U.S. patent application number 09/792297 was filed with the patent office on 2002-01-31 for pcr compatible nucleic acid sieving medium.
Invention is credited to Mehta, Tammy Burd.
Application Number | 20020012971 09/792297 |
Document ID | / |
Family ID | 26886426 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012971 |
Kind Code |
A1 |
Mehta, Tammy Burd |
January 31, 2002 |
PCR compatible nucleic acid sieving medium
Abstract
Sieving mediums comprising less than about 0.5% polymer, less
than about 0.4% polymer, and 0.35% polymer or less are used to
perform nucleic acid separations and PCR. The low polymer
concentration does not inhibit PCR reactions and is sufficient for
performing nucleic acids separations. Microfluidic devices are used
to perform nucleic acids separations and PCR reactions in the
sieving mediums described.
Inventors: |
Mehta, Tammy Burd; (San
Jose, CA) |
Correspondence
Address: |
LAW OFFICES OF JONATHAN ALAN QUINE
P O BOX 458
ALAMEDA
CA
94501
|
Family ID: |
26886426 |
Appl. No.: |
09/792297 |
Filed: |
February 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60190773 |
Mar 20, 2000 |
|
|
|
Current U.S.
Class: |
435/91.2 ;
435/287.2; 435/6.19 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12N 15/1006 20130101; C12Q 1/686 20130101; C12Q 2565/137 20130101;
C12Q 2565/629 20130101; C12N 15/101 20130101 |
Class at
Publication: |
435/91.2 ; 435/6;
435/287.2 |
International
Class: |
C12Q 001/68; C12P
019/34; C12M 001/34 |
Claims
What is claimed is:
1. A microfluidic device for performing PCR and nucleic acid
separations, the device comprising at least one microscale channel
and a sieving medium, which sieving medium is disposed within the
at least one microscale channel and comprises a polymer solution,
which polymer solution comprises less than about 0.5% polymer.
2. The microfluidic device of claim 1, wherein the polymer solution
comprises less than about 0.4% polymer.
3. The microfluidic device of claim 1, wherein the polymer solution
comprises about 0.35% polymer or less.
4. The microfluidic device of claim 1, wherein the polymer solution
comprises acrylamide.
5. The microfluidic device of claim 4, wherein the acrylamide
comprises linear acrylamide, polyacrylamide,
polydimethylacrylamide, or polydimethylacrylamide/coacrylic
acid.
6. The microfluidic device of claim 1, wherein the polymer solution
comprises agarose, methyl cellulose, polyethylene oxide,
hydroxycellulose, or hydroxy ethyl cellulose.
7. The microfluidic device of claim 1, further comprising one or
more proteins, nucleic acids, PCR reaction components, or PCR
products disposed within the at least one microfluidic channel.
8. The microfluidic device of claim 7, wherein the PCR reaction
components comprise one or more of: a thermostable DNA polymerase,
a plurality of nucleotides, a nucleic acid template, a primer which
hybridizes to the nucleic acid template, or Mg.sup.++.
9. A method of separating polynucleotides, the method comprising:
(i) providing two or more polynucleotides; (ii) providing a sieving
medium, which sieving medium comprises a polymer solution, which
polymer solution comprises less than about 0.5% polymer; (iii)
allowing the two or more polynucleotides to migrate through the
sieving medium, thereby separating the two or more
polynucleotides.
10. The method of claim 9, wherein the polynucleotides comprise one
or more PCR products, RNA, or DNA.
11. The method of claim 9, wherein the polymer solution comprises
less than about 0.4% polymer.
12. The method of claim 9, wherein the polymer solution comprises
about 0.35% polymer or less.
13. The method of claim 9, wherein the polymer solution comprises
acrylamide.
14. The method of claim 13, wherein the acrylamide comprises linear
acrylamide, polyacrylamide, polydimethylacrylamide, or
polydimethylacrylamide/coacrylic acid.
15. The method of claim 9, wherein the polymer solution comprises
agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or
hydroxy ethyl cellulose.
16. The method of claim 9, further comprising introducing the
sieving medium into a microfluidic channel and allowing the two or
more polynucleotides to migrate through the sieving medium in the
microfluidic channel.
17. The method of claim 9, wherein the separating comprises
electrophoretically separating the two or more polynucleotides.
18. A method of performing PCR and separating one or more PCR
products, the method comprising: (i) mixing one or more PCR
reaction components with a sieving medium to provide a PCR sieving
medium, wherein the sieving medium comprises a polymer solution,
which polymer solution comprises less than about 0.5% polymer; and
(ii) thermocycling the PCR sieving medium to produce one or more
PCR products; and, (iii) separating the one or more PCR products by
flowing the one or more PCR products through the sieving
medium.
19. The method of claim 18, wherein the polymer solution comprises
less than about 0.4% polymer.
20. The method of claim 19, wherein the polymer solution comprises
about 0.35% polymer or less.
21. The method of claim 18, wherein the polymer solution comprises
acrylamide.
22. The method of claim 21, wherein the acrylamide comprises linear
acrylamide, polyacrylamide, polydimethylacrylamide, or
polydimethylacrylamide/coacrylic acid.
23. The method of claim 28, wherein the polymer solution comprises
agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or
hydroxy ethyl cellulose.
24. The method of claim 18, wherein the one or more PCR reaction
components comprise one or more of: a thermostable DNA polymerase,
a plurality of nucleotides, a nucleic acid template, a primer which
hybridizes to the nucleic acid template, or Mg.sup.++.
25. The method of claim 18, comprising mixing the PCR reaction
components with the sieving medium in a microfluidic channel.
26. The method of claim 25, further comprising separating the one
or more PCR products by flowing the one or more PCR products
through the sieving medium in the microfluidic channel.
27. The method of claim 26, wherein separating comprises
electrophoretically separating.
28. A nucleic acid sieving medium, which medium comprises one or
more polynucleotides, one or more PCR reagents, and a polymer
solution, which polymer solution comprises less than about 0.5%
polymer.
29. The sieving medium of claim 28, wherein the polymer solution
comprises less than about 0.4% polymer.
30. The sieving medium of claim 29, wherein the polymer solution
comprises about 0.35% polymer or less.
31. The sieving medium of claim 28, wherein the polymer solution
comprises acrylamide.
32. The sieving medium of claim 31, wherein the acrylamide
comprises linear acrylamide, polyacrylamide,
polydimethylacrylamide, or polydimethylacrylamide/coacrylic
acid.
33. The sieving medium of claim 28, wherein the polymer solution
comprises agarose, methyl cellulose, polyethylene oxide,
hydroxycellulose, or hydroxy ethyl cellulose.
34. The sieving medium of claim 28, wherein the one or more PCR
reaction components comprise one or more of: a thermostable DNA
polymerase, a plurality of nucleotides, a nucleic acid template, a
primer which hybridizes to the nucleic acid template, or
Mg.sup.++.
35. The sieving medium of claim 28, wherein the one or more
polynucleotides comprise DNA, RNA, or PCR products.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn. 119(e) and any other applicable
statute or rule, the present application claims benefit of and
priority to U.S. Ser. No. 60/190,773, entitled "PCR COMPATIBLE
NUCLEIC ACID SIEVING MEDIUM," filed Mar. 20, 2000 by Burd
Mehta.
BACKGROUND OF THE INVENTION
[0002] Manipulating fluidic reagents and assessing the results of
reagent interactions are central to chemical and biological
science. Manipulations include mixing fluidic reagents, assaying
products resulting from such mixtures, separation or purification
of products and reagents, and the like. A single experiment may
involve hundreds of fluidic manipulations, product separations,
recording processes and the like, each of which involve different
types of laboratory equipment and conditions.
[0003] One particularly labor intensive biochemical series of
laboratory fluidic manipulations is nucleic acid synthesis and
analysis. A variety of in vitro amplifications methods for
biochemical synthesis of nucleic acids are available, such as the
polymerase chain reaction (PCR). See, Mullis et al., (1987) U.S.
Pat. No. 4,683,202 and PCR protocols: A Guide to Methods and
Applications (Innis et al. eds., Academic Press Inc. San Diego
Calif. (1990). PCR methods typically require the use of specialized
machinery for performing thermocycling reactions for DNA synthesis
followed by the use of special machinery for the electrophoretic
analysis of synthesized nucleic acids.
[0004] Various strategies have been used to increase laboratory
throughput. For example, microscale devices for high throughput
mixing and assaying small fluid volumes have been developed. See,
e.g., Parce et al., U.S. Pat. No. 5,942,443, "High Throughput
Screening Assay Systems in Microscale Devices," which provides
pioneering technology related to microscale devices. In particular,
U.S. Ser. No. 09/093,832 by Burd Mehta et al., "Microfluidic Matrix
Localization Apparatus and Methods," provides methods of performing
PCR and nucleic acid separations in the same microfluidic
device.
[0005] Improved methods for performing PCR and nucleic acid
separations including improved sieving mediums are desirable,
particularly those which take advantage of high-throughput, low
cost microfluidic systems. The present invention provides these and
other features by providing nucleic acid sieving mediums, methods
of performing PCR and nucleic acid separations along with high
throughput microscale systems and many other features that will be
apparent upon complete review of the following disclosure.
SUMMARY OF THE INVENTION
[0006] The present invention provides nucleic acid sieving mediums,
microfluidic devices, and methods for performing nucleic acid
separations and PCR. The sieving mediums provided are compatible
with both nucleic acid separations and PCR because they provide
baseline nucleic acid separation and do not inhibit PCR.
[0007] In one aspect, a microfluidic device for performing both PCR
and nucleic acid separations is provided. The device comprises at
least one microscale channel and a sieving medium. The sieving
medium is disposed within the at least one microscale channel and
comprises a polymer solution, which polymer solution comprises less
than about 0.5% polymer, less than about 0.4% polymer, or about
0.35% polymer or less.
[0008] Typical polymers include acrylamide, such as linear
acrylamide, polyacrylamide, polydimethylacrylamide,
polydimethylacrylamide/coacrylic acid, and the like. Other polymers
include, but are not limited to, agarose, methyl cellulose,
polyethylene oxide, hydroxycellulose, hydroxy ethyl cellulose, and
the like.
[0009] The devices also optionally comprise one or more proteins,
nucleic acids, PCR reaction components, or PCR products disposed
within the at least one microfluidic channel. PCR reaction
components include, but are not limited to, a polymerase, e.g., a
thermostable DNA polymerase, a plurality of nucleotides, a nucleic
acid template, a primer which hybridizes to the nucleic acid
template, and Mg.sup.++.
[0010] In a second aspect, methods of separating polynucleotides
are provided. In one embodiment, the method comprises providing two
or more polynucleotides and a sieving medium. The polynucleotides
typically comprise one or more PCR products, RNA, or DNA. The
sieving medium typically comprises a polymer solution as described
above. The two or more polynucleotides migrate through the sieving
medium, thereby separating the two or more polynucleotides.
[0011] In another embodiment, the sieving medium is introduced into
a microfluidic channel and the two or more polynucleotides migrate
through the sieving medium in the microfluidic channel. For
example, the two or more polynucleotides are optionally separated
by electrophoresis in the sieving medium.
[0012] In a third aspect, methods of performing PCR and separating
one or more PCR products are provided. The methods comprise mixing
one or more PCR reaction component as described above with a
sieving medium to provide a PCR sieving medium, wherein the sieving
medium comprises a polymer solution as described above. The PCR
sieving medium is then thermocycled to produce one or more PCR
products, which are separated by flowing in the sieving medium.
[0013] In another embodiment, PCR is performed in a microfluidic
device by mixing the PCR reaction components with the sieving
medium in a microfluidic channel. The one or more PCR products are
separated, e.g., electrophoretically, by flowing the one or more
PCR products through the sieving medium in the microfluidic
channel.
[0014] In a fourth aspect, nucleic acid sieving mediums are
provided. The sieving mediums comprise one or more polynucleotides,
such as DNA, RNA, PCR products, or the like, one or more PCR
reagents as described above, and a polymer solution as described
above.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIG. 1 is a schematic drawing of a microfluidic device for
performing PCR and nucleic acid separations.
[0016] FIG. 2 is a schematic drawing of a microchannel for joule
heating.
[0017] FIG. 3 is a graph providing nucleic acid separation data
obtained in a sieving medium comprising 0.35% polymer.
DETAILED DISCUSSION OF THE INVENTION
[0018] DNA separations are normally performed in sieving mediums
that have a certain concentration of sieving medium to achieve
separation of various sizes of DNA fragments. The concentration of
polymers used in sieving media typically inhibits the polymerase
chain reaction (PCR), thereby preventing the two assays from being
carried out in one medium. The present invention provides a mixture
compatible with PCR that also serves as a DNA separation medium.
The sieving mediums of the present invention comprise lower
concentrations of polymer than are normally used to achieve DNA
separation. The lower concentrations of polymer do not inhibit PCR
and still provide separation of polynucleotides.
[0019] A typical sieving polymer concentration used in DNA
separations is about 3%, e.g., 3% acrylamide. This concentration is
known to inhibit polymerase chain reactions (PCR). However, lower
levels of the same polymer are not inhibitory. The present
invention provides a PCR compatible mixture with a low amount of
polymer used to suppress electroosmotic flow in the channels of a
microfluidic device. The concentration of polymer provided in the
present invention is typically less than about 0.5%, more typically
less than about 0.4% and preferably about 0.35% or less. Since this
is below the usual threshold of sieving, the polymer here was
initially used as an agent to eliminate bulk movement of the fluid
by acting as a dynamic coating for the channel walls.
[0020] However, the present sieving medium also provided nucleic
acid separations in the same low polymer medium in which PCR was
performed. While, the exact mechanism of DNA sieving is not known,
one advantage of the present medium is to provide a single fluid
compatible with PCR and nucleic acid separations. Thus, the present
invention provides, with a relatively simple loading or fabrication
procedure, multi-step assays, e.g., PCR and subsequent product
separation, using the present sieving medium.
[0021] The present invention provides a nucleic acid sieving medium
compatible with nucleic acid separations and PCR as well as devices
for performing nucleic acid separations and PCR, e.g., devices
comprising a sieving medium with a low polymer concentration.
Methods of separating nucleic acids and performing PCR using low
concentration sieving mediums are also provided.
[0022] I. PCR Compatible Sieving Mediums
[0023] A nucleic acid sieving medium that is compatible with PCR
and nucleic acid separations is provided. The sieving medium
comprises a low concentration of polymer, which low concentration
does not inhibit PCR. Typically DNA is separated in sieving mediums
with higher polymer concentrations than those provided herein.
However, nucleic acids are optionally separated using the lower
polymer concentrations provided herein thereby providing methods
for performing nucleic acid separation and PCR in the same
medium.
[0024] The sieving medium typically comprises one or more
polynucleotides, one or more PCR reagents, and a polymer solution,
which polymer solution comprises less than about 0.5% polymer, less
than about 0.4% polymer, or about 0.35% polymer or less. The
polymer solution is thereby used to provide PCR and nucleic acid
separations in the same matrix. In some embodiments, the polymer
solution is eliminated and optionally replaced with an
electroosmotic flow suppressor.
[0025] The one or more PCR reaction components comprise one or more
of: a thermostable polymerase, a thermostable DNA polymerase, a
plurality of nucleotides, a nucleic acid template, a primer which
hybridizes to the nucleic acid template, Mg.sup.++, and the like.
The one or more polynucleotides typically comprise DNA, RNA, or PCR
products.
[0026] Typical polymer solutions of the invention comprise low
concentrations of one or more of the following: acrylamide,
agarose, methyl cellulose, polyethylene oxide, hydroxycellulose,
hydroxy ethyl cellulose, or the like. Combinations of any of these
polymers are also optionally used. Various types of acrylamide are
used, including, but not limited to, linear acrylamide,
polyacrylamide, polydimethylacrylamide,
polydimethylacrylamide/coacrylic acid, or the like.
[0027] A wide variety of alternative sieving mediums are available,
and are optionally used in methods of the invention, e.g., at low
concentrations to provide a medium compatible with both PCR and
nucleic acid separations. For example, a variety of sieving
matrixes and the like are available from Supelco, Inc. (Bellefonte,
Pa.; see, 1997 Suppleco catalogue). Common matrixes which are
useful in the present invention include those generally used in low
pressure liquid chromatography, gel electrophoresis and other
liquid phase separations; matrix materials designed primarily for
non-liquid phase chromatography are also useful in certain
contexts, as the materials often retain separatory characteristics
when suspended in fluids. For a discussion of electrophoresis see,
e.g., Weiss (1995) Ion Chromatography VCH Publishers Inc.; Baker
(1995) Capillary Electrophoresis John Wiley and Sons; Kuhn (1993)
Capillary Electrophoresis: Principles and Practice Springer Verlag;
Righetti (1996) Capillary Electrophoresis in Analytical
Biotechnology CRC Press; Hill (1992) Detectors for Capillary
Chromatography John Wiley and Sons; Gel Filtration: Principles and
Methods (5th Edition) Pharmacia; Gooding and Regnier (1990) HPLC of
Biological Macromolecules: Methods and Applications (Chrom. Sci.
Series, volume 51) Marcel Dekker and Scott (1995) Techniques and
Practices of Chromatography Marcel Dekker, Inc.
[0028] Alternate separation matrix media include, but are not
limited to, low pressure liquid chromatography media include, e.g.,
non-ionic macroreticular and macroporous resins which adsorb and
release components based upon hydrophilic or hydrophobic
interactions such as Amberchrom resins (highly cross-linked
styrene/divinylbenzene copolymers suitable for separation of
peptides, proteins, nucleic acids, antibiotics,
phytopharmacologicals, and vitamins); the related Amberlite XAD
series resins (polyaromatics and acrylic esters) and amberchroms
(polyaromatic and polymethacrylates) (manufactured by Rohm and
Haas, available through Suppleco); Diaion (polyaromatic or
polymethacrylic beads); Dowex (polyaromatics or substituted
hydrophilic functionalized polyaromatics) (manufactured by Dow
Chemical, available through Suppleco); Duolite (phenol-formaldehyde
with methanolic functionality), MCI GEL sephabeads, supelite DAX-8
(acrylic ester) and Supplepak (polyaromatic) (all of the preceding
materials are available from Suppleco). For a description of uses
for Amberlite and Duolite resins, see, Amberlite/Duolite Anion
Exchange Resins (Avaliable from Suppleco, Cat No. T412141). Gel
filtration chromatography matrixes are also suitable, including
sephacryl, sephadex, sepharose, superdex, superose, toyopearl,
agarose, cellulose, dextrans, mixed bead resins, polystyrene,
nuclear resins, DEAE cellulose, Benzyl DEA cellulose, TEAE
cellulose, and the like (Suppleco).
[0029] Other electrophoresis media include silica gels such as
Davisil Silica, E. Merck Silica Gel, Sigma-Aldrich Silica Gel (all
available from Suppleco) in addition to a wide range of silica gels
available for various purposes as described in the Aldrich
catalogue/handbook (Aldrich Chemical Company (Milwaukee, Wis.)).
Preferred gel materials include agarose based gels, various forms
of acrylamide based gels, Genescan polymers (reagents available
from, e.g., Suppleco, SIGMA, Aldrich, SIGMA-Aldrich and many other
sources), colloidial solutions such as protein colloids (gelatins)
and hydrated starches. Various forms of gels are discussed further
below.
[0030] A variety of affinity media for purification and separation
of molecular components are also available, including a variety of
modified silica gels available from SIGMA, Aldrich and
SIGMA-Aldrich, as well as Suppleco, such as acrylic beads, agarose
beads, cellulose, sepharose, sepharose CL, toyopearl, or the like
chemically linked to an affinity ligand such as a biological
molecule. A wide variety of activated matrixes, amino acid resins,
avidin and biotin resins, carbohydrate resins, dye resins,
glutathione resins, hydrophobic resins, immunochemical resins,
lectin resins, nucleotide/coenzyme resins, nucleic acid resins, and
specialty resins are available, e.g., from Suppleco, SIGMA, Aldrich
or the like. See also, Hermanson et al. (1992) Immobilized Affinity
Ligand Techniques Academic Press and optionally used in the
channels of the invention.
[0031] Other media commonly used in chromatography are also
adaptable to the present invention, including activated aluminas,
carbopacks, carbosieves, carbowaxes, chromosils, DEGS, Dexsil,
Durapak, Molecular Sieve, OV phases, pourous silica, chromosorb
series packs, HayeSep series, Porapak series, SE-30, Silica Gel,
SP-1000, SP-1200, SP-2100, SP-2250, SP-2300, SP2401, Tenax, TCEP,
supelcosil LC-18-S and LC-18-T, Methacrylate/DVBm,
polyvinylalcohols, napthylureas, non-polar methyl silicone,
methylpolysiloxane, poly (ethylene glycol) biscyanopropyl
polysiloxane and the like.
[0032] Other types of separation matrices are also optionally used
and discussed in U.S. patent application Ser. No. 09/093,832 filed
Jun., 8, 1998, entitled "Microfluidic Matrix Localizations
Apparatus and Methods," by Mehta and Kopf-Sill. For a review of
chromatography techniques and media, see, e.g., Affinity
Chromatography--A Practical Approach, Dean et al., (Eds.) IRL
Press, Oxford (1985); and, Chromatographic Methods, 5.sup.th
Edition, Braithwaite et al., (1996).
[0033] Many of the materials used to provide the sieving mediums of
the invention are supplied in a liquid or fluidic phase and then
polymerized to provide a sieving matrix. In one embodiment, the
fluid polymerizes upon exposure to light (i.e., the fluid comprises
a "photopolymerizable" polymer). The fluid is then selectively
exposed to light (e.g., using photomasking techniques) in those
regions where a polymerized gel is desired. Unpolymerized fluid is
then optionally washed out of the unselected regions of the
microfluidic device, or into a waste reservoir using electrokinetic
flow or pressure.
[0034] A wide variety of free-radical polymerizable monomers
photopolymerize to form gels, or can be made photopolymerizeable by
the addition of, e.g., energy transfer dyes. For example,
free-radical polymerizable monomers can be selected from acrylate,
methacrylate and vinyl ester functionalized materials. They can be
monomers and/or oligomers such as (meth)acrylates
(meth)acrylamides, acrylamides, vinyl pyrrolidone and azalactones.
Such monomers include mono-, di-, or poly-acrylates and
methacrylates such as methyl acrylate, methyl methacrylate, ethyl
acrylate, isopropyl methacrylate, isooctyl acrylate, isobornyl
acrylate, isobornyl methacrylate, acrylic acid, n-hexyl acrylate,
stearyl acrylate, allyl acrylate, glycerol diacrylate, glycerol
triacrylate, ethylene glycol diacrylate, diethyleneglycol
diacrylate, triethyleneglycol dimethacrylate, 1,6-hexanediol
diacrylate, 1,3-propanediol diacrylate, 1,3-propanediol
dimethacrylate, trimethanol triacrylate, 1,2,4-butanetriol
trimethylacrylate, 1,4-cyclohexanediol diacrylate, pentaerythritol
triacrylate, pentaerythritol tetraacrylate, pentaerythritol
tetramethacrylate, sorbitol hexacrylate,
bis[1-(2-acryloxy)]-p-ethoxyphenyl-dimethylmethane,
bis[1-(3-acryloxy-2-hydroxy)]-propoxyphenyl dimethylmethane,
tris-hydroxyethyl isocyanurate trimethacrylate; the bis-acrylates
and bis-methacrylates of polyethylene glycols of molecular weight
200-500, copolymerizable mixtures of acrylated monomers, acrylated
oligomers, PEG diacrylates, etc. Strongly polar monomers such as
acrylic acid, acrylamide, itaconic acid, hydroxyalkyl acrylates, or
substituted acrylamides or moderately polar monomers such as
N-vinyl-2-pyrrolidone, N-vinyl caprolactam, and acrylonitrile are
also useful.
[0035] Proteins such as gelatin, collagen, elastin, zein, and
albumin, whether produced from natural or recombinant sources,
which are made by free-radical polymerization by the addition of
carbon-carbon double or triple bond-containing moieties, including
acrylate, diacrylate, methacrylate, ethacrylate, 2-phenyl acrylate,
2-chloro acrylate, 2-bromo acrylate, itaconate, oliogoacrylate,
dimethacrylate, oligomethacrylate, acrylamide, methacrylamide,
styrene groups, and other biologically acceptable
photopolymerizable groups, can also be used, e.g., in low
concentration, to form sieving matrixes.
[0036] Dye-sensitized polymerization is well known in the chemical
literature. For example, light from an argon ion laser (514 nm), in
the presence of an xanthin dye and an electron donor, such as
triethanolamine, to catalyze initiation, serves to induce a free
radical polymerization of acrylic groups in a reaction mixture
(Neckers, et al., (1989) Polym. Materials Sci. Eng., 60:15;
Fouassier, et al., (1991) Makromol. Chem., 192:245-260). After
absorbing laser light, the dye is excited to a triplet state. The
triplet state reacts with a tertiary amine such as the
triethanolamine, producing a free radical which initiates a
polymerization reaction. Polymerization is extremely rapid and is
dependent on the functionality of the composition, its
concentration, light intensity, and the concentration of dye and,
e.g., amine.
[0037] Dyes are also optionally used which absorb light having a
frequency between 320 nm and 900 nm, form free radicals, are water
soluble, etc. There are a large number of photosensitive dyes that
are optionally used to optically initiate polymerization, such as
ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy-2-phenyl
acetophenone, 2-methoxy,2-phenylaceto- phenone, camphorquinone,
rose bengal, methylene blue, erythrosin, phloxime, thionine,
riboflavin, methylene green, acridine orange, xanthine dye, and
thioxanthine dyes.
[0038] Cocatalysts useful with photoinitiating dyes are typically
nitrogen based compounds capable of stimulating a free radical
reaction. Primary, secondary, tertiary or quaternary amines are
suitable cocatalysts, as are nitrogen atoms containing
electron-rich molecules. Cocatalysts include triethanolamine,
triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl
benzylamine, dibenzyl amine, N-benzyl ethanolamine, N-isopropyl
benzylamine, tetramethyl ethylenediamine, potassium persulfate,
tetramethyl ethylenediamine, lysine, ornithine, histidine and
arginine. Examples of the dye/photoinitiator system include ethyl
eosin with an amine, eosin Y with an amine,
2,2-dimethoxy-2-phenoxyacetophenone- ,
2-methoxy-2-phenoxyacetophenone, camphorquinone with an amine, and
rose bengal with an amine.
[0039] In some cases, dye may absorb light and initiate
polymerization, without any additional initiator such as an amine.
In these cases, only the dye and a monomer need be present to
initiate polymerization upon exposure to light. The generation of
free radicals is terminated when the laser light is removed. Some
photoinitiators, such as 2,2-dimethoxy-2-phenylacetophenone, do not
require any auxiliary amine to induce photopolymerization; in these
cases, the presence of dye, monomer, and an appropriate wavelength
of light is sufficient for photopolymerization.
[0040] Preferred light sources include various lamps and lasers
such as those which have a wavelength of about 320-800 nm. This
light can be provided by any appropriate source able to generate
the desired radiation, such as a mercury lamp, longwave UV lamp,
He-Ne laser, an argon ion laser, etc. In a preferred embodiment, a
UV source is used to polymerize a UV photopolymerizeable gel.
Similarly, the light source used is typically selected based upon
the chemistry which is to be affected by the source.
[0041] Similarly, a variety of gels or polymers are selectively
polymerized by exposure to heat. As described herein, selective
heat control using applied current is easily performed in the
microfluidic apparatus of the invention, providing for simplified
control of gel polymerization through thermal processes. Examples
include initiation by thermal initiators, which form free radicals
at moderate temperatures, such as benzoyl peroxide, with or without
triethanolamine, potassium persulfate, with or without
tetramethylethylenediamine, and ammonium persulfate with sodium
bisulfite.
[0042] In another embodiment, the fluid is polymerized by
selectively exposing it to an activator or cross-linker. For
example, where the fluid is polyacrylamide, the activator/cross
linker can be TEMED and/or APS.
[0043] The above polymers, e.g., in solution format, gel format, or
the like, and typically polymerized as described above, are
typically used in microfluidic devices to provide devices useful
for both PCR and separation of nucleic acids.
[0044] II. Separating nucleic acids in a polymer sieving medium
[0045] A mixture of polynucleotides, e.g., DNA or RNA molecules or
fragments thereof, PCR products, sequencing reaction products, and
the like, are separated by size and/or charge in a sieving medium,
e.g., a sieving medium as described above. For example, the
separation is typically an electrophoretic separation. The
separated products are detected, often as they pass a detector
(nucleic acids are typically labeled with radioactive nucleotides
or fluorophores; accordingly appropriate detectors include
spectrophotometers, fluorescent detectors, microscopes (e.g., for
fluorescent microscopy) and scintillation counting devices). If the
separated components are the products of a sequencing reaction,
e.g., a chain termination method of sequencing, detection of size
separated products is used to compile sequence information for the
region being sequenced.
[0046] Typically electrophoretic separation is used to separate the
mixture of components in the sample. Electrophoretic separation is
the separation of substances achieved by applying an electric field
to samples in a solution or gel, e.g., a polymer solution. In its
simplest form, it depends on the different velocities with which
the substances or components move in the field. The velocities
depend, e.g., on the charge and size of the substances.
[0047] In a preferred embodiment, the polynucleotides are separated
in a microscale separation channel or capillary. The separation
channels or regions typically comprise a separation matrix, e.g., a
polymerized sieving medium as discussed above. When the sample is
flowed through the separation matrix, the components are separated,
e.g., based on physical or chemical properties, such as molecular
weight or charge. In the present invention, the sieving medium
optionally comprises a gel or solution. In the present invention,
the concentration of the polymer in the separation gel or solution
is less than about 0.5%. Typically the concentration of the sieving
medium is less than about 0.4% and more typically about 0.35% or
less. In other embodiments, the polymer concentration can be
greater than 0.5%, e.g., 0.55%, 0.6% or higher. See e.g., U.S. Ser.
No. 09/093,832 by Burd Mehta et al.
[0048] Preferably, the channel, such as channel 103 in FIG. 1, is a
polyacrylamide gel filled channel or a
polydimethylacrylamide/co-acrylic acid polymer filled channel on
which the mixture of components is electrophoretically separated
based on charge/mass ratio or molecular weight. Polyacrylamide used
as a separation matrix in a microfluidic channel is optionally
cross-linked or non-cross-linked. Preferably it is linear
polyacrylamide, i.e., polydimethylacrylamide,
polydimethylacrylamide/co-acrylic acid, or the like. Other polymers
include cellulose, agarose, Genescan polymers, and the like.
[0049] For a review of electrophoretic separation techniques and
polyacrylamide gels, see, e.g., The Encyclopedia of Molecular
Biology, Kendrew (ed.) (1994); and, Gel Electrophoresis of
Proteins: A Practical Approach, 2.sup.nd edition Hames and Rickwood
(Eds.) IRL Press, Oxford England, (1990).
[0050] A detector is optionally positioned so that it detects the
polynucleotides, e.g., polynucleotides that are stained in the gel
with a fluorescent nucleic acid stain. If the components are
detected as they exit the separation region, the components are
optionally identified by their retention times. The retention time
of the oligonucleotides as they are electrophoresed through the
sieving medium is used, e.g., in combination with markers to
measure the molecular weight of and identify the
polynucleotides.
[0051] FIG. 3 demonstrates baseline separation of polynucleotides
achieved using the low polymer concentration sieving mediums of the
present invention, e.g., a sieving medium comprising 0.35%
polymer.
[0052] III. Performing PCR in a sieving medium
[0053] One aspect of the present invention is the surprising
discovery that PCR can be performed in the same sieving matrix used
to separate nucleic acids. For example, PCR is optionally performed
in the presence of a low polymer sieving matrix, and the products
of the PCR reaction are separable in the same sieving matrix, e.g.,
in a microfluidic channel.
[0054] Accordingly, in one aspect, the invention provides new
methods of performing PCR. In the methods, components of a PCR
reaction mixture (i.e., the molecules which participate in a PCR
reaction, such as PCR extension primers, nucleotide triphosphates,
thermostable enzymes, ions and buffer components such as Mg.sup.++,
template DNAs, etc.) are mixed with a sieving medium comprising
less than about 0.5% polymer to provide a PCR sieving medium.
Typically, the sieving medium comprises less than about 0.4%
polymer. In a preferred embodiment, the sieving medium comprises
about 0.35% polymer or less. In other embodiments, the sieving
medium can comprise more than 0.5% polymer. The resulting mixture,
e.g., the PCR sieving medium, is then repetitively thermocycled as
described below to produce one or more PCR products, which are
separated, e.g., electrophoretically, in the same sieving medium.
Sieving mediums of use in performing PCR, e.g., with nucleic acid
separation of the products, are described above.
[0055] Bench scale in vitro amplification techniques suitable for
amplifying sequences to provide a nucleic acid e.g., as a
diagnostic indicator for the presence of the sequence, or for
subsequent analysis, sequencing or subcloning are known.
[0056] In brief, the most common form of in vitro amplification,
i.e., PCR amplification, generally involves the use of one strand
of the target nucleic acid sequence, e.g., the sequence to be
amplified, as a template for producing a large number of
complements to that sequence. Generally, two primer sequences
complementary to different ends of a segment of the complementary
strands of the target sequence hybridize with their respective
strands of the target sequence, and in the presence of polymerase
enzymes and nucleoside triphosphates, the primers are extended
along the target sequence through the action of the polymerase
enzyme (in asymmetric PCR protocols, a single primer is used). The
extensions are melted from the target sequence by raising the
temperature of the reaction mixture, and the process is repeated,
this time with the additional copies of the target sequence
synthesized in the preceding steps. PCR amplification typically
involves repeated cycles of denaturation, hybridization and
extension reactions to produce sufficient amounts of the target
nucleic acid, all of which are carried out at different
temperatures. Typically, melting of the strands, or heat
denaturation, involves temperatures ranging from about 90.degree.
C. to 100.degree. C. for times ranging from seconds to minutes. The
temperature is then cycled down, e.g., to between about 40.degree.
C. and 65.degree. C. for annealing of primers, and then cycled up
to between about 70.degree. C. and 85.degree. C. for extension of
the primers along the target strand. This process if referred to
herein as "thermocycling."
[0057] Examples of techniques sufficient to direct persons of skill
through in vitro amplification methods at benchtop scales,
including the polymerase chain reaction (PCR) the ligase chain
reaction (LCR), Q.beta.-replicase amplification and other RNA
polymerase mediated techniques (e.g., NASBA) are found in Berger,
Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat.
No. 4,683,202; PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
(Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The
Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc.
Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35,
1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt
(1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4,
560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek
(1995) Biotechnology 13: 563-564. Improved methods of cloning in
vitro amplified nucleic acids are described in Wallace et al., U.S.
Pat. No. 5,426,039. Improved methods of amplifying large nucleic
acids by PCR are summarized in Cheng et al. (1994) Nature 369:
684-685 and the references therein, in which PCR amplicons of up to
40 kb are generated. One of skill will appreciate that essentially
any RNA can be converted into a double stranded DNA suitable for
restriction digestion, PCR expansion and sequencing using reverse
transcriptase and a polymerase. See, Ausbel, Sambrook and Berger,
all supra.
[0058] In the present invention, the PCR reactants are mixed with a
sieving medium comprising a low polymer concentration, e.g., 0.5%
or less. The products are then directly separated, e.g., in a
capillary, using the same sieving medium.
[0059] In preferred embodiments, the components of the PCR reaction
mixture, e.g., a polymerase, nucleotides, and the like, are mixed
with a sieving medium, e.g., with a low polymer concentration, in a
microfluidic channel, e.g., a channel on a LABCHIP.TM., as
described in more detail below. The apparatus optionally includes
one or more additional channels crossing the microfluidic channel
and optionally includes fluid (or joule heating) means such as an
electrokinetic controller for thermocycling and fluid direction
systems, e.g., electrokinetic controllers and/or pressure sources
such as vacuum sources, for flowing materials and reagents through
the channels. The PCR products are typically electrophoresed
through the channels in the same sieving medium used to achieve
product separation. Detection regions in the channels and
corresponding detectors are also used, e.g., to detect the
separated products.
[0060] IV. Microfluidic devices comprising a PCR compatible nucleic
acid sieving medium
[0061] The sieving medium of the invention is typically used in a
microfluidic device and the separation and PCR methods described
above are preferably performed in a microfluidic device. The
sieving medium is typically polymerized in a microscale channel of
a microfluidic device, e.g., after the PCR reaction. Alternatively,
dynamic sieving mediums, such as Genescan polymers, are used that
do not require polymerization in the channel. PCR and separation of
the products are both optionally performed in a sieving medium of
the present invention in the channels of a microfluidic device.
[0062] A variety of microfluidic devices are optionally adapted for
use in the present invention, e.g., by designing and configuring
the channels as discussed below. These devices are described in
various PCT applications and issued U.S. Patents by the inventors
and their coworkers, including U.S. Pat. Nos. 5,699,157 (J. Wallace
Parce) issued Dec. 16, 1997, 5,779,868 (J. Wallace Parce et al.)
issued Jul. 14, 1998, 5,800,690 (Calvin Y. H. Chow et al.) issued
Sep. 1, 1998, 5,842,787 (Anne R. Kopf-Sill et al.) issued Dec. 1,
1998, 5,852,495 (J. Wallace Parce) issued Dec. 22, 1998, 5,869,004
(J. Wallace Parce et al.) issued Feb. 9, 1999, 5,876,675 (Colin B.
Kennedy) issued Mar. 2, 1999, 5,880,071 (J. Wallace Parce et al.)
issued Mar. 9, 1999, 5,882,465 (Richard J. McReynolds) issued Mar.
16, 1999, 5,885,470 (J. Wallace Parce et al.) issued Mar. 23, 1999,
5,942,443 (J. Wallace Parce et al.) issued Aug. 24, 1999, 5,948,227
(Robert S. Dubrow) issued Sep. 7, 1999, 5,955,028 (Calvin Y. H.
Chow) issued Sep. 21, 1999, 5,957,579 (Anne R. Kopf-Sill et al.)
issued Sep. 28, 1999, 5,958,203 (J. Wallace Parce et al.) issued
Sep. 28, 1999, 5,958,694 (Theo T. Nikiforov) issued Sep. 28, 1999,
and 5,959,291 (Morten J. Jensen) issued Sep. 28, 1999; and
published PCT applications, such as, WO 98/00231, WO 98/00705, WO
98/00707, WO 98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO
98/45929, WO 98/46438, and WO 98/49548, WO 98/55852, WO 98/56505,
WO 98/56956, WO 99/00649, WO 99/10735, WO 99/12016, WO 99/16162, WO
99/19056, WO 99/19516, WO 99/29497, WO 99/31495, WO 99/34205, WO
99/43432, and WO 99/44217.
[0063] In particular, the use of sieving mediums in PCR and nucleic
acid separations in microfluidic devices is described in U.S. Ser.
No. 09/093,832, by Burd Mehta, filed Jun. 8, 1998. In addition,
various other elements are optionally included in the device, such
as particle sets, separation gels, antibodies, enzymes, substrates,
and the like. These optional elements are used in performing
various assays, such as nucleic acid sequencing. For example, the
use of particle sets in nucleic acid sequencing is described, e.g.,
in WO 00/50172, published Aug. 31, 2000, entitled "Manipulation of
Microparticles In Microfluidic Systems," by Burd Mehta et al.
[0064] Complete integrated systems with fluid handling, signal
detection, sample storage and sample accessing are also available.
For example WO 98/00231 (supra) provides pioneering technology for
the integration of microfluidics and sample selection and
manipulation.
[0065] One aspect of the invention is the placement of the sieving
medium in selected channels or channel regions of a microfluidic
substrate. These materials (or precursors of the materials, e.g.,
monomers polymerized in the device as discussed above) are loaded
into microfluidic components by electrokinesis, by pressurized
pumping, by centrifugal force, or capillary flow. The present
invention provides methods of DNA separation and PCR that are both
compatible with the sieving mediums of the invention. Therefore,
the sieving medium is typically loaded throughout the entire
device. However if other assays are steps are desired, e.g., DNA
sequencing reactions, certain channels are optionally filled with
reagents for the desired assay or left empty.
[0066] Several methods of providing fluidic regions in selected
regions of a channel, or selected channels are provided. In a first
aspect, multiple microfluidic regions are filled with a sieving
medium, e.g., in an unpolymerized solution that, upon
polymerization, forms a sieving matrix. Elements of the
microfluidic device such as microfluidic channels are filled with
the sieving medium by forcing the fluid into the channel under
pressure, or by moving the fluid into the channel
electrokinetically. In one embodiment, the first fluid polymerizes
upon exposure to light (i.e., the fluid comprises a
"photopolymerizable" polymer). The fluid is then selectively
exposed to light (e.g., using photomasking techniques) in those
regions where a polymerized gel is desired. Unpolymerized fluid is
then optionally washed out of the unselected regions of the
microfluidic device, or into a waste reservoir using electrokinetic
flow or pressure.
[0067] For either the thermal or photopolymerization methods
herein, monomer is pumped, e.g., in aqueous buffer, into a channel
or channel region using electroosmotic flow, or using a pressure
gradient. After selective exposure to light or heat, as
appropriate, unpolymerized materials are removed, typically using
electroosmotic flow, but optionally using a pressure gradient, from
regions where monomer material is undesirable.
[0068] In another embodiment, a sieving matrix is deposited
throughout a channel or channels of a microfluidic device in a form
which is subject to electroosmosis (i.e., the matrix moves
electrokinetically in the channel).
[0069] In an additional embodiment, a sieving medium is loaded into
multiple channels of a microfluidic device, e.g., under pressure,
and polymerized in place. Selective components which solubilize the
polymerized gel are then loaded (e.g., electrokinetically or under
pressure) into channel regions where polymerized product is not
desired, i.e., channels in which other assays are to be performed.
The selected components dissolve the polymerized gel. Example of
solubilization compounds include acids, bases and other chemicals.
In one preferred embodiment, at least two compounds are used to
dissolve polymerized products, where both products need to be
present to dissolve the polymer. This provides for fine control of
dissolution, e.g., where each chemical is under separate
electrokinetic control. An example of such a chemical pair is
DTT(N,N'-bis(acrylol)cystamine or (1,2-dihydroxyethylene-bis-acry-
lamide) [DHEBA] and sodium periodiate or calcium alginate+EDTA or
TCEP-HCL and N,N'-bis(acryloyl)cystamine. A variety of such
materials are known.
[0070] The sieving medium of the invention is particularly useful
for performing experimental or diagnostic procedures which combine
nucleic acid amplification and product separatory aspects. For
example, PCR is performed in a microfluidic channel comprising a
sieving medium as described herein, followed by separation in the
same sieving medium, e.g., in the same or in a different channel
region.
[0071] In one aspect, PCR or other thermal reaction reagents (e.g.,
LCR reagents) such as thermostable polymerase, DNA template,
primers, nucleotides and buffers are mixed, e.g., with a sieving
medium, in a microchannel or chamber, with the entire microfluidic
substrate (e.g., a LABCHIP.TM. from Caliper Technologies) being
subject to repeated cycles of heating and cooling, e.g., on a
thermocycler or by switching between a hot plate and a heat
sink.
[0072] In a second more preferred embodiment, variations in channel
thickness and/or voltage are used selectively to heat selected
regions of a channel which contain a PCR reaction. PCR
amplification is particularly well suited to this use in the
apparatus, methods and systems of the invention. Thermocycling
amplification methods, including PCR and LCR, are conveniently
performed in microscale devices, making iterative fluidic
operations involving PCR well suited to use in methods and devices
of the present invention (see also, U.S. Pat. Nos. 5,498,392 and
5,587,128 to Willingham et al.). Accordingly, in one preferred
embodiment, generation of amplicons such as sequencing templates
using PCR, or direct sequencing of nucleic acids by PCR (e.g.,
using nuclease digestion as described supra) is performed with the
integrated systems and devices of the invention.
[0073] Thermocycling in microscale devices, including thermocycling
by joule heating is described in WO 99/12016, entitled "ELECTRICAL
CURRENT FOR CONTROLLING FLUID PARAMETERS IN MICROCHANNELS"
published Mar. 11, 1999 by Calvin Chow, Anne R. Kopf-Sill and J.
Wallace Parce; in 08/977,528, filed Nov. 25, 1997; in WO 98/45481,
entitled "CLOSED-LOOP BIOCHEMICAL ANALYZERS," published Oct. 15,
1998; and in 09/287,069, entitled INEFFICIENT FAST PCR, filed Apr.
6, 1999 by Kopf-Sill. In brief, energy is provided to heat fluids,
e.g., samples, analytes, buffers and reagents, in desired locations
of the substrates in an efficient manner by application of electric
current to fluids in microchannels. Thus, the present invention
optionally uses power sources that pass electrical current through
a first channel region for heating purposes, as well as for
material transport. In exemplary embodiments, fluid passes through
a channel of a desired cross-section (e.g., diameter) to enhance
thermal transfer of energy from the current to the fluid. The
channels can be formed on almost any type of substrate material
such as, for example, amorphous materials (e.g., glass, plastic,
silicon), composites, multi-layered materials, combinations
thereof, and the like.
[0074] In general, electric current passing through the fluid in a
channel produces heat by dissipating energy through the electrical
resistance of the fluid. Power dissipates as the current passes
through the fluid and goes into the fluid as energy as a function
of time to heat the fluid. The following mathematical expression
generally describes a relationship between power, electrical
current, and fluid resistance:
POWER=I.sup.2R
[0075] where
[0076] POWER=power dissipated in fluid;
[0077] I=electric current passing through fluid; and
[0078] R=electric resistance of fluid.
[0079] The above equation provides a relationship between power
dissipated ("POWER") to current ("I") and resistance ("R"). In some
of the embodiments, which are directed toward moving fluid in
channels, e.g., to provide mixing, electrophoretic separation, or
the like, a portion of the power goes into kinetic energy of moving
the fluid through the channel. However, it is also possible to use
a selected portion of the power to controllably heat fluid in a
channel or selected channel regions. A channel region suitable for
heating is often narrower or smaller in cross-section than other
channel regions in the channel structure, as a smaller
cross-section provides higher resistance in the fluid, which
increases the temperature of the fluid as electric current passes
through. Alternatively, the electric current is increased across
the length of the channel by increased voltage, which also
increases the amount of power dissipated into the fluid to
correspondingly increase fluid temperature.
[0080] To selectively control the temperature of fluid at a region
of the channel, a power supply applies voltage and/or current in
one of many ways. For instance, a power supply can apply direct
current (i.e., DC) or alternating current (AC), which passes
through the channel and into a channel region which is smaller in
cross-section, thereby heating fluid in the region. This current is
selectively adjusted in magnitude to complement any voltage or
electric field that is applied to move fluid in and out of the
region. AC current, voltage, and/or frequency can be adjusted, for
example, to heat the fluid without substantially moving the fluid.
Alternatively, a power supply can apply a pulse or impulse of
current and/or voltage, which passes through the channel and into a
channel region to heat fluid in the region. This pulse is
selectively adjusted to complement any voltage or electric field
that is applied to move fluid in and out of the region. Pulse
width, shape, and/or intensity can be adjusted, for example, to
heat the fluid substantially without moving the fluid or to heat
the fluid while moving the fluid. Still further, the power supply
can apply any combination of DC, AC, and pulse, depending upon the
application. In practice, direct application of electric current to
fluids in the microchannels of the invention results in extremely
rapid and easily controlled changes in temperature.
[0081] A controller or computer such as a personal computer
monitors the temperature of the fluid in the region of the channel
where the fluid is heated. The controller or computer receives
current and voltage information from, for example, the power supply
and identifies or detects temperature of fluid in the region of the
channel. Depending upon the desired temperature of fluid in the
region, the controller or computer adjusts voltage and/or current
to meet the desired fluid temperature. The controller or computer
also can be set to be "current controlled" or "voltage controlled"
or "power controlled" depending upon the application.
[0082] The region which is heated can be a "coil" which is
optionally in a planar arrangement. Transfer of heat from the coil
to a reaction channel through a substrate material is used to heat
the reaction channel. Alternatively, the coil itself is optionally
the reaction channel.
[0083] A voltage is applied between regions of the coil to direct
current through the fluid for heating purposes. In particular, a
power supply provides a voltage differential between regions of the
coil. Current flows between the regions and traverses a plurality
of coils or coil loops (which can be planar), which are defined by
a substrate. Shape and size of the coils can influence an ability
of current to heat the fluid in the coil. As current traverses
through the fluid, energy is transferred to the fluid for heating
purposes. Cooling coils can also be used. As a cooling coil, a
fluid traverses from region to region in the coil, which can be
placed to permit heat transfer through a substrate from a sample.
The cooling fluid can be a variety of substances including liquids
and gases. As merely an example, the cooling fluid includes aqueous
solutions, liquid or gaseous nitrogen, and others. The cooling
fluid can be moved between regions using any of the techniques
described herein, and others. Further details are found in Chow et
al., supra.
[0084] The introduction of electrical current into fluid causes
heat (this procedure is referred to as "Joule heating"). In the
examples of fluid movement herein where thermal effects are not
desired, the heating effect is minimal because, at the small
currents employed, heat is rapidly dissipated into the chip itself.
By substantially increasing the current across the channel, rapid
temperature changes are induced that can be monitored by
conductivity. At the same time, the fluid can be kept static in the
channel by using alternating instead of direct current. Because
nanoliter volumes of fluid have tiny thermal mass, transitions
between temperatures can be extremely short. Oscillations between
any two temperatures above 0.degree. C. and below 100.degree. C. in
100 milliseconds have been performed.
[0085] Joule heating in microchannels is an example of how benchtop
methods can be dramatically improved in the formats provided
herein. PCR takes hours to perform currently, primarily because it
takes a long time for conventional heating blocks to oscillate
between temperatures. In addition, reagent cost is an obstacle to
massive experimentation. Both these parameters are altered by
orders of magnitude in the LabChip.TM. format.
[0086] In one aspect, PCR reaction conditions are controlled as a
function of channel geometry. Microfabrication methods permit the
manufacture of channels that have precise variations in cross
sectional area. Since the channel resistance is inversely
proportional to the cross sectional area, the temperature varies
with the width and depth of the channel for a given flow of
current. As fluid moves through a structure of varying cross
sectional area, its temperature will change, depending on the
dimensions of the channel at any given point. The amount of time it
experiences a given temperature will be determined by the velocity
of the fluid flow, and the length of channel with those dimensions.
This concept is illustrated in FIG. 2. Nucleic acids of typical
lengths have a low diffusion coefficient (about 10.sup.-7
cm/sec.sup.2). Thus over the time frame necessary to affect
amplification, DNA will only diffuse a few hundred microns. In a
given channel, reactions of a few nanoliters will occupy a few
millimeters. Thus in devices of convenient length (a few
centimeters), many PCR reactions can be performed concurrently
yielding new amplification products every few seconds per channel.
In parallel formats, hundreds of separate reactions can be
performed simultaneously. Because of its simplicity, throughput,
and convenience, this amplification unit is a preferred feature of
many assays herein.
[0087] In FIG. 2, amplification reactions are performed
concurrently in series using biased alternating current to heat the
fluid inside the channel and move material through it. The time for
each step of the reaction is controlled by determining the speed of
movement and the length of channel having particular widths. Flow
can be reversed to allow a single small channel region to be used
for many separate amplifications.
[0088] As depicted, several samples are run simultaneously in
channel 210. Sample 215 is in narrow channel region 220; in
operation, this region is heated to, e.g., 95.degree. C. (hot
enough to denature nucleic acids present in sample 215, but cool
enough that thermostable reagents such as Taq DNA polymerase are
relatively stable due to the relative size of region 220 and the
applied current. Concurrently, wide channel region 230 is heated,
e.g., to 60.degree. C. (cool enough for binding of primers in
sample 225 and initiation of polymerase extension), due to the
relative size of region 230 and the applied current. Concurrently,
intermediate channel region 235 is heated, e.g., to 72.degree. C.
(hot enough to prevent unwanted non-specific primer-target nucleic
acid interactions in sample 240 and cool enough to permit continued
polymerase extension), due to the relative size of region 235 and
the applied current. This process can be concurrently carried out
with a plurality of additional channel regions such as narrow
region 245, wide region 250 and intermediate region 255, with
samples 260, 265 and 270.
[0089] Where possible, direct detection of amplified products can
be employed. For example, differentially labeled competitive probe
hybridization is used for single nucleotide discrimination.
Alternatively, molecular beacons or single nucleotide polymerase
extension can be employed. Homogeneous detection by fluorescence
polarization spectroscopy can also be utilized (fluorescence
polarization has been used to distinguish between labeled small
molecules free in solution or bound to protein receptors).
[0090] The present invention provides the ability to integrate
complex functions such as PCR and nucleic acid separation in a
single format, e.g., in a microfluidic device. PCR and nucleic acid
separations are optionally performed in the same sieving medium,
therefore making it possible to perform both functions in a single
channel of a microfluidic device.
[0091] For example, a Caliper LabChip.TM. is optionally used to
load DNA template, run the PCR reaction, and then size the
resulting PCR product by gel separation. FIG. 1 provides a
schematic of an example device. Device 100 is optionally filled
with a sieving medium as described above, e.g., a sieving gel. The
sieving medium provides a continuous fluid phase throughout the
device, such that PCR is optionally carried out by joule heating in
one channel, e.g., channel 101, and product separation in another
channel, e.g., channel 103. Alternatively, the entire device is
thermocycled to perform PCR. PCR is thereby carried out in the
sieving medium. PCR reagents and a target nucleic acid are added to
the sieving medium if not already incorporated therein, e.g., into
channel 101. Cross channel 101 is optionally a PCR channel as
described herein and shown in FIG. 2. The loaded device is
typically placed on a thermocycler (MJ Research) and the
temperature is typically cycled to amplify the target nucleic acid.
At the end of the cycling procedure, the chip is typically placed
on a microscope detection station and the product is
electrokinetically injected into separation channel 103. For
example, voltages are applied at wells 107 and 109 to
electrokinetically separate the product nucleic acids. An example
spectrum showing separated nucleic acid peaks is provided in FIG.
3.
[0092] It will be appreciated that separations chips comprising a
single sieving matrix are produced as described above. However,
additional fluidic phases are optionally placed in additional
channels or channel regions in fluid communication with a channel
region comprising the PCR sieving mixture for electrophoretic or
electroosmotic movement of the PCR components or products in the
chips. For example, in some aspects a PCR reaction product is
selected for further manipulations such as cloning, sequencing or
the like, all of which are optionally performed in the same device
(see also, U.S. Ser. No. 60/068,311, entitled "Closed Loop
Biochemical Analyzer" by Knapp, filed Dec. 19, 1997 and "Closed
Loop Biochemical Analyzers" by Knapp et al. U.S. Ser. No.
09/054,962, filed Apr. 3, 1998).
[0093] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above may be used in various
combinations. All publications, patents, patent applications, or
other documents cited in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application, or
other document were individually indicated to be incorporated by
reference for all purposes.
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