U.S. patent application number 11/937567 was filed with the patent office on 2008-05-01 for methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to William Bedingham, Vicky L. Morris, Ranjani V. Parthasarathy, Raj Rajagopal, Barry W. Robole.
Application Number | 20080103297 11/937567 |
Document ID | / |
Family ID | 21836445 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080103297 |
Kind Code |
A1 |
Parthasarathy; Ranjani V. ;
et al. |
May 1, 2008 |
METHODS AND DEVICES FOR REMOVAL OF ORGANIC MOLECULES FROM
BIOLOGICAL MIXTURES USING A HYDROPHILIC SOLID SUPPORT IN A
HYDROPHOBIC MATRIX
Abstract
Methods and devices for removing small negatively charged
molecules from a biological sample mixture that uses a solid-phase
extraction material that includes a hydrophilic solid support at
least partially embedded within a hydrophobic matrix.
Inventors: |
Parthasarathy; Ranjani V.;
(Woodbury, MN) ; Rajagopal; Raj; (Woodbury,
MN) ; Morris; Vicky L.; (North Branch, MN) ;
Bedingham; William; (Woodbury, MN) ; Robole; Barry
W.; (Woodville, WI) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
21836445 |
Appl. No.: |
11/937567 |
Filed: |
November 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10027226 |
Dec 20, 2001 |
|
|
|
11937567 |
Nov 9, 2007 |
|
|
|
Current U.S.
Class: |
536/25.4 ;
422/255; 428/323 |
Current CPC
Class: |
Y10T 436/255 20150115;
Y10T 428/25 20150115; Y10T 436/25125 20150115; Y10T 436/25375
20150115; C12N 15/1006 20130101; Y10T 436/25 20150115 |
Class at
Publication: |
536/025.4 ;
422/255; 428/323 |
International
Class: |
C07H 21/00 20060101
C07H021/00; B01J 19/00 20060101 B01J019/00; B32B 5/16 20060101
B32B005/16 |
Claims
1. An analytical receptacle comprising one or more reservoirs and a
surface with a cover film adhered to the surface and enclosing the
one or more reservoirs; wherein the cover film comprises a backing
and an adhesive disposed on at least one major surface of the
backing and in contact with the receptacle surface; wherein at
least a portion of the adhesive has a solid-phase extraction
material disposed thereon; wherein the solid-phase extraction
material comprises hydrophilic particles at least partially
embedded in the adhesive.
2. The analytical receptacle of claim 1 wherein the particles have
an average particle size of at least about 5 nm.
3. The analytical receptacle of claim 1 wherein the particles have
an average particle size of no greater than about 500 microns.
4. The analytical receptacle of claim 1 wherein the particles are
disposed on the layer of adhesive at a density of about 0.1 mg per
12 mm.sup.2 surface area to about 5 mg per 12 mm.sup.2 surface
area.
5. The analytical receptacle of claim 1 wherein the particles are
pattern coated on the adhesive.
6. The analytical receptacle of claim 1 wherein the particles have
a surface area of from about 50 square meters per gram (m.sup.2/g)
to no greater than about 200 m.sup.2/g.
7. The analytical receptacle of claim 1 wherein the particles
comprise active groups at a density of at least about 0.01
micromoles per 1735 square millimeters (mmoles/1735 mm.sup.2).
8. The analytical receptacle of claim 1 wherein the particles
possess a fixed positive charge between pH 8.0 and pH 9.0.
9. The analytical receptacle of claim 1 wherein the particles
separate at least about 70% of negatively charged unincorporated
polymerase chain reaction (PCR) reagents from desired PCR
products.
10. The analytical receptacle of claim 1 wherein the negatively
charged unincorporated PCR reagents comprise unincorporated
residual primers, unincorporated dye molecules, or unincorporated
dNTPs.
11. The analytical receptacle of claim 10 wherein the particles
separate at least about 90% of the unincorporated residual primers
and/or at least about 70% of the unincorporated dNTPs from the
desired PCR products.
12. The analytical receptacle of claim 10 wherein the particles
separates at least about 95% of the unincorporated residual
primers, unincorporated dye molecules, and unincorporated dNTPs
from the desired PCR products.
13. The analytical receptacle of claim 1 wherein at least a portion
of the particles is coated with a negatively charged polymer.
14. The analytical receptacle of claim 13 wherein the particles,
the negatively charged polymer, or both are pattern coated.
15. The analytical receptacle of claim 1 wherein the particles bind
less than 30% of the desired PCR products.
16. The analytical receptacle of claim 10 wherein the particles
comprise active groups at a density of at least about 0.01
micromoles per 1735 square millimeters (mmoles/1735 mm.sup.2).
17. The analytical receptacle of claim 1 wherein the particles
separate at least about 70% of negatively charged unincorporated
DNA sequencing reagents from desired DNA sequencing products.
18. The analytical receptacle of claim 17 wherein the negatively
charged unincorporated DNA sequencing reagents comprise
unincorporated residual primers, unincorporated dye molecules,
unincorporated dNTPs, unincorporated ddNTPs, or unincorporated dye
terminators.
19. The analytical receptacle of claim 18 wherein the particles
separate at least about 95% of the unincorporated residual primers,
unincorporated dye molecules, unincorporated dNTPs, or
unincorporated dye terminators from the desired DNA sequencing
products.
20. The analytical receptacle of claim 18 wherein the particles
separate at least about 95% of the unincorporated residual primers,
unincorporated dye molecules, unincorporated dye terminators,
unincorporated dNTPs, and unincorporated ddNTPs from the desired
DNA sequencing products.
21. The analytical receptacle of claim 17 wherein at least a
portion of the particles is coated with a negatively charged
polymer.
22. The analytical receptacle of claim 21 wherein the particles,
the negatively charged polymer, or both are pattern coated.
23. The analytical receptacle of claim 17 wherein the particles
comprise active groups at a density of at least about 0.01.mu.
moles/1735 mm.sup.2.
24. The analytical receptacle of claim 17 wherein the particles
bind less than 30% of the desired DNA sequencing products.
25. A method of removing small negatively charged organic molecules
from a biological sample mixture, the method comprising: providing
an analytical receptacle comprising one or more reservoirs and a
surface with a cover film adhered to the surface and enclosing the
one or more reservoirs; wherein the cover film comprises a backing
and an adhesive disposed on at least one major surface of the
backing and in contact with the receptacle surface, wherein at
least a portion of the adhesive has a solid-phase extraction
material disposed thereon, wherein the solid-phase extraction
material comprises hydrophilic particles at least partially
embedded in the adhesive; providing a biological sample mixture;
and contacting the biological sample mixture with the solid-phase
extraction material to remove at least a portion of the small
negatively charged organic molecules from the biological sample
mixture.
26. An analytical receptacle comprising one or more reservoirs
comprising a surface with an adhesive disposed on at least a
portion of the surface; wherein at least a portion of the adhesive
has a solid-phase extraction material disposed thereon; wherein the
solid-phase extraction material comprises hydrophilic particles at
least partially embedded in the adhesive.
27. A method of removing small negatively charged organic molecules
from a biological sample mixture, the method comprising: providing
an analytical receptacle comprising one or more reservoirs
comprising a surface with an adhesive disposed on at least a
portion of the surface, wherein at least a portion of the adhesive
has a solid-phase extraction material disposed thereon, wherein the
solid-phase extraction material comprises hydrophilic particles at
least partially embedded in the adhesive; providing a biological
sample mixture; and contacting the biological sample mixture with
the solid-phase extraction material to remove at least a portion of
the small negatively charged organic molecules from the biological
sample mixture.
28. An analytical receptacle comprising one or more reservoirs
comprising a solid phase extraction material comprising solid
hydrophilic particles at least partially embedded in a hydrophobic
matrix, wherein the hydrophobic matrix is disposed on at least a
portion of a surface of the one or more reservoirs, and wherein the
solid support material comprises molecules that are different than
the hydrophobic matrix.
29. A method of removing small negatively charged organic molecules
from a biological sample mixture, the method comprising: providing
an analytical receptacle comprising one or more reservoirs
comprising a solid phase extraction material comprising solid
hydrophilic particles at least partially embedded in a hydrophobic
matrix, wherein the hydrophobic matrix is disposed on at least a
portion of a surface of the one or more reservoirs, and wherein the
solid support material comprises molecules that are different than
the hydrophobic matrix; providing a biological sample mixture; and
contacting the biological sample mixture with the solid-phase
extraction material to remove at least a portion of the small
negatively charged organic molecules from the biological sample
mixture.
30. A film comprising: a substrate; an adhesive disposed on at
least one major surface of the substrate; and particles comprising
hydrophilic solid-phase extraction material at least partially
embedded in at least a portion of the adhesive.
31. A method of removing small negatively charged organic molecules
from a biological sample mixture, the method comprising: providing
a substrate, an adhesive disposed on at least one major surface of
the substrate, and particles comprising hydrophilic solid-phase
extraction material at least partially embedded in at least a
portion of the adhesive; providing a biological sample mixture; and
contacting the biological sample mixture with the solid-phase
extraction material to remove at least a portion of the small
negatively charged organic molecules from the biological sample
mixture.
32. A film comprising: a substrate; a hydrophobic matrix disposed
on at least a portion of the substrate; and particles comprising
hydrophilic solid-phase extraction material at least partially
embedded in at least a portion of the hydrophobic matrix, wherein
the hydrophilic solid-phase extraction material comprises molecules
that are different than the hydrophobic matrix.
33. A method of removing small negatively charged organic molecules
from a biological sample mixture, the method comprising: providing
a solid-phase extraction material comprising a hydrophilic solid
support at least partially embedded within a hydrophobic matrix,
wherein the hydrophilic solid support comprises molecules that are
different than the hydrophobic matrix; providing a biological
sample mixture; and contacting the biological sample mixture with
the solid-phase extraction material to remove at least a portion of
the small negatively charged organic molecules from the biological
sample mixture.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/027,226, filed Dec. 20, 2001, now allowed, the disclosure of
which is incorporated by reference in their entirety herein.
BACKGROUND
[0002] Water-soluble dyes (e.g., fluorescent, chemiluminescent,
visible, and near-IR) are used routinely in molecular biology to
label and monitor components of biological reactions. Frequently,
residual dyes as well as other organic molecules should be removed
before proceeding with many downstream applications. Thus, the
present invention is directed to removing dyes and other organic
molecules from biological mixtures, particularly in low volume,
microfluidic devices.
[0003] There is a significant need for high throughput, low volume,
integrated microfluidic devices in order to increase sample
throughput and reduce the amount of reagents used per sample
(thereby reducing cost per sample) in biological reactions. Small
volume Polymerase Chain Reaction (PCR) and nucleic acid cycle
sequencing reactions are examples of standard molecular biology
techniques that are suitable for incorporation into miniaturized
formats. In both applications, removal of residual primers, nucleic
acid templates, dyes, and other organic molecules are generally
necessary prior to any further downstream applications.
[0004] One example where such removal methods are used is in the
preparation of a finished sample (e.g., purified nucleic acid
materials) from a starting sample (e.g., a raw sample such as
blood, bacterial lysate, etc.). For example, to obtain a purified
sample of the desired materials in high concentrations, the
starting sample is typically prepared for PCR after which the PCR
process is performed to obtain a desired common PCR reaction
product. The common PCR reaction product can then be used in a
variety of molecular biological applications, including, for
example, sequencing, cloning, genotyping, and forensic
applications.
[0005] In fluorescence-based DNA sequencing applications,
unincorporated dye terminators (i.e., dye-labeled dideoxy
terminators such as dideoxynucleotide triphosphates (ddNTPs))
should preferably be removed from the reaction mixture prior to
analysis of the DNA sequence fragments. Failure to sufficiently
reduce the concentration of dye terminator molecules leads to dye
artifacts (i.e., other dye-containing molecules such as dye-labeled
dideoxy terminators such as dideoxynucleotide diphosphates
(ddNDPs), dideoxynucleotide monophosphates (ddNMPs), and
dideoxynucleosides) that can significantly obscure DNA sequence
information. Sequencing reaction purification is a desired step in
the preparation of samples prior to sequence analysis, particularly
when using a capillary electrophoresis (CE) sequencer.
[0006] Conventionally, after completion of the PCR or cycle
sequencing reaction, the product is generally purified by either
alcohol (ethanol or isopropanol) precipitation or gel filtration
chromatography. Other protocols using polyalkylene glycol and
biotin-streptavidin interactions have also been utilized for
sequencing reaction clean-up. Ultrafiltration membranes,
phenol/chloroform extraction, and enzymatic treatments are other
methods that are commonly used for purification of PCR and
sequencing reaction mixtures.
[0007] Such conventional technologies for the purification of PCR
and nucleic acid sequencing reactions have not proven to be
suitable for incorporation into a microfluidic device. Alcohol
precipitation utilizes volatile and flammable reagents. Hydrogels
(e.g., crosslinked dextrans), commonly used in size exclusion
chromatography, require large bed volumes (10.times. relative the
volume of sample) for efficient separation of impurities from
product. Gels are first swollen with a relatively large volume of
water, centrifuged, and loaded substantially immediately, because,
upon dehydration, these materials are prone to cracking.
Biotin-streptavidin mediated purifications require the use of
custom biotinylated primers for the efficient capture of product.
Biotinylated products are generally captured onto
streptavidin-treated paramagnetic particles and physically
separated from impurities with the use of a magnet. Alternatively,
hybridization based purification (HBP) of the PCR or nucleic acid
sequencing product can be accomplished by utilizing primers
containing specially designed capture tags. Separation of the
nucleic acid fragment from the biological matrix can be achieved by
hybridization of the capture tag to a complementary strand bound to
a solid support. Both the biotin and HBP strategies would require a
rinsing step followed by elution of the sequencing or PCR product
from the substrate. Although biotin-streptavidin and HBP
purification methods yield clean PCR and sequencing fragments, both
approaches require customized primers, which can be cumbersome and
expensive.
[0008] An alternative approach for the removal of residual dye
terminators from DNA sequencing reactions involves treating the
reaction mixture with an enzyme (e.g., shrimp alkaline phosphatase)
to dephosphorylate residual nucleotide triphosphates. Although
cleavage of the phosphate groups(s) from the dye-labeled
dideoxynucleotide triphosphates alters the mobility of the
dye-labeled nucleotides in the sequencing gel, residual dye
moieties are not removed from the reaction mixture by this
procedure and must still be eliminated prior to injection of the
sample into the sequencer. This is generally accomplished by
subsequent alcohol precipitation of the digested product.
[0009] PCR and sequencing products can also be effectively purified
by adsorption of nucleic acid fragments onto beads and silica gel
membranes using chaotropic agents. Impurities (e.g., residual
primers, dyes, and salts) can be rinsed from the substrate and the
purified product eluted. This multi-step bind/rinse/elute
purification scheme may also prove to be cumbersome within the
context of a microfluidic device.
[0010] Yet another method of removing unwanted materials (e.g.,
dyes) from cycle sequencing (e.g., Sanger cycling) reaction
mixtures involves the use of paramagnetic particles. One example of
suitable paramagnetic particles incorporating dye terminator
removal materials is available under the trade designation
RAPXTRACT from Prolinx Inc., Bothell, Wash. Further examples of
these materials (and their methods of use) may be found in U.S.
patent application Ser. No. 09/894,810 filed on Jun. 28, 2001 and
entitled ENHANCED SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS
(U.S. Pat. Application Publication No. 2002/0047003 (Bedingham et
al.)). Unfortunately, however, with such particles, the particles
must remain in a hydrated state, which limits the ability to
prefabricate particle-loaded devices.
[0011] Thus, methods are needed for the removal of dyes and other
organic molecules from biological mixtures, such as nucleic acid
amplification reaction mixtures (e.g., PCR or cycle sequencing
reaction mixtures).
SUMMARY OF THE INVENTION
[0012] The present invention provides a solid-phase extraction
material that includes a hydrophilic solid support (typically in
the form of particles) at least partially embedded within a
hydrophobic matrix. The present invention also provides methods for
processing biological mixtures, i.e., samples containing a
biological material such as peptide- and/or nucleotide-containing
material, using such solid-phase material. Specifically, the
present invention provides methods for the removal of negatively
charged organic molecules (e.g., dyes, primers, probes, dNTPs, dye
terminators such as ddNTPs, ddNDPs, ddNMPs, and nucleosides) from
biological sample mixtures using hydrophilic particles at least
partially embedded within a hydrophobic matrix. These methods are
based on solid-phase extraction techniques. They are advantageous
because they can be incorporated into high throughput, low volume,
integrated microfluidic devices, if desired, particularly those
being developed for PCR and DNA sequencing.
[0013] The present invention provides methods for removing small
negatively charged organic molecules (i.e., unwanted molecules)
from a biological sample mixture. Preferably, the biological sample
mixture is a biological sample mixture such as a nucleic acid
amplification reaction mixture (e.g., a PCR reaction mixture or a
nucleic acid sequencing reaction mixture).
[0014] Herein, "removal" of unwanted molecules involves adhering
such molecules to the solid-phase material and allowing desirable
products to remain in solution. This is in contrast to conventional
elution methods that involve adhering the desirable products to the
solid-phase material, washing away the unwanted molecules, and
eluting the desirable products to remove them from the solid-phase
material.
[0015] In one embodiment, a method includes: providing a
solid-phase extraction material that includes a hydrophilic solid
support (preferably, particles) at least partially embedded within
a hydrophobic matrix (preferably, an adhesive); providing a
biological sample mixture; and contacting the biological sample
mixture with the solid-phase extraction material to remove at least
a portion of the small negatively charged organic molecules from
the biological sample mixture. Preferably, the hydrophilic solid
support is in the form of particles pattern coated on a layer, and
at least partially embedded therein, of the hydrophobic matrix
(preferably, a silicone, polyvinyl butyral, polyolefin, fluorinated
polymer, acrylate, epoxy, natural or synthetic rubber, or
combinations (e.g., mixtures, copolymers, terpolymers, etc.)
thereof.
[0016] In another embodiment, a method includes: providing a device
that includes at least one process array that includes a
solid-phase extraction material, wherein the solid-phase extraction
material includes a hydrophilic solid support (preferably,
particles) at least partially embedded within a hydrophobic matrix
(preferably, an adhesive); providing a biological sample mixture in
the at least one process array; and transferring the biological
sample mixture within the at least one process array, wherein the
biological sample mixture and the solid-phase extraction material
remain in contact for a sufficient time to remove at least a
portion of the small negatively charged organic molecules from the
biological sample mixture. Preferably, the hydrophilic solid
support is in the form of particles pattern coated on a layer of
the hydrophobic matrix.
[0017] In yet another embodiment, a method includes: providing a
device that includes at least one process array that includes a
solid-phase extraction material, wherein the solid-phase extraction
material includes hydrophilic particles disposed on a layer of a
hydrophobic matrix and at least partially embedded therein;
providing a biological sample mixture in the at least one process
array; and transferring the biological sample mixture within the at
least one process array, wherein the biological sample mixture and
the solid-phase extraction material remain in contact for a
sufficient time to remove at least a portion of the small
negatively charged organic molecules from the biological sample
mixture. Preferably, the hydrophilic particles are pattern coated
on a layer of the hydrophobic matrix.
[0018] When the biological sample mixture is a sequencing reaction
mixture, the small negatively charged molecules are typically
selected from the group consisting of dye-labeled terminators,
primers, degraded dye molecules, deoxynucleotide triphosphates, and
mixtures thereof. Preferably, for such a sample, the method is
carried out under conditions effective to remove substantially all
the dye-labeled terminators from the biological sample mixture.
[0019] When the biological sample mixture is a PCR reaction
mixture, the small negatively charged molecules are typically
selected from the group consisting of primers, degraded dye
molecules, deoxynucleotide triphosphates, and mixtures thereof.
Preferably, for such a sample, the method is carried out under
conditions effective to remove substantially all the primers from
the biological sample mixture.
[0020] The present invention also provides devices that can be used
to carry out methods of the present invention. Such devices include
analytical receptacles, such as microfluidic devices and microtiter
plates, for example.
[0021] In one embodiment, the present invention provides a device
that includes: a plurality of process arrays that include: a
plurality of process chambers, each of the process chambers
defining a volume for containing a biological sample mixture; and
at least one distribution channel connecting the plurality of
process chambers; and a solid-phase extraction material within at
least one of the process arrays that includes a hydrophilic solid
support (preferably, particles) at least partially embedded within
a hydrophobic matrix (preferably, adhesive). Preferably, the device
further includes a plurality of valves, wherein at least one of the
valves is located along the at least one distribution channel.
Preferably, the hydrophilic solid support is in the form of
particles pattern coated on a layer of the hydrophobic matrix.
[0022] The present invention also provides an analytical receptacle
that includes one or more reservoirs and a surface with a cover
film adhered to the surface and enclosing the one or more
reservoirs. The cover film includes a backing and an adhesive
disposed on at least one major surface of the backing and in
contact with the receptacle surface. At least a portion of the
adhesive has a solid-phase extraction material disposed thereon;
wherein the solid-phase extraction material includes hydrophilic
particles at least partially embedded in the adhesive.
[0023] In another embodiment, the present invention provides an
analytical receptacle that includes a plurality of reservoirs
adapted for receipt of a biological sample mixture. At least one
reservoir includes a solid-phase extraction material that includes
hydrophilic particles at least partially embedded in a hydrophobic
matrix.
[0024] These and other features and advantages of the methods of
the invention are described below with respect to illustrative
embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a top plan view of one device that can be used in
connection with the present invention.
[0026] FIG. 2 depicts an alternative device that can be used in
connection with the present invention.
[0027] FIG. 3 is an enlarged view of one process array on the
device of FIG. 2.
[0028] FIG. 4 is a cross-sectional view of a portion of the process
array of FIG. 3, taken along line 4-4 in FIG. 3.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0029] The methods of the present invention utilize solid-phase
extraction techniques for processing biological sample mixtures to
remove at least a portion of small organic molecules (e.g.,
molecules having a molecular weight of less than about 6,000)
included in such mixtures. These small molecules are typically
negatively charged and the solid-phase extraction material
typically includes a hydrophilic solid support at least partially
embedded within a hydrophobic material. The biological sample
mixture (i.e., a sample containing a biological material such as
peptide- and/or nucleotide-containing material) is preferably a
biological reaction mixture (e.g., a PCR or cycle sequencing or
other nucleic acid amplification reaction mixture). The small
organic molecules are preferably residual or unincorporated
materials (including degradation products) in biological reactions
(e.g., dyes, primers, probes, dNTPs, dye terminators such as
ddNTPs, ddNDPs, ddNMPs, and nucleosides). Significantly, using the
solid-phase extraction materials of the present invention, the
undesirable molecules preferably preferentially adhere to the
solid-phase material and the desirable products remain in the
biological sample solution.
[0030] Examples of nucleic acid amplification reaction mixtures
suitable for use in the present invention include, but are not
limited to: a) polymerase chain reaction (PCR); b) target
polynucleotide amplification methods such as self-sustained
sequence replication (3SR) and strand-displacement amplification
(SDA); c) methods based on amplification of a signal attached to
the target polynucleotide, such as branched chain DNA
amplification; d) methods based on amplification of probe DNA, such
as ligase chain reaction (LCR) and QB replicase amplification
(QBR); e) transcription-based methods, such as ligation activated
transcription (LAT) and nucleic acid sequence-based amplification
(NASBA); f) cycle sequencing reactions such as Sanger sequencing;
and g) various other amplification methods, such as repair chain
reaction (RCR) and cycling probe reaction (CPR).
[0031] Such methods are particularly desirable for use in the
clean-up of PCR reaction mixtures, nucleic acid sequencing reaction
mixtures, nucleic acid labeling reaction mixtures, or hybridization
reaction mixtures, particularly PCR, nucleic acid sequencing, and
nucleic acid labeling reaction mixtures, and more particularly PCR
or nucleic acid cycle sequencing or other amplification reaction
mixtures. That is, the methods of the present invention are
particularly desirable for removing residual reactants and
degradation products thereof (e.g., undesirable dye-containing
molecules such as ddNDPs and the like) from the desired
amplification reaction products (e.g., PCR or sequencing reaction
products). The removal of residual dyes (including near-IR,
fluorescent, chemiluminescent, UV, and visible) or dye-containing
molecules and other small organic molecules may be important in
numerous other genomics and proteomics applications as well (e.g.,
ligation reactions and protein or peptide affinity binding
reactions).
[0032] These methods are based on solid-phase extraction
techniques, and can be desirably incorporated into high throughput,
low volume, integrated microfluidic devices, particularly those
being developed for PCR and DNA sequencing. Some desirable
qualities of a solid-phase extraction method for PCR or DNA
sequencing reaction clean-up for use in an integrated microfluidic
device include, for example: 1) the use of high surface area to bed
volume ratio porous or nonporous materials that can be incorporated
into a spin column, titer well plate, or a well or channel within a
flow-through microfluidic device; 2) the use of non-hydrogel based
materials that do not require hydration/swelling and are not prone
to cracking upon dehydration; 3) no need for specially designed
primers or multi-step binding/rinsing/elution protocols; 4) no
volatile or corrosive solvents; 5) no leachables that could
contaminate DNA products or compromise the structure of the device;
and 6) the ability to remove dyes and other residual reactants
while not removing a significant amount of PCR or sequencing
reaction products.
[0033] The methods of the present invention use solid-phase
extraction materials effective for selective removal of negatively
charged small molecules (e.g., molecules having a molecular weight
of less than about 6,000, such as dye terminators), while retaining
the larger product molecules (e.g., sequencing ladders), which are
often negatively charged as well. Herein, "small organic molecules"
refer to molecules in a biological sample mixture, such as a PCR or
sequencing reaction mixture or other amplification reaction
mixture, that are not the desired product molecules. Typically, the
small organic molecules that are removed from biological sample
mixtures are smaller than the desired products. Preferably, the
small organic molecules that are removed from biological sample
mixtures have a molecular weight of less than about 6,000. Such
small molecules tend to adhere (i.e., adsorb, absorb, or otherwise
bind) to the solid-phase extraction materials of the present
invention, whereas molecules with a molecular weight of greater
than about 8,000 generally do not. For molecules of intermediate
molecular weight, the smaller the molecule, the greater the
tendency to adhere (i.e., adsorb, absorb, or otherwise bind),
whereas the larger the molecule, the less the tendency to adhere
(adsorb, absorb, or otherwise bind). Typically, the desired PCR
amplicons have greater than about 50 base pairs and molecular
weights of greater than about 33,000. Typically, the desired
sequencing ladders have greater than about 18 bases and molecular
weights of greater than about 6,000.
[0034] The solid-phase extraction materials are typically in the
form of particles that include a hydrophilic core as the solid
support and a hydrophobic matrix, although this is not a necessary
limitation. Suitable hydrophilic solid supports can be porous and
nonporous; however, porous materials have the advantage of large
surface area to bed volume ratios and are particularly useful in
flow-through applications.
[0035] Suitable solid supports can be in the form of beads,
particles, spheres, films, membranes, frits, foams, microreplicated
articles, monoliths, plates, tubes, dipsticks, strips, pads, disks,
a ceramic surface deposited on an organic film using, for example,
plasma deposition techniques, etc. Solid supports with higher
surface areas enhance contact area, which can improve processing
efficiency. The solid support can be treated for improved
adhesion/surface area by a variety of treatments such as oxygen
plasma, diamond-like glass deposition, corona treatment, e-beam or
uv radiation, heat, as well as other similar techniques.
[0036] Thus, the term "solid support" refers to a porous or
nonporous, water-insoluble material. The surface of the solid
support can be neutral or charged in nature (preferably, neutral or
basic) and is hydrophilic. The solid support can be composed of
inorganic particles such as silica, magnesium sulfate, alumina,
zirconia, titania, diatomaceous earth, molecular sieves such as
zeolites (i.e., sodium and calcium aluminosilicates),
hydroxyapatite, iron oxide, and the like; naturally occurring
organic polymeric materials, particularly cellulosic materials and
materials derived from cellulose, such as fiber-containing papers,
e.g., filter paper, chromatographic paper, etc.; synthetic or
modified naturally occurring organic polymers, such as
nitrocellulose, cellulose acetate, polyacrylamide, crosslinked
dextran, amphoteric nylon, and agarose; vitreous materials such as
glass, quartz, ceramics, and silicon nitride; or plastics that are
intrinsically hydrophilic or have been rendered hydrophilic by the
presence of hydrophilic functional groups, such as polyacrylates,
polyethylenes, polypropylenes, polystyrenes, polyvinyl chlorides
modified with hydrophilic functional groups such as carboxyl,
amino, carboxamido, phosphonate, sulfonate, or hydroxyl groups.
These materials can be used by themselves or in conjunction with a
structural support composed of other materials such as glass,
ceramics, metals, plastics, and the like. Other hydrophobic
materials (e.g., carbon particles) can be included with the
hydrophilic particles as well for various applications.
[0037] Preferably, the solid support is in the form of particles.
Preferably, the particles have an average particle size (i.e., the
largest dimension, typically the diameter) of at least about 5
nanometers (nm), and more preferably, at least about 3 micrometers
(i.e., microns or .mu. or .mu.m). Preferably, the particles have an
average particle size of no greater than about 500 microns, and
more preferably, no greater than about 100 microns.
[0038] The surface area of the particles can vary widely depending
on whether the solid support is porous or nonporous. For example,
the average surface area can be about 50 square meters per gram
(m.sup.2/g) to about 200 m.sup.2/g.
[0039] The hydrophobic matrix includes a material that will absorb
less than about 0.5 percent of its weight in water under common
hydrophilicity test conditions. A common measure of hydrophobicity
of polymers is water absorption by the bulk polymer within 24 hours
or at equilibrium as set out in ASTM D570 (standard method to
measure water absorption by polymers). There is, however, no
commonly agreed definition of hydrophobic and hydrophilic
materials. For purposes of this invention, a hydrophobic material
is one absorbing less than 0.5 percent of its weight of water
within 24 hours, and 4 percent or less at equilibrium. The surface
of a solid piece of such a material will typically not wet, and a
water drop placed on such an inclined surface will roll off without
tailing.
[0040] Alternatively, contact angle measurements can be used to
determine the hydrophobicity of a material. Hydrophobic materials
are those that have a solid-liquid contact angle of greater than
about 70.degree..
[0041] Suitable hydrophobic materials include numerous silicones,
such as silicone elastomer, room temperature vulcanizable (RTV)
silicone rubber, heat vulcanizable silicone rubber,
polydimethylsiloxane, poly(vinyl siloxane), silicone-polycarbonate
copolymer, polyvinyl butyral, polyolefins (including
poly-alpha-olefins), fluorinated polymers such as those disclosed
in U.S. Pat. No. 5,380,901 (Antonucci et al.) and perfluorinated
(polyether) urethanes, and block copolymers such as those disclosed
in U.S. Pat. No. 5,834,583 (Hancock et al.). These may or may not
be adhesive formulations.
[0042] Other hydrophobic materials include various adhesive
compositions including pressure sensitive adhesive and structural
adhesives. Such adhesive compositions include acrylates, silicones,
natural or synthetic rubbers (e.g., styrene-containing block
copolymers), epoxies, and the like. Suitable examples are described
in U.S. Pat. Nos. 4,780,367 (Lau et al.), 5,183,705 (Birkholz et
al.), 5,294,668 (Babu), 6,063,838 (Patnode et al.), 6,277,488 (Kobe
et al.), as well as International Publication Nos. WO 00/45180 and
WO 00/68336.
[0043] The hydrophobic material preferably includes a polymer
selected from the group consisting of a silicone, polyvinyl
butyral, polyolefin, fluorinated polymer, acrylate, epoxy, natural
or synthetic rubber, or combinations (e.g., mixtures, copolymers,
terpolymers, etc.) thereof.
[0044] Suitable solid-phase extraction materials can be prepared
using a variety of techniques. For example, for embodiments in
which hydrophilic particles are at least partially embedded within
a layer of an adhesive, the hydrophilic particles can be roll
coated (or they can be spray coated, dip coated, knife coated,
brush coated, or electrostatically deposited, for example) onto a
film having a layer of an adhesive thereon. The construction is
heated to better anchor the particles and to provide a generally
uniform deposition of particles. This processing temperature is
dependent on the type of adhesive. Typically, the more particles
deposited, the more wettable is the surface, which provides better
wicking action, better contact, and often shorter processing
times.
[0045] Preferably, for embodiments in which hydrophilic particles
are at least partially embedded within a layer of an adhesive, the
particles are disposed on a layer of adhesive at a coating density
of at least about 0.1 milligram (mg) per 12 square millimeters
(mm.sup.2) surface area, more preferably at a density of least
about 0.3 mg/12 mm.sup.2 surface area, and most preferably at a
density of least about 0.5 mg/12 mm.sup.2 surface area. Preferably,
the particles are disposed on a layer of adhesive at a coating
density of no greater than about 5 mg/12 mm.sup.2 surface area,
more preferably at a density of no greater than about 1.5 mg/12
mm.sup.2 surface area, even more preferably at a density of no
greater than about 1.3 mg/12 mm.sup.2 surface area, and most
preferably at a density of no greater than about 0.9 mg/12 mm.sup.2
surface area.
[0046] The materials (type of solid support and hydrophobic matrix)
and the coating density are selected to provide selective capture
of small organic molecules, particularly dye-containing molecules.
The solid-phase extraction materials of the present invention
provide sites for relatively strong binding of the residual small
organic molecules (e.g., dye terminators) while repelling larger
negatively charged molecules (e.g., DNA sequencing ladders) based
on charge and size effects, thereby allowing for selective
clean-up.
[0047] The solid-phase extraction materials of the present
invention can be used effectively for purification of nucleic acid
amplicons after the polymerase chain reaction (PCR), for example.
As is well known, PCR allows for analysis of extremely small
amounts of nucleic acid (e.g., DNA). Briefly, a nucleic acid
molecule (e.g., DNA template) is repeatedly synthesized using a
polymerase enzyme (such as Taq DNA polymerase), an excess of two
oligonucleotide primers (capable of flanking the region to be
amplified and acting as a point of initiation of synthesis when
placed under conditions in which synthesis of a primer extension
product that is complementary to a target nucleic acid strand is
induced), and free deoxynucleotide triphosphates (dNTPs, e.g.,
dGTP, dATP, dCTP and dTTP), which results in the amplification of a
particular sequence by a millionfold or more. The resultant
extension or amplification products are typically referred to as
"PCR products" or "PCR amplicons."
[0048] Preferably, the PCR products incorporate a detectable label
or tag, as can other materials in the PCR reaction mixture (e.g.,
primers and dNTPs). Thus, PCR amplification of target nucleic acid
is preferably accomplished by utilizing at least one primer
containing a detectable tag. For example, ultraviolet, visible, or
infrared absorbing tags could be used that would produce specific
ultraviolet, visible, or infrared signals. Examples of a wide
variety of tags (a chemical moiety that is used to uniquely
identify a nucleic acid of interest) are disclosed in International
Publication No. WO 97/27325. Particularly preferred such tags are
fluorescent or chemiluminescent agents. These are typically dye
compounds that emit visible radiation in passing from a higher to a
lower electronic state, typically in which the time interval
between adsorption and emission of energy is relatively short,
generally on the order of about 10.sup.-8 to about 10.sup.-3
second. Suitable fluorescent or chemiluminescent compounds can
include fluorescein, rhodamine, luciferin, as well as a wide
variety of others known to one of skill in the art.
[0049] In clean-up of PCR reaction mixtures after PCR has occurred,
the undesired negatively charged small molecules include residual
primers (labeled or unlabeled), degraded dye molecules (i.e., dye
molecules or fragments thereof severed from the dye-labeled
primers), and dNTPs (labeled or unlabeled). Of these molecules it
is particularly important to remove the primers.
[0050] Preferably, using the methods of the present invention at
least a portion of one or more of these unincorporated materials
can be separated from the PCR products (i.e., removed from the PCR
reaction mixture). Typically, the smaller molecules are removed
(e.g., dNTPs) more easily than the larger molecules (primers). More
preferably, at least about 90% of all the residual primers and/or
at least about 70% of the dNTPs are removed from a PCR reaction
mixture using the methods of the present invention. Even more
preferably, substantially all (i.e., at least about 95%) of one or
more of the residual primers, degraded dye molecules, and dNTPs,
are separated from the desired PCR products. Most preferably,
substantially all (i.e., at least about 95%) of all the residual
primers, degraded dye molecules, and dNTPs, are separated from the
desired PCR products. The level of removal of primers can be
determined by the OLIGREEN ssDNA quantitation reagent (Molecular
Probes, Eugene, Oreg.), high pressure liquid chromatography (HPLC),
and capillary electrophoresis (CE). The level of removal of dNTPs
can be determined by absorbance at 1260 nanometers (nm), HPLC, and
CE.
[0051] Preferably, using the methods of the present invention, at
least about 30% of the desired PCR product (e.g., DNA amplicon) is
recovered from a PCR reaction mixture. More preferably, at least
about 50% of the desired PCR product is recovered from a PCR
reaction mixture. Even more preferably, at least about 70% of the
desired PCR product is recovered from a PCR reaction mixture. Most
preferably, at least about 90% of the desired PCR product is
recovered from a PCR reaction mixture. The level of PCR product
recovery can be determined by Agilent 2100 Bioanalyzer available
from Agilent Technologies, Palo Alto, Calif.
[0052] For certain methods of PCR reaction mixture clean-up, the
clean-up is preferably carried out at room temperature, although
higher temperatures could be used if desired. A typical time for
clean-up is less than about 5 minutes.
[0053] The solid-phase extraction materials of the present
invention can also be used effectively for purification of nucleic
acid (e.g., DNA) sequencing ladders after, for example, Sanger
cycling. As is well known, sequencing, such as Sanger sequencing,
produces a nested set of fragments from a template strand (e.g., a
DNA template) by replicating the template strand to be sequenced.
Briefly, a nucleic acid molecule (e.g., DNA template) of unknown
sequence is combined with a nucleic acid polymerase, a primer, free
deoxynucleotide triphosphates (dNTPs, e.g., dGTP, dATP, dCTP and
dTTP), and one of the four free dideoxynucleotide triphosphates (a
dideoxynucleotide cannot bond to other nucleotides because its 3'
end is modified, thus, when dideoxynucleotides are incorporated,
strand synthesis stops) to produce a random sample of varying
length segments of nucleic acid. Thus, sequencing mixtures contain
salts, enzymes, unincorporated deoxynucleotide triphosphates
(dNTPs), template nucleic acid, primers, and the resultant nucleic
acid sequencing ladders. Various of these materials (e.g., primers
and dNTPs) can be labeled with dye molecules or unlabeled. Such
mixtures also include unincorporated dye-labeled dideoxynucleotide
terminators such as dye-labeled dideoxynucleotide triphosphates
(ddNTPs), which can be hydrolyzed (e.g., treated enzymatically with
a phosphatase such as shrimp alkaline phosphatase to
dephosphorylate residual nucleotide triphosphates) to form
dye-labeled artifacts such as dye-labeled dideoxynucleotide
diphosphates (ddNDPs), dye-labeled dideoxynucleotide monophosphates
(ddNMPs), and dye-labeled dideoxynucleosides. As described in
International Publication No. WO 01/25490, such unincorporated
dye-labeled terminators typically have to be removed from the DNA
sequencing ladders prior to electrophoresis. Herein, the
"dye-labeled terminators" are also referred to as "dye terminators"
and include ddNTPs, ddNDPs, ddNMPs, and dideoxynucleosides.
Particularly preferred such dyes are fluorescent or
chemiluminescent agents and include fluorescein, rhodamine,
luciferin, etc.
[0054] In clean-up of sequencing reaction mixtures after cycling
has occurred, the undesired negatively charged small molecules
include residual primers (labeled or unlabeled), degraded dye
molecules (i.e., dye molecules or fragments thereof severed from
the dye-labeled terminators), dNTPs (labeled or unlabeled), and dye
terminators. Of these, it is particularly important to remove the
dye terminators. Preferably, using the methods of the present
invention at least a portion of one or more of these unincorporated
materials can be separated from the sequencing products (i.e.,
removed from the sequencing reaction mixture). Typically, the
smaller molecules are removed (e.g., dNTPs) more easily than the
larger molecules (primers) and the more highly charged molecules
are removed more easily than the less highly charged molecules
(e.g., the ease of removal decreases from ddNTPs to ddNDPs to
ddNMPs to nucleosides). More preferably, substantially all (i.e.,
at least about 95%) of one or more of the residual primers,
degraded dye molecules, dNTPs, and dye terminators are separated
from the sequencing products. Most preferably, substantially all
(at least about 95%) of all the residual primers, degraded dye
molecules, dNTPs, and ddNTPs are separated from the sequencing
products. Significantly and preferably, using the methods of the
present invention, at least about 95%, more preferably, at least
about 98%, and most preferably, 100%, of all the dye terminators
are separated from sequencing products. Such products can then be
analyzed by sequencing. The level of removal of dye terminators can
be determined by fluorescence, CE, or HPLC.
[0055] Preferably, using the methods of the present invention, at
least about 30% of the desired sequencing product (e.g., DNA
ladder) is recovered from a cycle sequencing reaction mixture. More
preferably, at least about 50% of the desired sequencing product is
recovered from a cycle sequencing reaction mixture. Most
preferably, at least about 70% of the desired sequencing product is
recovered from a cycle sequencing reaction mixture. The level of
product recovery can be determined by CE, for example.
[0056] For certain methods of sequencing reaction mixture clean-up,
the clean-up is preferably carried out at room temperature,
although higher temperatures could be used if desired. A typical
time for clean-up is less than about 5 minutes.
[0057] The solid-phase extraction materials of the present
invention can be incorporated into flow-through devices or
non-flow-through formats. If a non-flow-through format is used, the
reaction mixture can be incubated with or without mixing,
preferably with mixing, for a given period of time and the
resultant supernatant containing at least partially purified
product (e.g., DNA amplicons) can be removed and analyzed.
[0058] Diffusion of small molecules to the solid-phase extraction
material can be improved by providing adequate mixing of the
reactants. This can be accomplished by vortexing, shaking, heating,
sonicating, etc. Providing intimate mixing can result in shorter
times for processing (e.g., clean-up), better product recovery
levels, and/or better reproducibility.
[0059] The solid-phase extraction materials described herein can be
incorporated into a variety of devices, particularly analytical
receptacles. As used herein, analytical receptacles are devices
that receive a sample, reagent, or solvent into one or more
reservoirs, which may or may not designed for filtration. Examples
include assay plate arrays (e.g., microtiter plates), discrete or
continuous (e.g., strip or tape) structures containing a plurality
of wells, channels, or other reservoirs, and arrays of the type
used in 96-well filter plate assemblies (e.g., of the type
described in U.S. Pat. No. 5,620,663 (Aysta et al.)).
[0060] Preferred analytical receptacles, without further
modification, provide an open system of one or more reservoirs
(e.g., wells or channels) to which fluids may be added directly. A
cover film is typically applied along the length and width of an
analytical receptacle to seal the receptacle, preferably the
reservoir(s) of the receptacle, and create a closed system.
Preferably, this results in producing individually sealed
enclosures, which can be substantially continuous or discrete
(i.e., discontinuous) structures.
[0061] A cover film, which acts as a sealing membrane, can include
an adhesive, preferably, a pressure sensitive adhesive, disposed on
a backing (preferably, a transparent backing). The adhesive is
selected such that it adheres well to materials of which
conventional analytical receptacles are made (preferably
polyolefins, polystyrene, polycarbonate, or combinations thereof),
maintains adhesion during high and low temperature storage (e.g.,
about -80.degree. C. to about 200.degree. C.) while providing an
effective seal against sample evaporation, and does not
substantially dissolve in or otherwise react with the components of
the biological sample mixture. Thus, the type of adhesive is not
critical as long as it does not interfere (e.g., bind DNA,
dissolve, etc.) with the removal of unwanted materials from a
biological sample mixture. Preferred adhesives include those
typically used on cover films of analytical devices in which
biological reactions are carried out. These include poly-alpha
olefins and silicones, for example, as described in International
Publication Nos. WO 00/45180 and WO 00/68336.
[0062] The solid-phase extraction material described herein can be
incorporated into the analytical receptacle in a variety of ways.
For example, it can be disposed to the walls of one or more
reservoirs, it can be in the form of a flow-through membrane placed
in one or more reservoirs, it can be disposed to a film (which can
be continuous or discontinuous or in the form of a plurality of
pieces) placed in one or more reservoirs, or it can be disposed on
the cover film.
[0063] The solid-phase extraction material described herein is
particularly well suited for use in a high throughput microfluidic
device resulting in reagent and time savings, as well as
elimination of the need to elute in the conventional sense (i.e.,
washing away the unwanted components from the bound desired
products followed by removing the desired products). Such devices
typically require low bed volume clean-up media for the
purification of small volume reactions. The hydrophilic particles
can be incorporated into a microfluidic device in a variety of
manners.
[0064] In one embodiment, an adhesive-coated cover film of a
microfluidic device can be coated with, preferably pattern coated
with, the hydrophilic particles. This coating, particularly pattern
coating, can be accomplished by a variety of methods such as spray
drying, dip coating, brush coating, knife coating, roll coating,
ink-jet coating, screen printing, electrostatic deposition, etc. An
unpurified biological sample mixture, e.g., a PCR or DNA sequencing
reaction mixture, can be spun into a clean-up chamber containing
the pattern-coated hydrophilic particles on either or both the top
and bottom surfaces of the chamber. The speed of this reaction can
be enhanced by intimate contact of the solution with the chamber
walls by mixing, vortexing, shaking, or through compression of the
walls (made of a compliant material) of the device, etc. The
purified reaction mixture is collected and ready for subsequent
analysis (e.g., by injection into a DNA sequencing instrument).
[0065] In another embodiment, the hydrophilic particles having a
hydrophobic matrix coated thereon can be positioned within a
microfluidic compartment or channel. For example, a device having
at least one process array that includes two connected process
chambers, at least one of the process chambers and/or at least one
volume defined by a connection (i.e., distribution channel) between
at least two process chambers can include the solid-phase material.
In this arrangement, if the solid-phase material includes a porous
material, an unpurified biological sample solution, e.g., PCR or
DNA sequencing reaction mixture, passes through the solid-phase
material, allowing sufficient residence time to trap the
undesirable components (e.g., excess unincorporated dye
terminators). Alternatively, if the solid-phase material includes a
nonporous material, the unpurified biological sample solution
passes by the material. The contact area of the sample with the
solid-phase material can be enhanced upon selection of a
solid-phase material within larger surface area. The purified
reaction mixture is collected and ready for subsequent analysis,
such as occurs, for example, upon injection into a DNA sequencing
instrument.
[0066] Although the methods of the present invention can be used in
a variety of devices, a variety of illustrative embodiments of some
suitable devices may be described in, e.g., U.S. patent application
Ser. No. 09/894,810 filed on Jun. 28, 2001 and entitled ENHANCED
SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS (U.S. Pat.
Application Publication No. 2002/0047003 (Bedingham et al.)) and
U.S. patent application Ser. No. 09/895,010 filed on Jun. 28, 2001
and entitled SAMPLE PROCESSING DEVICES METHODS (U.S. Pat.
Application Publication No. 2002/0064885 (Bedingham et al.)). Other
useable device constructions may be found in, e.g., U.S.
Provisional Patent Application Ser. No. 60/214,508 filed on Jun.
28, 2000 and entitled THERMAL PROCESSING DEVICES AND METHODS; U.S.
Provisional Patent Application Ser. No. 60/214,642 filed on Jun.
28, 2000 and entitled SAMPLE PROCESSING DEVICES, SYSTEMS AND
METHODS; U.S. Provisional Patent Application Ser. No. 60/237,072
filed on Oct. 2, 2000 and entitled SAMPLE PROCESSING DEVICES,
SYSTEMS AND METHODS; U.S. Provisional Patent Application Ser. No.
60/260,063 filed on Jan. 6, 2001 and titled SAMPLE PROCESSING
DEVICES, SYSTEMS AND METHODS; U.S. Provisional Patent Application
Ser. No. 60/284,637 filed on Apr. 18, 2001 and titled ENHANCED
SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS; and U.S. patent
application Ser. No. 09/895,001 filed Jun. 28, 2001 and entitled
SAMPLE PROCESSING DEVICES AND CARRIERS METHODS (U.S. Pat.
Application Publication No. 2002/0048533 (Harms et al.)).
[0067] The methods described herein can be used in a variety of
different processes requiring at least partial removal of dyes or
other organic molecules from biological reaction mixtures contained
in the process arrays of microfluidic devices. Examples of such
processes involve the clean-up of chemical reaction mixtures, e.g.,
nucleic acid amplification, which may or may not also be carried
out in process arrays of the device. Some or all of the required
reagents for clean-up may be present in the device as manufactured,
they may be loaded into the process arrays after manufacture of the
device, they may be loaded in the process arrays just before
introduction of the sample, or they may be mixed with sample before
loading into the process arrays.
[0068] A preferred method involves the use of a device with a
plurality of process arrays such as those illustrated in U.S.
patent application Ser. No. 09/894,810 filed on Jun. 28, 2001 and
entitled ENHANCED SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS
METHODS (U.S. Pat. Application Publication No. 2002/0047003
(Bedingham et al.)). Each of the process arrays includes a number
of chambers (e.g., loading chambers and process chambers such as
reaction chambers or clean-up chambers) that are preferably
arranged generally radially on a device (such that centrifugal
forces can move fluids sequentially from chamber to chamber, for
example). The chambers within each of the arrays are in fluid
communication using channels or other conduits that may, in some
embodiments, include valve structures to control the movement as
desired.
[0069] Using such a device, starting sample material, e.g., lysed
blood cells, is provided in a loading chamber. A filter is
preferably provided to filter the starting sample material as it
moves from the loading chamber to a first process chambers. The
first process chambers preferably include suitable PCR primers as
supplied, e.g., dried down in each of the chambers. Each of the
chambers may include the same primer or different primers depending
on the nature of the investigation being performed on the starting
sample material. One alternative to providing the primers in the
process chambers before loading the sample is to add a suitable
primer to the loading chamber with the starting sample material
(provided that the primer is capable of passing through the filter,
if present).
[0070] After locating the starting sample material and any required
primers in the process chambers, the materials in the process
chambers are thermally cycled under conditions suitable for PCR
amplification of the selected genetic material.
[0071] After completion of the PCR amplification process, the
materials in each of the first process chambers may be moved
through another filter chamber (one filter chamber for each process
chamber) to remove unwanted materials from the amplified materials,
e.g., PCR primers, unwanted materials in the starting sample that
were not removed by filter, etc. The filter chambers contain the
solid-phase extraction materials (e.g., hydrophilic particles)
described above for sample clean-up (e.g., dye removal). The area
in which the solid-phase extraction material is included in such
devices can be a chamber or in the volume defined by a connection
between two chambers or both.
[0072] After clean-up of the sample materials in the filter
chambers, the filtered PCR amplification products from each of the
first process chambers are moved into second process chambers for,
e.g., sequence cycling of the genetic materials amplified in the
first process chambers through appropriate control of the thermal
conditions encountered in second process chambers.
[0073] After completion of the sequence cycling (e.g., Sanger
sequencing) process, the materials in each of the second process
chambers may be moved through another filter chamber (one filter
chamber for each process chamber) to remove unwanted materials from
the sequencing ladders (e.g., sequencing primers, ddNTPs, etc.).
The filter chambers contain the solid-phase extraction materials
(e.g., hydrophilic particles with hydrophobic matrix) described
above for sample clean-up (e.g., dye removal). Again, the
solid-phase extraction material can be in a chamber or between
chambers in a channel.
[0074] The present invention also provides devices for processing
(e.g., clean-up) of sample mixtures. The sample materials may be
located in a plurality of chambers in the device which, in various
aspects, may include one or more of: a reflective layer (e.g., a
metallic layer); baffle structures to enhance cooling during
rotation of the device; capture plugs to capture filtering
materials; valve mechanisms capable of being selectively opened,
thermal indicators for monitoring/controlling the temperatures in
process chambers, absorptive materials in the process chambers to
enhance energy absorption, etc. In various embodiments, the devices
may include reagents, filters, and other sample processing
materials in the process chambers.
[0075] Among the thermal control advantages of the devices of the
present invention are chamber-to-chamber temperature uniformity,
comparable chamber-to-chamber temperature transition rates, and the
increased speed at which thermal energy can be added or removed
from the process chambers. Among the device features than can
contribute to these thermal control advantages are the inclusion of
a reflective layer (e.g., metallic) in the device, baffle
structures to assist in removing thermal energy from the device,
and low thermal mass of the device. By including thermal indicators
in the devices, enhanced control over chamber temperature may be
achieved even as the device is rotated during processing.
[0076] One illustrative device manufactured according to the
principles of the present invention is illustrated in FIG. 1. The
device 10 is preferably in the shape of a circular disk as
illustrated in FIG. 1, although any other shape that can be rotated
could be used in place of the preferred circular disc. The device
10 of FIG. 1 is a multi-layered composite structure including a
substrate, first layer, and a second layer.
[0077] The device includes a plurality of process chambers 50, each
of which defines a volume for containing a sample and any other
materials that are to be thermally cycled with the sample. The
illustrated device 10 includes ninety-six process chambers 50,
although it will be understood that the exact number of process
chambers provided in connection with a device manufactured
according to the present invention may be greater than or less than
ninety-six, as desired.
[0078] The process chambers 50 in the illustrative device 10 are in
the form of wells, although the process chambers in devices of the
present invention may be provided in the form of capillaries,
passageways, channels, grooves, or any other suitably defined
volume. The process chambers 50 are in fluid communication with
distribution channels 60 that, together with the common loading
chamber 62, provide a distribution system for distributing samples
to the process chambers 50. Introduction of samples into the device
10 through the loading chamber 62 may be accomplished by rotating
the device 10 about a central axis of rotation such that the sample
materials are moved outwardly due to centrifugal forces generated
during rotation. Before the device 10 is rotated, the sample can be
introduced into the loading chamber 62 for delivery to the process
chambers 50 through distribution channels 60. The process chambers
50 and/or distribution channels 60 may include ports through which
air can escape and/or features to assist in distribution of the
sample materials to the process chambers 50. Alternatively, it may
be possible to provide a closed distribution system, i.e., a system
in which materials may be introduced through an opening through
which air within the process chambers 50 and/or distribution
channels 60 also escapes during the distribution process. In
another alternative, sample materials could be loaded into the
process chambers 50 under the assistance of vacuum or pressure.
[0079] The process chamber 50, associated distribution channels 60,
and loading chamber 62 all combine to form a number of process
arrays on the device 10, with each process array including one of
the process chambers 50, the distribution channels 60 connecting
the process chamber 50 to the loading chamber 62, and the loading
chamber 62 itself. The process arrays may preferable be arranged
radially on the device 10.
[0080] Referring to FIGS. 2-4, an alternative device 110 with a
different arrangement of process arrays is depicted that can be
used in place of the device 10 of FIG. 1. The device 110 seen in
FIG. 2 includes a number of independent process arrays, each of
which includes distribution channels 160a and 160b connecting a
loading chamber 162 and process chambers 150a, 150b and 150c. The
process arrays on the device 110 are independent in the sense that
the different process arrays are not in fluid communication with
each other as are the process arrays on the device 10 of FIG. 1,
but are, instead, separate and distinct from each other.
[0081] It is preferred that the process arrays be arranged radially
from the center of the device 110. As a result, rotation of the
device can be used to move sample materials successively through
the chambers and distribution channels. The depicted device 110
includes sixteen process arrays, although it will be understood
that devices used in connection with the present invention can
include any desired number of process arrays. Furthermore, although
each of the process arrays of device 110 includes a loading chamber
and three process chambers connected sequentially by distribution
channels, it should be understood that a process array of the
present invention may include as few as two interconnected
chambers.
[0082] FIG. 3 is an enlarged view of one process array on device
110 and FIG. 4 is a cross-sectional view of a portion of the
process array of FIG. 3. Each process array includes a loading
chamber 162 connected to a first process chamber 150a through a
distribution channel 160a. The first process chamber 150a is, in
turn, connected to a second process chamber 150b through a
distribution channel 160b. The second process chamber 150b is
connected to a third process chamber 150c, that, in the depicted
process array, is located furthest from the loading chamber 162. If
materials are to be moved within the process array from the loading
chamber 162 towards the third process chamber 150c, it may be
preferred that the loading chamber 162 be located closer to the
axis of rotation of the device than the process chambers 150a, 150b
or 150c.
[0083] The cross-sectional view of FIG. 4 depicts a number of other
features of one potential construction of a device that could be
used in connection with the present invention. The construction
includes a core 180 in which the features of the device are formed.
One surface 182 of the core 180 may include a cover film 184
attached thereto. The cover film 184 may be of any suitable
construction, although the adhesive cover films described herein
may be preferred.
[0084] The bottom of the process chamber 150a also includes a cover
186 attached to the surface 188 of the core 180 to enclose the
volume of the process chamber 150a. Like the cover 184, it may be
preferred that the cover 186 be attached to and seal with the core
180 using an adhesive, e.g. a pressure sensitive adhesive as
described herein. It may be preferred that the cover 186 be
provided in the form of a metallic layer that enhances thermal
energy transfer into and out of the process chamber 150a. In some
embodiments, the cover 186 may be provided in the form of a
ring-shaped structure as described in, e.g., U.S. patent
application Ser. No. 09/894,810 filed on Jun. 28, 2001 and entitled
ENHANCED SAMPLE PROCESSING DEVICES, SYSTEMS AND METHODS (U.S. Pat.
Application Publication No. 2002/0047003 (Bedingham et al.)).
[0085] The first process chamber 150a includes a valve structure in
the form of a lip 170a that protrudes into the boundaries of what
would otherwise be a generally circular first process chamber 150a.
The lip 170a is in the form of an undercut extension into the
volume of the process chamber 150a as seen in, e.g., FIG. 4. When
an opening is provided in the lip 170a, sample materials in the
process chamber 150a can move into the distribution channel 160b
for delivery to the second process chamber 150b is desired. In the
absence of an opening in the lip 170a, movement of materials into
the second process chamber 150b through distribution channel 160b
is prevented by the lip 170a which otherwise seals against the
cover 184 to prevent the flow of sample materials from the first
process chamber 150a into the distribution channel 160b.
[0086] The lip 170a may preferably include an area 172a of reduced
thickness. This may be seen best in the cross-sectional view of
FIG. 4. When the area 172a is, e.g., pierced or otherwise deformed
to include an opening formed therethrough, any sample materials
located in the volume of the process chamber 150a can move from the
chamber into the distribution channel 160b for delivery to the
second process chamber 150b.
[0087] Although it is not required, the reduced thickness of the
area 172a may provide a number of advantages. It may, for example,
limit the location or locations in which the lip 170a may be easily
pierced to provide the desired opening, i.e., the thicker portions
of the lip 170a surrounding the area 172a may be more resistant to
piercing by any of the techniques that could be used to pierce the
lip 170a to form an opening therethrough. The techniques that could
be used to pierce the lip 170a may include, e.g., mechanical
piercing (using, e.g., a pin, needle, etc.), laser ablation, etc.
Another potential advantage of the area 172a of reduced thickness
is that it can be molded into the core layer 180 along with, e.g.,
the process chambers and distribution channels.
[0088] Although devices such as those described herein may be
well-suited to performing processes such as e.g., PCR, Sanger
sequencing, etc., devices of the invention may be limited to
clean-up of the products of such processes which may be performed
off of the devices.
[0089] Rotation of any device including process arrays such as
those depicted in FIGS. 1-4 may be used to facilitate mixing
through mechanical agitation of the sample materials and any other
materials (e.g., reagents, etc.) present in the process chambers.
The mechanical agitation may be accomplished by oscillating the
device in opposite directions about the axis of rotation. The
oscillations may vary in frequency and/or magnitude depending on a
variety of factors, e.g., the size/shape of the process chambers,
the amount of materials in the process chambers, viscosities,
temperatures, stability of the sample materials, etc. For example,
it may be useful to accomplish mixing by oscillating the device 10
at a frequency of about 1 Hertz (Hz) to about 100 Hertz. The
magnitude of the oscillations may be, e.g., from about 5 degrees to
about 360 degrees.
[0090] The mechanical agitation can be carried out during, for
example, PCR, Sanger cycling, clean-up of the PCR reaction mixture,
clean-up of the sequencing reaction mixture, as well as during
various other processes that can be carried out in the microfluidic
devices described herein. Similarly, mechanical agitation by
rotation, or other means, can be carried out on any of the devices
described herein.
EXAMPLES
[0091] Objects and advantages of this invention are further
illustrated by the following examples, but the particular materials
and amounts thereof recited in these examples, as well as other
conditions and details, should not be construed to unduly limit
this invention.
Example 1
Preparation of Alumina/Adhesive Composite
[0092] A tape with a hydrophobic adhesive was immersed face down in
a bed of ceramic particles. The alumina particles ranged in size
from 3 micrometers (micron or .mu. or .mu.m) to 6.mu. (ICN, Costa
Mesa, Calif.). Pressure was applied to the top surface to achieve
maximum efficiency of particle packing. This assembly was placed in
an oven for 30 minutes at temperatures ranging from 40.degree. C.
to about 120.degree. C. depending on the adhesive. The assembly was
then allowed to cool to room temperature in a chemical hood. Excess
particles were removed using air. The alumina/adhesive composite
was then used for clean-up of unpurified sequencing reaction.
[0093] The adhesive tapes used were SCOTCH Brand 143 Mailing Tape,
Stock Number 34-8501-9760-6, 3M, St. Paul, Minn. 55144; SCOTCH
Brand 3561-C Packaging Tape, Stock Number 34-8506-0549-3, St. Paul,
Minn. 55144; SCOTCH Brand 2020 Masking Tape, Stock Number
CV-0001-8259-8, St. Paul, Minn. 55144; and TIMEMED Brand TSI-501
Autoclave Tape, Fisher Catalog Number 11-875-52, Pittsburgh,
Pa.
[0094] A goniometer (model 100-00 115; Rame-Hart, Inc., Mountain
Lakes, N.J.) was used to measure the contact angle between the
alumina/adhesive composite (solid surface) and a sessile drop of
water (liquid) at ambient temperatures. The alumina/adhesive
composite was laid flat upon a stage and a cross beam of light was
passed through a 5 microliters (.mu.L) water drop placed on the
surface of the material. The solid-liquid (SL) angle was determined
and the values were used to gauge surface property changes of the
adhesive before and after embedding of alumina particles into the
adhesive. Generally, adhesives have a high SL contact angle of
greater than 90.degree.. After introduction of alumina particles
onto the adhesive and heating, the contact angle of water dropped
significantly, typically to between 10.degree. and 40.degree.,
making the surface highly wettable. The heat processing helped to
embed the particles into the adhesive matrix and provide a uniform
surface that resulted in reproducible performance of the composite
material for clean-up of amplification reactions. Scanning electron
microscope (SEM) micrographs were used to investigate the
topography of the alumina/adhesive composite. Micrographs were
taken at 1000.times., 2000.times., and 3000.times. on a series of
alumina particles embedded into various adhesives. Typical scanning
electron micrographs indicated that the particles were uniformly
distributed on the tape.
Example 2
Preparation of a Microfluidic Disk and Sequencing Reaction
Clean-Up
[0095] A simplified microfluidic disk was used that consisted of
eight duplicate processing lanes arranged radially in a laminated
polypropylene disk (80 millimeter (mm) diameter, 0.030 inch thick
(762 .mu.m thick). Each processing lane consisted of a single
combined input and clean-up chamber (circular well, 7.11 mm
diameter, located on a 16.5 mm radius) that was connected to an
output chamber (circular well, 4 mm diameter, located on a 29.0 mm
radius) by a single channel (0.010 inch deep (254 .mu.m deep),
0.015 inch wide (381 .mu.m wide)).
[0096] A 4-mm disk (12 mm.sup.2 surface area) was punched from the
alumina/adhesive composite and adhered onto the clean up chamber of
the microfluidic disc. These 4-mm discs were positioned onto an
adhesive cover film (9795R Advanced Sealing Tape, 3M Medical
Specialties, St. Paul, Minn.) which was laminated onto a
microfluidic disk with the 4-mm disk registered such that it
covered the top and/or bottom clean-up chambers. Ten microliters
(10 .mu.L) quarter strength BIGDYE Terminators v 2.0 (Applied
Biosystems, Inc., Foster City, Calif.) cycle sequencing reaction
mixtures containing 2 .mu.L BIGDYE mix, 200 nanograms (ng) DNA
template, and 1.6 picomoles of a primer were thermocycled according
to manufacturers instructions in a GENEAMP PCR System 9700
thermocycler (Applied Biosystems Inc.). Five microliters (5 .mu.L)
of unpurified sequencing reaction were introduced into the clean-up
chamber and allowed to contact the particles by shaking the disk at
a frequency of 12-14 Hz with an angular displacement of 20 degrees
for 2 to 5 minutes to achieve active mixing of the solution with
the ceramic surface. During this time, the smaller molecules in the
unpurified sequencing reaction mixture (containing dye terminators
and their hydrolysis products, DNA template, Taq polymerase,
buffer, dNTPs, and primer) were bound to the alumina/adhesive
composite and the DNA ladder was left in solution. Removal of the
solution from the clean-up chamber followed by dilution to 10 to 20
.mu.L in sterile water yielded a sample that was ready for further
analysis as described below in Examples 3 and 4.
Example 3
Screening of Alumina/Adhesive Composite for Clean-up of BIGDYE
Terminators v 2.0 Using Capillary Electrophoresis and Reverse Phase
HPLC
[0097] Capillary electrophoresis (CE) and reverse phase high
pressure liquid chromatography (HPLC) were used as analytical tools
to evaluate the performance of materials for clean-up of sequencing
reactions, specifically removal of dye terminators and sequencing
ladder recovery, prior to sequencing analysis by ABI PRISM 3100
Genetic Analyzer (Applied Biosystems, Inc.).
[0098] Capillary Electrophoresis (CE). Capillary electrophoresis
analyses of purified sequencing reactions were done with Beckman
P/ACE MDQ Capillary Electrophoresis instrument (Beckman Coulter,
Fullerton, Calif.) with a fluorescence detector (488 nm excitation,
530 to 700 nm emission) using a 75 micrometer ID, 30 cm long (20 cm
to the detector) fused silica capillary. Runs were performed at 500
volts per centimeter (V/cm) (15 KV total) using 50 nanomolar (mM)
Tris-HCl/1 mM EDTA (pH 8.5) as the running buffer. Sample injection
was done at 690 pascals for 5 seconds. The conditions used gave
good separation of the dye terminators (all the four ddNTPs
corresponding to the four bases, A, T, G, C as one peak with a
retention time of 3.2 minutes) and its degradation products (as one
peak with a retention time of 2.2 minutes) and the combined
sequencing ladder (as one peak with a retention time of 4.1
minutes). Sequencing ladder and dye terminator concentrations were
obtained by integrating the peak area of the various analytes. For
each of the samples, the baseline was subtracted from the analyte
values and the resulting analyte concentration was represented as a
percentage of the starting sequencing reaction. The data revealed
that the dye terminator removal and sequencing ladder recovery was
dependent on time of contact of the solution with the
alumina/adhesive composite and uniform distribution of particles
onto the surface, but not significantly on the type of adhesive or
particle density. For particularly good sequencing data, the
samples fulfilled the following two conditions: 1) dye terminator
removal was greater than 98%; and 2) recovery of sequencing ladder
was at least 30%.
[0099] Reverse Phase HPLC. HPLC analysis of purified sequencing
reactions was performed on a Waters ALLIANCE 2690 (Waters
Corporation, Milford, Mass.) separations module with a Waters 474
Scanning Fluorescence detector and Waters 996 Photodiode array
detector and a C18 column. The mobile phase used was 70%
triethylamine acetate (TEAaC) and 30% acetonitrile with a flow rate
of 0.1 milliliter per minute (mL/min). A 2.5-.mu.L sample of
purified sequencing reaction was injected and the run time was for
15 minutes. Analysis was done using the fluorescence detector and
the conditions used gave good separation of the dye terminators
(all the four ddNTPs corresponding to the four bases, A, T, G, C as
one peak) and their degradation products (as one peak). The dye
terminator concentrations were obtained by integrating the peak
area of the various analytes. For each of the samples, the baseline
was subtracted from the analyte values and the resulting analyte
concentration was represented as a percentage of the starting
sequencing reaction. Again, the data revealed that the dye
terminator removal and sequencing ladder recovery was dependent on
time of contact of the solution with the alumina/adhesive composite
and uniform distribution of particles onto the surface, but not
significantly on the type of adhesive or particle density and to
obtain good sequencing data. For particularly good sequencing data,
the dye terminator removal was greater than 98%. These results
correlated well with the results obtained using capillary
electrophoresis.
Example 4
BIGDYE Terminators v 2.0 Sequencing Reaction Clean-up Using 3-6.mu.
Aumina Particle/Adhesive Composite
[0100] An alumina/adhesive composite using 3-6.mu. particles (ICN)
was prepared as described in Example 1 on various adhesive tapes.
Five microliters (5 .mu.L) of unpurified BIGDYE Terminators v 2.0
quarter strength sequencing reaction was cleaned up using a
microfluidic disk containing the alumina/adhesive composite in the
clean-up chambers as described in Example 2. The purified
sequencing reaction samples were analyzed under standard sequencing
conditions using an ABI PRISM 3100 Genetic Analyzer (Applied
Biosystems, Inc.). The resulting electropherograms were manually
checked for quality (like dye blobs, number of Ns) and compared
against the reference sequence using the BLAST program available
through GenBank.
[0101] In addition, the electropherograms were analyzed using Phred
base calling program (Codoncode Corp., Dedham, Mass.). The Phred
program generates highly accurate, base-specific quality scores and
the quality scores are an ideal tool to assess the quality of
sequences. The Phred quality scores generated for sequencing
reactions purified using alumina/adhesive composites on different
adhesive tapes are shown below in Table 1. The results indicated
that the alumina/adhesive composites gave good sequence data and
was comparable to data generated using CENTRISEP columns (Princeton
Separations, Adelphia, N.J.) which is considered an industry
standard. Average read length of successful reactions was 500 bases
with 97% to 98% base-calling accuracy. TABLE-US-00001 TABLE 1
Scores .gtoreq. Scores .gtoreq. Scores .gtoreq. Classifi- Sample 20
30 40 cation 3-6.mu. SCOTCH 488 380 266 Good Brand 143 Mailing Tape
3-6.mu. TIMEMED 462 339 275 Good Brand TSI-501 Autoclave Tape
3-6.mu. SCOTCH 365 237 163 Good Brand 2020 Masking Tape 3-6.mu.
SCOTCH 504 395 276 Good Brand 3561-C Packaging Tape CENTRISEP 610
418 291 Good
Example 5
BIGDYE Terminators v 2.0 Sequencing Reaction Clean-up Using Scotch
Brand 143 Mailing Tape with Various Size Alumina Particle/Adhesive
Composite
[0102] Alumina/adhesive composites using particles ranging in size
from 3 to 6.mu. to 32 to 63.mu. (ICN) and 20 to 100.mu. (Sigma
Aldrich, St. Louis, Mo.) were prepared on the SCOTCH brand 143
Mailing Tape as described in Example 1. Five microliters (5 .mu.L)
of unpurified BIGDYE Terminators v 2.0 quarter strength sequencing
reaction was cleaned up using a microfluidic disk containing the
alumina/adhesive composites in the clean-up chambers as described
in Example 2. The purified sequencing reaction samples were
analyzed under standard sequencing conditions using an ABI PRISM
3100 Genetic Analyzer, Applied Biosystems, Inc. The resulting
electropherograms were manually checked for quality and base
calling accuracy using BLAST and Phred program as described in
Example 4. The Phred quality scores generated for sequencing
reactions purified using different size alumina particle/adhesive
composite on mailing tape are shown below in Table 2. The results
indicated that the alumina/adhesive composites gave good sequence
data and was comparable to data generated using CENTRISEP columns.
Average read length of successful reactions was 500 to 600 bases
with 97% to 98% base-calling accuracy. TABLE-US-00002 TABLE 2
Scores .gtoreq. Scores .gtoreq. Scores .gtoreq. Classifi- Sample 20
30 40 cation 3-6.mu. 511 400 294 Good 7-12.mu. 534 416 297 Good
10-18.mu. 508 389 294 Good 18-32.mu. 530 396 301 Good 32-63.mu. 505
368 244 Good 50-200.mu. neutral 494 350 233 Good 100.mu. neutral
500 359 228 Good 100.mu. acidic 426 328 271 Good 100.mu. basic 549
412 338 Good CENTRISEP 638 436 330 Good
Example 6
Polymerase Chain Reaction (PCR) Clean-up Using Alumina/Adhesive
Composite
[0103] Briefly, a typical PCR reaction contains 200 nM of each of
the two primers and 200 .mu.M of each of the four dNTPs (dGTP,
dATP, dCTP, and dTTP). Most of the residual primers and dNTPs after
a thermocycling reaction need to be removed as the residual dNTPs
and primers can interfere in subsequent down-stream applications
such as sequencing reaction. At the same time sufficient amount of
PCR product needs to be recovered for further processing. The
ability of alumina/adhesive composites to remove primers and dNTPs
and recover PCR amplicons and clean-up a PCR reaction for further
processing was analyzed.
[0104] Primer removal: A known amount (1 to 10 picomoles) of an
oligonucleotide (M13/PUC sequencing primer (-47) (24-mer), New
England Biolabs, Beverly, Mass.) in 5 .mu.L of water (distilled,
deionized, and autoclaved) was added to a 32-63.mu. alumina
particles coated on the SCOTCH Brand 3561-C Packaging Tape
(occupying a space of 4 mm diameter) adhered to the clean-up
chamber of the microfluidic disk as described in Example 2. The
solution was allowed to contact the particles by shaking the disk
at a frequency of 12-14 Hz with an angular displacement of 20
degrees for 2 to 5 minutes to achieve active mixing of the solution
with the alumina/adhesive composite surface and the clean solution
was removed from the clean-up chambers for further analysis. The
amount of primers remaining in the solution was determined using
the OLIGREEN ssDNA quantitation reagent (Molecular Probes, Eugene,
Oreg.). This material was very effective in removing about 80% of
the primers even at a concentration of 10 picomoles within 5
minutes of incubation.
[0105] dNTP removal: A known amount of dNTPs (8 nanomoles with
equal amounts of each of the four dNTPs) in 5 .mu.L of water
(distilled, deionized, and autoclaved) was added to a 32-63.mu.
alumina particles coated on the SCOTCH brand 3561-C Packaging Tape
(occupying a space of 4 mm diameter) adhered to the clean-up
chamber of the microfluidic disk as described in Example 2. The
solution was allowed to contact the particles by shaking the disk
at a frequency of 12-14 Hz with an angular displacement of 20
degrees for 2 to 5 minutes to achieve active mixing of the solution
with the alumina/adhesive composite surface and the clean solution
was removed from the clean-up chambers for further analysis. The
amount of dNTPs remaining in the solution was determined using
absorbance at 260 nm in a spectrophotometer. Again, this material
was effective in removing about 80% of the dNTPs within 5 minutes
of shaking.
[0106] PCR product purification and recovery in a microfluidic
disk: A known amount of PCR amplicon (10 to 17 ng) ranging in size
from 150 to 930 base pairs (bp) in 5 .mu.L of water (distilled,
deionized, and autoclaved) was added to a 32-63.mu. alumina
particles/adhesive composite on the SCOTCH Brand 3561-C Packaging
Tape (occupying a space of 4 mm diameter) adhered to the clean-up
chamber of a microfluidic disk as described in Example 2. The
solution was allowed to contact the particles by shaking the disk
at a frequency of 12-14 Hz with an angular displacement of 20
degrees for 2 to 5 minutes to achieve active mixing of the solution
with the alumina/adhesive composite surface and the clean solution
was removed from the clean-up chambers for further analysis. The
amount of PCR amplicon remaining in the supernatant was determined
by Agilent 2100 Bioanalyzer using a DNA 7500 lab chip kit (Agilent
Technologies). The 32-63.mu. alumina particles/adhesive composite
gave good recovery, a minimum of 90%, independent of the size of
the PCR amplicon. The purified PCR amplicons were used for
sequencing and the sequencing reactions were purified with 3-6.mu.
alumina particle/adhesive composite on SCOTCH Brand 143 Mailing
Tape and analyzed by ABI PRISM 3100 Genetic Analyzer under standard
sequencing conditions (Applied Biosystems, Inc.). The resulting
electropherograms were evaluated as described in Example 4. The
electropherograms gave good sequencing data and yielded Phred
scores of >20 for 500 to 600 bases (for larger amplicons).
[0107] Patents, patent applications, and publications disclosed
herein are hereby incorporated by reference (in their entirety) as
if individually incorporated. It is to be understood that the above
description is intended to be illustrative, and not restrictive.
Various modifications and alterations of this invention will become
apparent to those skilled in the art from the foregoing description
without departing from the scope of this invention, and it should
be understood that this invention is not to be unduly limited to
the illustrative embodiments set forth herein.
* * * * *