake 278.
[0115] By connecting heating element 252, thermocouple 260 and door
motor 276 to an electronic control system, such as a computer
controller, this thermal cycler may perform precise air temperature
varying sequences. Additional heat is added when needed by heating
element 252 and heat is exhausted by opening door 274, with the
temperature result of either action monitored by thermocouple 260.
Exhausting circulating air through door 274 allows air within
internal passageway to drop in temperature at a rate greater than
10 degrees per second. The rapid temperature change combined with
the rapid transfer of heat to or from the capillaries allows for
efficient temperature cycling reactions. For example in reactions
using a thermostable polymerase, the denaturing of nucleic acid
strands and the annealing of primer to template strands each may
take place in one to five seconds. The extension of the primer will
require less time to effect since the rapidly circulating air and
the thin walls of the capillaries rapidly bring the internal volume
of the capillaries to the selected temperature. The thin walls of
the capillaries and the small capillary volume enable a rapid
temperature change and heat transfer throughout the internal
capillary volume. This greatly reduces the preparation time
required for each reaction, allowing more efficient use of the
thermal cycler and greater throughput in sample preparation.
Presently, a 30 cycle PCR amplification may be performed in under
30 minutes. It should be possible to reduce this time to under 8
minutes.
[0116] Once the thermal cycling reaction is complete, upper
platform 261 may be raised and capillary cassette 15 removed from
internal passageway 256. During the temperature cycling process,
the liquid within each capillary tube segment will expand somewhat
and some liquid will leak from the capillary and be carried away by
the rapidly flowing air. However, such loss is only a few percent
of the volume of the capillary tube segment and should not present
either a contamination problem or cause enough reaction product
loss to materially affect subsequent analysis. In addition, the
portion of capillary tube segments 12 located between substrate 10
and deformable membrane 264a will receive only poor air flow and
will be less likely to rapidly reach the denaturation temperature.
However since this length is short, the failure of this area to as
rapidly reach the denaturation temperature should not adversely
affect the ability of the remaining portion of the capillary from
producing sufficient reaction product for subsequent analysis.
[0117] An alternative device for sealing the ends of the capillary
is a capillary cassette holder which seals the ends of capillary
tube segments of a capillary cassette. With reference to FIG. 3B
the capillary cassette holder is comprised of a pair of parallel
deformable membranes 14a, 14b each secured onto a platform 16a,
16b. The deformable membranes may be silicon rubber seals,
Teflon.RTM., plastics or other resilient, deformable material. A
pair of parallel posts 9 extend from bottom platform 16a to top
support platform 24 where the posts are secured by internally
threaded nut 18. Post 9 passes through platform 24 and nut 18 is
retained on an annular lip of platform 24. Shoulder screws 20
extend through holes in support 24 and are secured to top platform
16b. Springs 22 bias the top platform 16b against the ends of
capillary tube segments 12 while allowing 16b to float. The
substrate 10 of capillary cassette 15 may be designed to have holes
which conform to the spacing and dimension of posts 18 such that
capillary cassette 15 may be more easily and securely held within
holder 23.
[0118] Once the ends of the capillary cassette are sealed in holder
23, the combined capillary cassette and holder may be exposed to
thermal cycles. The holder shown seals 16 capillaries. However, a
holder may be designed to hold capillary cassettes having 96
capillaries or higher densities of capillaries. In addition to
capillary cassettes, chips of other substrates may be used as the
reaction containers. FIG. 3E shows a chip substrate 70 comprised of
two bonded substrate layers 72, 74. One layer 72 has grooves 76
extending the length of the chip. The affixed top substrate 72
encloses a capillary dimension passage 76 with opposite open ends.
A liquid reaction mixture may be introduced into the inclosed
passage. The ends of these passages may be sealed by pressing the
ends against a deformable membrane, as was done with the capillary
cassettes. Temperature cycling may require longer times because of
greater mass material comprising the chip, but cycling times should
still be more rapid than conventional cycling.
[0119] For isothermal reactions, such as rolling cycle
amplification, temperature cycling is not required to effect the
reaction. Once an isothermal reaction mixture is combined and
introduced into a capillary cassette, incubation of the cassette at
a reaction temperature will allow the reaction to occur. With
reference to FIG. 1, the automated transfer device may transfer a
capillary cassette into incubator 124 where the capillary cassette
is incubated at a selected temperature. A set of deformable
membranes may be used to seal the ends of the capillaries during
incubation. As was seen in other system components, incubator 124
may be used at the same time as other system components.
[0120] In the case of PCR or chain termination sequencing reactions
it is necessary to expose the reaction mixture to temperature
cycles. In FIG. 1 the transfer head 104 moves the capillary
cassette into thermocycler 116. The thermocycling device may be any
device which can expose the capillary tube segments of the
capillary cassette to temperature cycles. Thermal cycling devices
which use water, electric field, heating blocks, or other means may
be used. Alternatively, air based thermal cycling devices are rapid
and adaptable to the low volume cycling of the present
invention.
[0121] A thermal cycling device which uses air as the temperature
transfer medium is shown in FIG. 6. The reaction mixture is
contained in capillary tube segments which have a high surface to
volume ratio and small material thickness. This allows very rapid
transfer of heat through the walls of the capillary and throughout
the liquid reaction mixture. An equilibrium temperature is reached
rapidly throughout the liquid in the capillary. The use of air as a
heat transfer medium enables the rapid ramping of temperature in
the reaction chamber. Rapid circulation of the air ensures rapid
and more uniform heating or cooling of the capillary segments and
their contents.
[0122] The capillary cassette 15 sealed within holder 8 is inserted
through opening 215 in housing 202 of the air based thermal cycler.
The holder 8 is supported by housing surface 215 of the thermal
cycling chamber 210. The capillary tubes 12 mounted to substrate 10
are exposed to the air of thermal cycling chamber 210 such that the
air may freely flow around capillary tube segments 12. Thermocouple
216 monitors the temperature of the air moving past capillary tubes
12.
[0123] In the air based thermal cycling device, paddle 208 driven
by motor 206 rapidly circulates air with reaction chamber 210. The
air is rapidly circulated past the capillaries 12 of capillary
cassette 15. Halogen bulb 220 acts as a heat source to heat the air
within the thermal cycling chamber 210. To effect a thermal cycling
reaction, the circulating air is held at a selected temperature for
a selected period of time. The thermocouple 216 transmits the
temperature of the capillary tube segment 12 to microprocessor 218.
To effect the needed temperature changes the microprocessor
instructs actuator 222 to open door 226 allowing air to pass
through vent 224. As air passes through vent 224 additional air is
drawn into the reaction chamber through air inlet 203 by fan blade
204. Fan blade 204 is driven by motor 206. The venting of hot air
and replacement with cooler ambient air, combined with the rapid
circulation of air by fan 208, a relatively small thermal cycling
chamber 210 and precise measurement of sample temperatures by
thermocouple 216 enables rapid temperature ramping. The time
required for effecting the thermal cycles is greatly reduced. A
typical thermal cycling reaction requires different temperatures
for denaturing of nucleic acid strands, annealing of a primer, and
extension of a polymerase. The denaturing and annealing steps occur
rapidly in a capillary tube where the small internal volume of
liquid will rapidly come to equilibrium, while the extension of the
DNA molecule takes less than 10 seconds for a 500 base extension.
The time required for each thermal cycle of the three temperatures
(annealing, extension, denaturing) may be reduced to under 15
seconds by using the rapid heat transfer of the air based thermal
cycling apparatus. A program of 30 cycles, each cycle exposing the
capillary to three temperatures for varying amounts of time
theoretically may be effected in under 8 minutes.
[0124] The use of the capillary cassette in combination with an air
based thermal cycler allows additional advantages. The capillary
cassette holder temporarily seals the capillary, allowing rapid and
simplified sealing of each capillary tube segment. The capillary
cassette contains a number of capillary tubes in parallel
arrangement, allowing for more efficient use of the thermal cycler
and allowing greater sample throughput. Once the thermal cycle is
completed the capillary cassette 15 contained with in holder 8 is
removed through opening 215. The capillary cassette 15 is released
from the holder and is subsequently dispensed.
[0125] The thermal cyclers of FIGS. 6 and 7A-C were illustrated as
being used with capillary cassettes. The same devices are adaptable
to other containers with opposing ends. For example, a chip-like
substrate with a plurality of passageways extending through the
chip (as seen in FIG. 3E) has, like a capillary cassette, evenly
spaced opposed open ends. Several chips could be placed into a
thermal cycler with the open ends temporarily sealed and exposed to
thermal cycles. The rapid temperature changes may be a bit slower
due to increased material thickness. Other containers with opposing
open ends may also be used with either temperature cycling
device.
[0126] C. Dispensing Completed Reaction Mixture
[0127] Following the completion of the thermal cycling reaction,
the prepared reaction mixture is dispensed into a substrate for
analysis by an analytical system. As noted above, the capillary
cassette may be dispensed by air displacement, centrifugal force,
vacuum or any other displacement method. The substrate into which
the reaction mixture is displaced may be the wells of a multiwell
plate, locations on a planar substrate, or wells which lead into an
analytical chip. The reaction mixture, though small, still may
produce enough amplified reaction product that dilution is
necessary.
[0128] Dispensing Completed Reaction Mixture Example 1:
[0129] Direct Dilution
[0130] In reference to FIG. 1, following completion of the
temperature cycling process, the capillary cassette may be removed
from air thermal cycler 116 by transfer head 104. The capillary
cassette may be moved by transfer head 104 to be placed in a plate
dispensed from finished sample hotel 112. The plate, located at
position c, may be a multiwell plate such as a 384 well microplate.
The wells of the plate contain a dilution liquid, such as
formamide, water, TBE or other selected buffer. The reaction
mixture may be dispensed from the capillary tube segments of the
capillary cassette by positive displacement, centrifugation, or
other dispensing means. The reaction may also be dispensed into a
solution for further chemical or biochemical reaction.
[0131] Dispensing Completed Reaction Mixture Example 2:
[0132] Ethanol Precipitation
[0133] Ethanol precipitation may be effected in a dispensing means
similar to the means of direct dilution. Transfer head 104 of FIG.
1 would again take the capillary cassette from air thermal cycler
116 and place the short ends of the capillaries in a multiwell
plate located at position c. In this case the wells of the plate
would contain an ethanol, such as 90% ethanol chilled to 4EC. The
reaction mixture would be dispensed from the capillary cassette
into the ethanol by centrifuge, positive displacement, or other
dispensing method. The ethanol could then be removed by aspiration
or other means. The precipitated DNA could then be resuspended in
formamide, water or other suitable diluent. Once the sample plate
is prepared, by either direct dilution or ethanol precipitation,
the plate may be transferred by transfer head 104 to analytical
stage 120. Analytical stage 120 may feed the sample plate directly
into an analytical device, for example a capillary array
electrophoresis system, such as MegaBACE.TM. produced by Molecular
Dynamics, Sunnyvale Calif. Alternatively, the analytical stage
could direct the product to other systems for further processing.
It is also possible to dispense the samples onto a substrate for
mass spectrometry analysis, calorimetric analysis, or other
analytical methods.
[0134] Dispensing Completed Reaction Mixture Example 3: Dispense
Directly into Analytical System
[0135] In the previous two examples the samples were dispensed into
multiwell plates. These plates could then be moved manually or
robotically onto a stage for analysis by an analytical system.
Alternatively the capillary cassette could be dispensed directly
into the wells of an analytical device, such as an electrophoresis
chip. For example a capillary cassette having 16 capillaries
disposed in the substrate in two parallel rows of eight capillaries
may dock with 16 wells in an analytical microchip. Such a microchip
would have an array of analytical lanes in fluid communication with
a sample port.
[0136] The capillary cassette may be designed such that the spacing
of the capillaries matches the spacing of the sample reservoir
inlets. For example, the capillary cassette illustrated in FIG. 3C
includes capillaries 12 extending through flexible strip 11.
Flexible strip 11 may be used alone or in combination with other
such strips. The orientation of the capillaries in an essentially
straight line may be altered by bending strip 11 to form an arc.
FIG. 3D illustrates strip 11 but allowing capillaries 12 to mate
with input ports which is disposed on a substrate in a circular
pattern. The liquid in capillaries 12 may then be
electrokinetically injected or otherwise dispensed from capillaries
12 into ports of an analytical chip if an appropriate electrode
array is used. Strip 13 may be positioned in the curved orientation
by pressing strip 13 against a curved form, such as a curved metal
block. This may be done by an automated strip mover incorporated
into an automated sample preparation system.
[0137] The capillary cassette could be dispensed by air
displacement or other dispensing means preferably selected to
minimize splattering and bubble formation. Prior to dispensing the
prepared reaction mixture into the wells for analysis, a small
amount of a dilutant could be added to each analytical microchip
well. When the capillary cassette is dispensed, the diluent will
dilute the samples in the sample wells. The sub-microliter volume
reaction mixtures prepared in the capillary cassette, such as a DNA
sequencing reaction product mixture, can readily be integrated with
the analytical microchip for sequencing.
[0138] D. Washing Capillary Cassette
[0139] Following each use of a capillary cassette, the capillary
cassette may be washed and reused. After the contents of the
capillary cassette have been dispensed or a capillary cassette has
otherwise been used, the capillary cassette is taken to cassette
washer 118 where the cassette is washed. Following washing, the
cassette is returned to the cassette hotel 106 where the cassette
may be reused.
[0140] With reference to FIG. 8A, capillary cassette washer 410 is
comprised of wash manifold 412 and wash tank stage 416. Between
wash manifold 412 and wash tank stage 416 is capillary cassette
platform 414. Extending from wash tank stage 416 is leg 419. In
this wash system, a wash liquid is pumped from one or more of
containers 452, 454, 456, 458 through respective tubes 1, 2, 3, 4
into respective router inputs 453, 455, 457, 459. The router
directs the selected wash fluid through router outflow 451 through
line 451a into the wash tank 440. The fluid is drawn from wash tank
440 through capillary tube segments of a capillary cassette. The
capillary cassette substrate is held between wash manifold 412 and
wash tank 440 such that if suction is applied to wash manifold 412,
wash fluid will be drawn through capillary tube segments from wash
tank 440. The wash solution is drawn by vacuum through wash
manifold 412 and into waste receptacle 490.
[0141] FIG. 8E provides a schematic of the working of the wash
station. Nitrogen tank 460 provides a pressure source to direct
fluid flow. Opening manual valve 462 allows gas to flow through
regulator 466 and through filter 468. Regulator 466 regulates the
pressure from the pressure source. Pressure sensor 464 monitors gas
pressure from the nitrogen source, and indicates if gas pressure is
below a selected pressure. The pressurized gas flows through filter
468 into line 470. Pressurized gas line 470 branches into the top
of sealed wash bottles 471, 472, 473, and 474. The pressurized
nitrogen pumps the wash liquid within each wash bottle into
respective fluid lines 471a, 472a, 473a and 474a respectively
through an intake filter 476 on each of said respective fluid
lines. Each of the sealed wash solution bottles may contain a
different wash solution, such as water, alcohol, a buffer or other
wash solution. Although four wash bottles are illustrated, the
system is adaptable for use with more or fewer wash fluids. In
addition, exchange of wash bottles simply requires venting N2
pressure on bottles 471, 472, 473, 474 at value 462, the removal of
the cap from the selected bottle and replacement of the cap with
attached pressure and fluid lines into a new or refilled wash fluid
bottle. Each of the fluid lines 471a, 472a, 473a and 474a terminate
in selector valve 478. According to a preset program, the selector
valve routes one of the selected fluids from the input line into
valve output line 480. The valve output line then transports the
pressurized liquid into wash tank 440.
[0142] The capillary tubes in the capillary cassette function as a
conduit for transport of fluid from the wash tank 440 into the wash
manifold interior 425. Vacuum source 496 provides a vacuum force
once valve 492 is open. When vacuum valve 498 is open, a vacuum
force is directed into waste bottle 490 creating negative pressure
within line 490a. When valve 495 is open, suction will be applied
through suction line 490a, suction line 495a and suction lines
424a. As suction is applied through suction ports 424 by suction
lines 424a the negative pressure through interior wash manifold 425
will draw liquid up through the capillary tube segments extending
into wash manifold interior 425. The liquid will travel through
suction passageways 424, into suction lines 424a, past valve 495,
through suction lines 495a and 490a and into waste bottle 490.
[0143] FIG. 8D illustrates a view of the wash manifold. The bottom
of the wash manifold contains holes 426 into which the capillaries
are inserted. Wash manifold interior 425 is comprised of lanes
joined at a first end to suction passageways 424 and at a second
end to purge passageways 423. When suction is applied through line
424a fluid will be drawn through capillaries into the lanes
comprising interior 425, through passageways 424 and into line
424a. When the purge valve is opened, air will pass through line
423a, through passageway 423, into interior 425, and into
passageway 424, clearing interior 425 of any liquid remaining in
interior 425.
[0144] Following a wash procedure, wash tank 440 is lowered
relative to the capillary cassette platform such that the ends of
the capillary tube segments are not in contact with the liquid in
wash tank 440. The liquid within wash tank 440 is drained through
drain 484 which transmits the fluid into drain line 484a when value
485 is opened and suction is applied through suction line 490a. The
fluid within wash tank 440 will then drain into waste bottle
490.
[0145] Before each wash solution is introduced into wash tank 440,
wash fluid supply line 480 and the wash tank distribution manifold
480a are purged to empty the line of any previous liquid. This is
effected by opening one of the valves in selector valve 478 and
flowing wash fluid through supply line 480 and through bleed lines
482. Opening valve 487 allows a vacuum force to be transmitted
through line 490a through line 488 providing suction which in
conjunction with fluid pressure is used to purge the distribution
manifold through bleed lines 482. Once wash fluid supply line 480
and distribution manifold are purged, valve 487 is closed and the
wash tank is raised and filled. The fill level of wash tank 440 is
controlled by a selected wash fluid fill time and wash fluid
pressure. Overflow port 486 acts as a safety drain to drain off
fluid overfill. If the fluid level within wash tank 440 is too
high, liquid will flow from wash tank 440 into overflow port 486
and into line 486a. When valve 487 is open, the suction force from
line 490a and 488 will draw overflow liquid from overflow port 486
into waste bottle 490. Restriction flow valve 441 limits liquid
fluid flow through lines 482.
[0146] FIG. 8F shows the top perspective of wash fluid tank 440. An
input line introduces a wash solution into wash fluid distribution
manifold 480a. This manifold supplies wash fluid ports 481 which
fill tank 440. The spacing of wash fluid ports 481 aids in uniform
filling across the width of tank 440. The fill time and fluid
pressure regulate the amount of fluid filling tank 440. If excess
fluid enters tank 440 it will drain from overflow port 486.
[0147] To empty the tank, the tank is lowered by the pneumatics as
described, and drain 484 is opened. The shape of tank 440 directs
fluid to drain 484 when the end of tank 440 containing drain 484 is
lowered. This configuration is designed for efficient filling,
emptying and purging of tank 440 and associated fill lines.
[0148] Again with reference to FIG. 8E, once a wash cycle has been
completed, any liquid remaining within wash manifold interior 425
may be eliminated by opening valve 491 while suction is applied
through the manifold. Opening valve 491 causes a pulse of air to be
drawn in through vent 493. The air is introduced into wash manifold
interior 425 through purge lines 423a and is removed by suction
lines 424a. If the manifold is in contact with a capillaries, the
relatively narrow bores of the capillary cassette provide a limited
capacity for drawing air through the wash manifold. By opening
valve 491, a much greater amount of air may be drawn through the
manifold through purge lines 423a which have a much greater
capacity for drawing air. This will result in a sudden rush of air
drawn through the manifold. This acts to clear the wash manifold of
any liquid remaining within the wash manifold interior 425.
Preferably manifold interior 425 is purged before and after raising
the wash manifold.
[0149] With reference to FIG. 8B, the wash station 410 is shown in
side view. The capillary cassette platform 414 is mounted on
support legs 445. The reservoir section, shown in internal cross
section has at a back lower end of the reservoir, drain outlet 484.
Upwardly positioned from the drain outlet at the back wall of the
tank is overflow outlet 486. Disposed at the front of the reservoir
is reservoir bleed outlet 446. Each outlet is associated with a
respective tube and valve, as described in conjunction with FIG.
8E. Each tube carries liquid flowing from an associated outlet when
the associated valve is opened and vacuum source applied.
[0150] Capillary cassette platform 414 is held in a fixed position
by support legs 445. Extending downward from the front of capillary
cassette platform 414 is hinge 418 with pivot 432. Attached to a
lower end of hinge 418 is wash tank stage 416. Extending from below
wash tank stage 416 is leg 419 which is attached at a lower end by
pivot 443 to pneumatic cylinder 429. At the back end of the
stationary capillary cassette platform 414, the wash manifold is
attached at pivot 420. When pneumatic cylinder 429 is extended from
the lower end, wash tank stage 416 will be lowered in an arc away
from stationary capillary platform. This occurs when no pressure is
applied to 429 and gravity causes the wash tank stage to pivot
down. When pneumatic cylinder 429 is extended from the upper end by
applied pressure, wash manifold 412 will be raised in an arc away
from capillary cassette platform 414.
[0151] Disposed above capillary cassette platform 414 is wash
manifold 412. The wash manifold has a purge passageway 423 disposed
at a front end and a suction passageway 424 disposed toward the
back end. The respective lines carrying air to the manifold or
removing gas or liquids from the manifold are described in
conjunction with FIG. 8E.
[0152] With reference to FIG. 8C, pneumatic cylinder 429 is shown
fully extended from a lower connection pivot 443 on leg 419,
through hole 333 in capillary cassette platform 414, to an upper
connection at pivot 428 on wash manifold 412. The extended height
of the wash manifold is limited by plate 430 which is secured to
the top of manifold 412. Plate 430 abuts pin 422 on capillary
cassette platform 414 when the wash manifold is raised to a
selected level and prevents the wash manifold 412 from being raised
beyond this level. When suction is applied to wash manifold
interior 425 by applying suction through suction passageway 424,
fluid is drawn through capillaries 12 from tank 440.
[0153] The front end of capillary cassette platform 414 is joined
at pivot 432 to hinge 418 and wash tank stage 416 and the back end
of capillary cassette platform 414 is joined at pivot 420 to wash
manifold 412. Extending through capillary cassette platform 414 is
cutout 434. The dimensions of cutout 434 are such that capillary
cassette 15, when placed on capillary cassette platform 414 has
associated capillary tube segments 12 extending through capillary
cassette platform 414 while the four edges of capillary cassette
substrate 10 are retained on the capillary cassette platform 414 on
the edge of cutout 434. Alignment pins may be added to capillary
cassette platform 414 to properly position the capillary
cassette.
[0154] To effect the cassette wash sequence, an electronic
controller implements a sequence of steps. The electronic
controller instructs associated controlled devices of the wash
station to carry out a programmed wash sequence. The programmed
sequence begins with the capillary cassette being placed on the
capillary cassette stage by the robotic transfer device. The wash
manifold lowers onto the capillary cassette such that the shorter
end of capillary tube segments extend into the wash manifold and
the opposite end of the capillary tube segments are within the wash
liquid in the wash tank once filled. The substrate provides a
partial seal between the wash manifold and cassette such that when
suction is applied to the capillary tube segments by the wash
manifold, fluid will be drawn up into the wash manifold through the
capillary tube segments. The wash solution supply line is purged
with the first selected solution to clear the previous solution
from the line. As noted in relation to FIG. 8E, the purge solution
is removed through distribution manifold to drain 484 and bleed
lines 482 to wash waste line 488 and 490a then into waste bottle
490. The wash tank 440 is then raised and filled with the selected
wash solution.
[0155] A vacuum is applied to the wash manifold causing the
solution in the wash tank to be drawn up through all of the
capillary tube segments in the capillary cassette. After the
programmed wash duration, the wash tank is drained and lowered. The
vacuum force is continued through the wash manifold, drawing air
through the capillary tube segments. Once the capillary tube
segments are dried, the vacuum line of the wash manifold is turned
off. The wash solution supply line is purged with the next wash
solution and the steps of raising and filling the wash tank,
drawing the wash solution through the capillary tube segments and
emptying the wash tank are repeated for each selected solution. The
specified sequence may repeat these steps for any number of wash
solutions. After the final wash has been completed and the tank
emptied, air is drawn through the capillaries by applying a vacuum
to the wash manifold, drying the capillary tube segments.
Periodically the purge valve 491 is opened and air is drawn through
vent 493 into purge lines 423a into purge inlets 423. This draws a
blast of air through wash manifold interior 425 and clears the wash
manifold interior of any remaining liquid, ensuring that any
remaining liquid within the wash manifold will not wick back into
the capillaries. The manifold vacuum is then shut off and the
manifold is raised, removing the manifold from the capillary
cassette. The manifold vacuum is again applied and the purge valve
491 is opened and air is drawn through vent 493 into purge line
423a into purge inlet 423. This ensures that any remaining liquid
is removed from the wash manifold interior. The vacuum is then shut
off. The washed and dried capillary cassette may then be moved by
the transfer robot to a capillary cassette hotel or other
location.
[0156] System Integration
[0157] The components of the system could be integrated in a
combined system which allows several elements of the complete
system of FIG. 1 to operate at the same time. For example,
electronic control device 123 may be used to send instructions to
the components of the integrated system. The electronic control
device may be a computer which sends electronic signals to various
system components to effect a programmed set of instructions.
Elements of the system could operate simultaneously, increasing
system efficiency. For example automated robot 102 could retrieve a
capillary cassette from cassette hotel 106, place the capillary
cassette in a sample plate at stage a. An amount of sample from the
plate is drawn into the capillary tubes by capillary action. The
capillary cassette could then be moved to be placed on top of a
nanotiter plate such that the short ends of the capillary tube
segments are in the wells of the nanotiter plate. The robot 102
could then transfer the combined nanotiter plate/capillary cassette
to dispense location 122 for dispensing. The movement of the robot
102, transfer head 104 and dispensing device located at location
122 are controlled by electronic control device 123.
[0158] At the same time that a reaction mixture is being assembled,
the electronic control device could also be sending electronic
signals to thermocycler 116. The vent door, heating element, and
thermocouple of thermocycler 116 could be linked to electronic
control device 123, allowing electronic control device 123 to
effect a selected temperature cycling procedure by regulating the
temperature at which air is cycling within the thermal cycler. This
precise monitoring allows the temperature cycling procedure to be
effected in a minimum amount of time. Once the thermal cycling
procedure is complete, the electronic control device 123 could
electronically instruct the thermal cycler to shut off the
thermocycler fan and heating element and open the lid pneumatically
to allow a capillary cassette to be removed from the interior of
the thermal cycler.
[0159] While automated robot 102 is moving capillary cassettes to
assemble a reaction mixture and the thermocycler is operating, the
cassette washer 118 could also be cleaning a capillary cassette.
Again the electronic control device 123 could instruct the cassette
washer 118 to perform a wash sequence in which a capillary cassette
is cleaned with a selected sequence of wash liquids and air
dried.
[0160] Electronic control device 123 enables each element of the
system to be used with maximum efficiency. A single set of
instructions to electronic control device 123 could allow assembly
of the reaction mixture, thermal cycling of the reaction mixture to
effect the desired reaction, dispensing of the completed reaction
mixture onto an analytical substrate, movement of the analytical
substrate to a stage for processing by an analytical instrument,
and cleaning of used capillary cassettes.
[0161] E. Reaction Preparation Examples
[0162] The following examples illustrate the use of the combined
reaction preparation systems. The examples are representative of
the many different types of reactions that could be effected with
the disclosed device or system and are described by 1) dye-primer
DNA sequencing, 2) dye-terminator DNA sequencing, 3) PCR
amplification, 4) PCR amplification, enzymatic purification, and
DNA sequencing, and 5) a general enzymatic reaction.
EXAMPLE 1
Dye-primer DNA Sequencing Analyzed by CAE
[0163] Dye-primer sequencing reactions were performed within a
capillary cassette comprised of 96 uncoated 2.8 cm long, 150 .mu.m
I.D., 360 O.D. fused-silica capillaries. Dye-primer sequencing
reactions were performed by amplifying template DNA with
emission-specific primers corresponding to ddT, ddA, ddC, and ddG
terminated reactions. The amplification of template was performed
as single reactions in each capillary and pooled into a common well
for post-reaction processing and analysis. The color-specific
primers were based on the M13 -40 FWD primer
(5'-FAM-GTTTTCCCAGT*CACGACG-3'), with 5-carboxyfluorescein (FAM) as
the donor dye, and a termination-specific fluor attached to the
indicated thymine (T*) as the acceptor dye. The thymine was labeled
with FAM for ddC-terminated reactions (C-FAM), 6-carboxyrhodamine
for ddA reactions (A-REG), N,N,N',N'-tetramethyl-5-carboxyrhodamine
for ddG reactions (G-TMR), and 5-carboxy-Xrhodamine for ddT
reactions (T-ROX). A master mix for 100 dye-primer sequencing
reactions was prepared by combining 65 :L reaction buffer (220 mM
Tris-HCl, pH 9.5, 33.2 mM MgCl.sub.2), 100 :L dye-primer solution
(either 1 .mu.M T-ROX, 1 .mu.M G-TMR, 0.5 .mu.M A-REG, or 0.5 .mu.M
C-FAM), 100 .PHI.L of the corresponding deoxy- and
dideoxynucleotide mix (0.94 mM DATP, dCTP, dTTP, 7-deaza-dGTP, with
3.1 uM dideoxynucleotide), 10 .PHI.L of enzyme (32 U/.PHI.L
ThermoSequenase), and 225 .PHI.L filtered deionized water. This
solution was aliquoted into a 96-well reagent plate prior to mixing
with template DNA. The general mixing scheme required the use of
two capillary cassettes and a 384-well "mix plate". The first
capillary cassette (transfer cassette) was dipped in a solution of
template DNA (20 ng/:L M13mp18), and then inverted onto the top of
a 384-well "mix plate" with the short ends of the capillaries
inserted into the wells. The inverted transfer cassette and mix
plate were placed inside a benchtop centrifuge. A balance plate was
added to balance the rotor and the centrifuge brought to
3,000.times.g for 5 seconds. The centrifugation uniformly dispensed
the contents of the transfer cassette into individual wells of the
384-well plate. After the centrifuge step, the transfer cassette
was transferred to the capillary cassette washer 410 for cleaning,
and the mix plate was used for a subsequent centrifuge step for
reagent addition.
[0164] To add reagents, a second capillary cassette, (the reaction
cassette), was dipped into the wells containing sequencing reagents
(prepared as described in the preceding paragraph) and inverted
over the wells of the same 384-well plate. The reaction cassette
and mix plate were placed in the centrifuge, spun at 3,000.times.g
for 5 seconds, and removed from the centrifuge. At this point each
well contained 500 nL of template DNA and 500 nL of sequencing
reagents to form the final reaction mixture. The second capillary
cassette (used to add reagents) was then dipped into the 1 :L
mixture contained in the mix plate, filling the capillaries of the
reaction cassette in 500 nL.
[0165] The capillary cassette was inserted into the internal
chamber of an air-based thermal cycler, as described herein, where
the ends of the capillary segments are sealed by depressing the
ends of the capillaries against deformable membrane. After 30
cycles of 95EC for 2 seconds, 55EC for 2 seconds, and 72EC for 60
seconds, the thermal cycler was opened, removing the ends of the
capillaries from contact with the deformable membranes. The
capillary cassette was removed and placed on top of a 384-well "mix
plate" with the short ends of the capillaries inserted into the
wells. The capillary cassette and mix plate were placed into a
centrifuge, with a balance plate. The reaction products were
dispensed by centrifugal force (.about.2500 g) into a microtiter
plate containing 40 .mu.L of 80% isopropyl alcohol. After an
initial reaction, the capillaries were washed as described herein.
After the four dye-primer reactions had been performed in four
individual capillary cassettes and the products pooled into the
wells of a microtiter plate, the samples were subsequently
centrifuged at 3000.times.g for 30 minutes. The alcohol was
decanted by a gentle inverted spin, and the samples were
resuspended in 5 .mu.L of ddH20 for electrokinetic injection and
analysis by capillary array electrophoresis.
[0166] Analysis of the DNA sequencing fragments was performed with
MegaBACE, a 96-capillary array electrophoresis instrument
(Molecular Dynamics, Sunnyvale, Calif.) using scanning confocal
laser-induced fluorescence detection. Separations were performed in
62 cm long, 75 .mu.m I.D., 200 .mu.m O.D. fused-silica capillaries
with a working separation distance of 40 cm. Electroosmotic flow
was reduced by Grignard coupling of a vinyl group to the capillary
surface and acrylamide polymerization. The capillaries were filled
with a fresh solution of 3% linear polyacrylamide (LPA)(MegaBACE
Long Read Matrix, Amersham Life Sciences, Piscataway, N.J.) which
was pumped through the capillaries under high-pressure from the
anode chamber to individual wells of a 96-well buffer plate
contained in the cathode chamber. Each well was filled with 100 :L
of Tris-TAPS running buffer (30 mM Tris, 100 mM TAPS, 1 mM EDTA, pH
8.0). The matrix was equilibrated for 20 minutes followed by
pre-electrophoresis for 5 minutes at 180 V/cm. Prior to sample
injection, the cathode capillary ends and electrodes were rinsed
with ddH.sub.2O to remove residual LPA prior to sample
injection.
[0167] DNA sequencing samples were electrokinetically injected at
constant voltage from a 96-well microtiter plate according to the
specified conditions; one preferred injection condition for 500 nL
samples is 40 seconds of injection at an applied voltage of 2 kV.
After injection, the capillary ends were rinsed with water, the
buffer plate was placed in the cathode chamber, and the
electrophoresis run was commenced. Separations were typically for
120 minutes at 8 kV. Computer controlled automation of the
instrument and data collection was performed using LabBench
software (Molecular Dynamics, Sunnyvale, Calif.). Specific
injection and run conditions were tailored to the reaction mixture
to be analyzed.
[0168] The reproducibility of the described method for
sub-microliter dye-primer cycle sequencing is shown in FIG. 9. This
histogram shows the percent success versus readlength window and
shows that the method is highly reproducible. Over 80 percent of
the sequenced DNA inserts had a readlength over 600 bases. Overall,
this plate yielded 55,000 high quality bases, with an average
readlength of 605 bases.
EXAMPLE 2
Dye-primer DNA Sequencing Analyzed by a CAE Microchip
[0169] In another analysis example, dye-primer reactions performed
in the same capillary cassette were analyzed by direct injection
into a microfabricated "chip-based" analyzer. In this example, a
dye-primer reaction terminated by ddT was performed as described
and dispensed into the sample wells of a microchip containing
1.5.div..mu.L of ddH.sub.2O. The electropherogram is featured in
FIG. 10 exemplifying microchip analysis of reactions performed in
the described system.
EXAMPLE 3
Dye-terminator Cycle-sequencing with Alcohol Precipitation
Purification
[0170] Dye-terminator cycle-sequencing was demonstrated using the
capillary cassette system and alcohol precipitation for cleanup
prior to capillary array electrophoresis. In this example, the
sequencing reaction mix was prepared by mixing 400 .mu.L of
sequencing reagents (Dyenamic ET terminator kit, Amersham Pharmacia
Biotech, Part 81600) with 100 mL of 5 pmol/.mu.L of M13 -28 FWD
primer (5'-TGT AAA ACG ACG GCC AGT-3'). The reaction mix was
distributed in 5 .mu.L aliquots to a 96-well "reagent" plate.
Mixing of template DNA and sequencing reagents was performed in the
same series of steps described in Example 1. A second cassette was
used to transfer 500 nL of sequencing reagents from the reagent
plate to the wells of the mix plate. This same cassette was then
filled by capillary action with the template/reagent mixture.
[0171] The capillary cassette was transferred to the air-based
thermal-cycler where the capillaries were sealed between the
deformable membranes within the thermal cycler. Thermal cycling was
achieved with 30 cycles of 95.degree. C. for 2 s, 55.degree. C. for
2 s, and 60.degree. C. for 60 seconds. After the thermal cycling,
the cassette was removed from the cycling chamber and the contents
of the capillaries dispensed by centrifugal force (3000.times.g)
into a 96-well plate containing 40 .mu.L of 80% ethanol. The
samples were centrifuged at 3000.times.g for 30 minutes. The
alcohol was decanted by a gentle inverted spin, and the samples
were resuspended in 5 .mu.L of ddH20 for electrokinetic injection
and analysis by capillary array electrophoresis. The cleanup of
dye-terminator reactions by alcohol precipitation, the
reproducibility of the technique, and the application to
"real-world" templates is represented as a histogram of percent
success versus readlength in FIG. 11. FIG. 11 demonstrates
excellent readlengths and success rates with M13 subclone inserts
prepared from the subclone library of a Mouse bacterial artificial
chromosome (BAC).
EXAMPLE 4
Dye-terminator Cycle Sequencing with Size-exclusion
Purification
[0172] In another example, dye-terminator reactions were performed
in 500 nL capillaries as described in Example 3, and the reaction
products dispensed into 15 .mu.L of ddH2O by centrifugal force. The
15 mL samples were transferred to a filter plate containing 45 mL
of hydrated Sephadex G-50. The samples were centrifuged through the
Sephadex matrix at 910.times.g for 5 minutes and the eluent
collected in a clean 96-well injection plate. The samples were
electrokinetically injected without further dehydration or
processing. For 16 samples, an average readlength of 650 bases was
obtained demonstrating the compatibility of sub-microliter
dye-terminator sequencing with alcohol and size-exclusion
purification.
EXAMPLE 5
PCR Amplification of Plasmid Insert DNA
[0173] The present technology uses the disclosed system for the
polymerase chain reaction (PCR) amplification of insert DNA (e.g.
subclone inserts from a DNA library). The PCR reaction mixture was
prepared by mixing 5 :L of 10 :M of M13 -40 FWD primer (5' GTT TTC
CCA GTC ACG AC 3') and 5 :L of 10 NM M13 -40 REV primer (5' GGA TAA
CAA TTT CAC ACA GG 3') with 25 :L of 10.times. GeneAmp buffer, 15
:L of 25 mM MgCl.sub.2, 5 :L of AmpliTaq Gold, 2.5 :L of 1 mg/mL
bovine serum albumin (BSA), and 67.5 :L of ddH.sub.2O. This mix was
aliquoted in equal volumes to sixteen 0.20 mL tubes.
[0174] The reaction was initiated by mixing template DNA with the
PCR cocktail using the two-capillary cassette and mix-plate method
described. The transfer cassette was dipped into the glycerol stock
solutions of a subclone library and dispensed by centrifugal force
into the wells of a 384-well plate. A second "reaction" cassette
was used to transfer 500 nL of PCR cocktail to the same wells by
centrifugal force. The capillaries were subsequently dipped into
the combined mixture of template DNA and PCR reagents, filling the
capillaries by capillary action. Amplification was effected by
placing the capillaries into the cycling chamber and thermally
cycling with an activation step of 95.degree. C. for 12 minutes
followed by 30 cycles of 64.degree. C. for 4.5 minutes and
95.degree. C. for 5 seconds.
[0175] The PCR products were analyzed by agarose gel
electrophoresis and compared with the same subclones amplified by
large-volume (25 :L) reactions performed in 0.20 mL tubes.
Nanoscale capillary cassette samples were dispensed into 4.5 :L of
ddH2O by centrifugal force. Equivalent volume aliquots of full
volume reactions were transferred manually using a low volume
pipettor. To each 5 :L sample, 1 :L of 6.times. loading dye was
added and the sample quantitatively transferred to the wells of an
agarose gel. Agarose gel electrophoresis was performed using a 0.7%
agarose gel with 1.times.Tris-acetate-EDTA buffer, pH 8.0. Samples
were separated for 40 minutes at 15 V/cm, stained with Sybr Green
II (Molecular Probes, Eugene, Oreg.), and imaged using a
two-dimensional fluorescence scanner (FluorImager, Molecular
Dynamics, Sunnyvale, Calif.). The scanned gel image is shown in
FIGS. 12A and 12B. It can be seen that samples prepared at
full-volume (FIG. 12A) and 500 nL amplification (FIG. 12B) have the
same molecular weight distribution. This example demonstrates
nanoscale sample preparation can be analyzed by traditional
macro-scale analysis such as agarose gel electrophoresis.
EXAMPLE 6
PCR Amplification and Cycle-sequencing
[0176] A preferred mode of preparing cycle sequencing samples using
the present invention is to prepare nanoscale PCR samples in the
capillary cassette and related instrumentation, perform macroscale
ExoI/SAP reactions, and then perform the cycle sequencing in the
capillary cassette and related instrumentation. Nanoscale PCR
template preparation for DNA sequencing was demonstrated by
performing PCR amplification from glycerol stock subclones.
Glycerol stock subclones were PCR amplified as described in Example
5. After PCR amplification, the contents of the capillaries were
dispensed by centrifugation into the wells of a 96-well plate
containing 4.5 :L of 7.5 mU of shrimp alkaline phosphatase (SAP)
and 37.5 mU of exonuclease I (ExoI). The PCR products and ExoI/SAP
solution were allowed to incubate at 37.degree. C. for 5 minutes to
digest the unincorporated primers and to dephosphorylate the
unincorporated nucleotides. After an initial incubation, the
enzymes were deactivated by heating the solution to 72.degree. C.
for 15 minutes.
[0177] The ExoI/SAP treated PCR products were aliquoted to a fresh
384-well mix plate with a transfer capillary cassette and
centrifugal dispensing. An equal aliquot of dye-terminator
sequencing reagents were added to the 500 nL of purified PCR
products using another capillary cassette and centrifugal
dispensing. The capillaries were then filled by dipping the
capillary cassette into the 1 :L reaction mixture. The template was
amplified according to Example 3, dispensed into 40 :L of 80%
ethanol and purified as described. Analysis of the sequencing
reactions was performed by MegaBACE using electrokinetic injection.
Portions of six base-called sequencing electropherograms from
subclone templates prepared by nanoscale PCR amplification from
glycerol stock solutions and by nanoscale cycle sequencing are
shown in FIG. 13. By performing PCR in a capillary cassette and
subsequently transferring the reaction mixture to a microplate, the
present system allows a simplified transition from nanoscale (less
than 1 .mu.L volumes) to greater than nanoscale reaction volumes.
The present system also allows a simplified transition from
macroscale (more than 1 .mu.L volumes) to nanoscale reaction
volumes, as shown by utilizing the Exo I/SAP reactions for cycle
sequencing in the capillary cassette.
[0178] E. Reaction Preparation Examples
EXAMPLE 7
[0179] Isothermal enzyme assay performed in sub-microliter
capillary cassette. The use of the described system for performing
general enzyme reactions was demonstrated with a fluoregenic assay
of #-galactosidase. The #-galactosidase (#-Gal) catalyzed
hydrolysis of resorufin-#-D-#-galactosidase (#-Gal) catalyzed
hydrolysis of resorufin-#-D-galactosidase (RBG) was performed
within the capillaries of a 96-capillary cassette in which #-Gal
hydrolyzes RBG to the fluorophore resorufin.
[0180] A stock solution of 350 micromolar RBG was prepared in 5 mL
of 100 mM Tris-HCL, 20 mM KCl, and 2 mM MgCl2 to 5 mg of RBG,
vortexing vigorously, and filtering the solution through a 0.40
micron filter. A one-half dilution curve of RBG was prepared from
the stock solution. To each 10 microliters of RBG solution prepared
in 0.20 mL tubes, 200 ug of #-Gal was added and after briefly
mixing, filled into a capillary cassette by capillary action. The
cassette was placed in an air-cycler and after 2 minutes at 37
degrees C., the capillary cassette was removed and the contents
centrifuged out of the capillaries into a 384-well scan plate
containing 5 microliters of 1M sodium bicarbonate. The wells of the
scan plate were subsequently filled with 50 microliters of ddH20
and the plate was read by a fluorescent plate reader (Typhoon,
Molecular Dynamics). A control aliquot from the enzyme reactions
performed in the 0.20 mL tubes was added to the scan plate.
[0181] Solid-phase capture of the #-Gal was also demonstrated with
this system by simply filling the cassette with a 20 ug/mL solution
of #-Gal, allowing the #-Gal to bind to the capillary surface
followed by removing the excess liquid and drying the cassette
using the described cassette wash-manifold. After #-Gal binding the
capillaries were filled with RBG solution by capillary action. The
reaction was performed for 2 minutes at 37.degree. C. and analyzed
by dispensing into 1M sodium bicarbonate, diluting the water and
scanning using a fluorescent plate reader. The results are
summarized in Figure XYZ showing the expected signal versus
substrate concentration for the tube reactions, and data points of
signal for the pre-mixed enzyme reaction performed in the capillary
cassette, and for the capillary-binding #-Gal assay. This example
serves to illustrate the compatibility of the described system for
performing a range of general enzyme activity and inhibition
assays.
* * * * *