U.S. patent application number 09/898404 was filed with the patent office on 2002-02-07 for pressure-enhanced extraction and purification.
This patent application is currently assigned to BBI BioSeq, Inc., a Masachusetts corporation. Invention is credited to Hess, Robert A., Laugharn, James A. JR., Tao, Feng.
Application Number | 20020016450 09/898404 |
Document ID | / |
Family ID | 25505646 |
Filed Date | 2002-02-07 |
United States Patent
Application |
20020016450 |
Kind Code |
A1 |
Laugharn, James A. JR. ; et
al. |
February 7, 2002 |
Pressure-enhanced extraction and purification
Abstract
The invention is based on the discovery that hyperbaric,
hydrostatic pressure reversibly alters the partitioning of
biomolecules between certain adsorbed and solvated phases relative
to partitioning at ambient pressure. The new methods and devices
disclosed herein make use of this discovery for highly selective
and efficient, low salt isolation and purification of nucleic acids
from a broad range of sample types, including forensic samples,
blood and other body fluids, and cultured cells. In one embodiment,
the invention features a pressure-modulation apparatus. The
apparatus includes an electrode array system having at least two
(i.e., two, three, four, or more) electrodes; and a conduit
interconnecting the electrodes. The conduit contains an
electrically conductive fluid in contact with a phase positioned in
a pressure chamber. The phase can be, for example, a binding medium
or stationary phase.
Inventors: |
Laugharn, James A. JR.;
(Winchester, MA) ; Hess, Robert A.; (Cambridge,
MA) ; Tao, Feng; (Boston, MA) |
Correspondence
Address: |
CHARLES J. BOUDREAU
Fish & Richardson P.C.
Suite 2800
45 Rockefeller Plaza
New York
NY
10111
US
|
Assignee: |
BBI BioSeq, Inc., a Masachusetts
corporation
|
Family ID: |
25505646 |
Appl. No.: |
09/898404 |
Filed: |
July 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09898404 |
Jul 3, 2001 |
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09016062 |
Jan 30, 1998 |
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6274726 |
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09016062 |
Jan 30, 1998 |
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08962280 |
Oct 31, 1997 |
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6111096 |
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Current U.S.
Class: |
536/23.1 ;
204/450; 536/25.4 |
Current CPC
Class: |
G01N 2030/522 20130101;
G01N 30/14 20130101; G01N 30/6047 20130101; B01L 2300/14 20130101;
G01N 30/52 20130101; B01L 2400/0688 20130101; G01N 27/44704
20130101; G01N 30/02 20130101; G01N 2030/8813 20130101; G01N 30/00
20130101; B01L 2400/0487 20130101; B01L 2200/10 20130101; B01L
3/50273 20130101; G01N 2030/009 20130101; G01N 30/14 20130101; B01L
2400/0421 20130101; B01L 3/5027 20130101; C12N 1/06 20130101; B01L
2400/0418 20130101; G01N 2030/009 20130101; G01N 30/02 20130101;
G01N 30/6047 20130101; B01L 2400/0406 20130101; G01N 30/02
20130101; C12N 15/101 20130101; B01D 15/365 20130101; G01N 2030/522
20130101; B01D 15/32 20130101; G01N 2030/285 20130101; B01L
2300/0816 20130101; B01L 2300/0681 20130101; G01N 2030/285
20130101; G01N 2030/522 20130101 |
Class at
Publication: |
536/23.1 ;
536/25.4; 204/450 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. A pressure-modulation apparatus, comprising: an electrode array
system comprising at least two electrodes; and a conduit
interconnecting said electrodes, wherein said conduit contains an
electrically conductive fluid in contact with a phase positioned in
a pressure chamber.
2. The apparatus of claim 1, further comprising at least one
reservoir in communication with the conduit to contain materials
transported by the conduit.
3. The apparatus of claim 2, wherein said reservoir is positioned
in the pressure chamber.
4. The apparatus of claim 1, wherein said conduit comprise an
electrically non-conducting tube.
5. The apparatus of claim 1, further comprising a
pressure-transmitting apparatus to transmit pressure to or from the
pressure chamber.
6. The apparatus of claim 1, comprising at least three
electrodes.
7. The apparatus of claim 6, wherein said electrodes defined at
least two axes.
8. A method for purifying nucleic acids from a sample, said method
comprising: contacting the sample with the phase of the apparatus
of claim 1 at an initial pressure, wherein said phase
non-specifically binds to nucleic acids with greater affinity than
said phase binds to non-nucleic acid components of the sample;
transporting at least some of the non-nucleic acid components
towards one of said electrodes; modifying the pressure to a level
sufficient to disrupt the binding of the nucleic acids to the
phase; and transporting the nucleic acids towards a second of said
electrodes.
9. The apparatus of claim 1, wherein said conduit comprises an
electrophoretic capillary.
10. A method for purifying nucleic acids from a sample, said method
comprising: contacting the sample with the phase of the apparatus
of claim 9 at an initial pressure, wherein said phase
non-specifically binds to nucleic acids with greater affinity than
said phase binds to non-nucleic acid components of the sample;
electrophoretically separating at least some of the non-nucleic
acid components from the nucleic acids; modifying the pressure to a
level sufficient to disrupt the binding of the nucleic acids to the
phase; and electrophoretically separating the nucleic acids from
the phase at the modified pressure.
11. The apparatus of claim 1, wherein said conduit comprises an
electroosmotic capillary.
12. A method for purifying nucleic acids from a sample, said method
comprising: contacting the sample with the phase of the apparatus
of claim 11 at an initial pressure, wherein said phase
non-specifically binds to nucleic acids with greater affinity than
said phase binds to non-nucleic acid components of the sample;
electroosmotically separating at least some of the non-nucleic acid
components from the nucleic acids; modifying the pressure to a
level sufficient to disrupt the binding of the nucleic acids to the
phase; and electroosmotically separating the nucleic acids from the
phase at the modified pressure.
13. The apparatus of claim 1, wherein said electrode array system
is configured on a microchip.
14. The apparatus of claim 1, wherein said phase comprises
hydroxyapatite.
15. The apparatus of claim 1, wherein said phase comprises an
immobilized nucleic acid molecule.
16. The apparatus of claim 1, wherein said phase comprises
silica.
17. The apparatus of claim 1, wherein said phase comprises an
anion-exchange resin.
18. A method for isolating and purifying nucleic acids from a
sample, said method comprising: applying the sample to a phase at
an initial pressure, wherein said phase non-specifically binds to
nucleic acids with greater affinity than said phase binds to
non-nucleic acid components of the sample; spatially separating at
least some of the non-nucleic acid components from the phase and
the nucleic acids; modifying the pressure to a level sufficient to
disrupt the binding of at least some of the nucleic acids to the
phase; and spatially separating the nucleic acids from the phase at
the modified pressure, wherein the applying and first spatially
separating steps are carried out within a single reaction
vessel.
19. The method of claim 18, wherein the first spatially separating
step comprises transporting non-nucleic acid components into a
reservoir.
20. The method of claim 19, wherein the reservoir contains a
binding material.
21. The method of claim 18, wherein the first spatially separating
step comprises electrophoresis.
22. The method of claim 18, wherein the first spatially separating
step comprises electroosmosis.
23. The method of claim 18, wherein said initial pressure is
ambient pressure and said modified pressure is an elevated
pressure.
24. The method of claim 23, wherein said elevated pressure is 500
to 100,000 psi.
25. The method of claim 18, wherein the sample comprises cells and
said method further comprises subjecting said sample to a
hyperbaric pressure sufficient to lyse the cells.
26. The method of claim 25, wherein the cells comprise external and
nuclear membranes, and the hyperbaric pressure is sufficient to
lyse the external membrane, but insufficient to lyse the nuclear
membranes.
27. The method of claim 18, wherein the sample comprises nucleic
acid-binding proteins and said method further comprises subjecting
said sample to a hyperbaric pressure sufficient to inactivate the
nucleic acid-binding proteins.
28. The method of claim 27, wherein the nucleic acid-binding
proteins comprise nuclease enzymes.
29. The method of claim 18, wherein the sample comprises various
sizes of nucleic acids, the modified pressure level is sufficient
only to disrupt the binding of relatively small nucleic acids to
the phase, and the method further comprises: further modifying the
pressure to a level sufficient to disrupt the binding of relatively
larger nucleic acids to the phase; and spatially separating the
nucleic acids from the phase at the further modified pressure.
30. The method of claim 25, wherein said sample comprises a
biological fluid.
31. The method of claim 25, wherein said sample comprises whole
blood.
32. The method of claim 25, wherein said sample comprises
serum.
33. The method of claim 25, wherein said sample comprises cultured
cells.
34. The method of claim 25, wherein said sample comprises tumor
biopsy tissue.
35. The method of claim 25, wherein said sample comprises plant
tissue.
36. The method of claim 25, wherein said sample comprises living
tissue.
37. The method of claim 18, wherein said nucleic acids comprise
DNA.
38. The method of claim 18, wherein said nucleic acids comprise
total RNA.
39. The method of claim 18, wherein said nucleic acids comprise
messenger RNA (mRNA).
40. The method of claim 18, wherein said nucleic acids comprise
viral RNA.
41. The method of claim 37, wherein said DNA is chromosomal
DNA.
42. The method of claim 37, wherein said DNA comprises a
vector.
43. The method of claim 37, wherein said DNA comprises viral
DNA.
44. The method of claim 18, wherein said modified pressure is
sufficient to elute vector DNA but not high enough to elute
chromosomal DNA.
45. The method of claim 18, wherein said modified pressure is
sufficient to elute RNA but not high enough to elute chromosomal
DNA.
46. The method of claim 18, wherein said method further comprises
adding a dicarbonyl compound to the sample to inactivate
nucleic-acid binding proteins.
47. The method of claim 18, wherein said phase comprises
hydroxyapatite.
48. The method of claim 18, wherein said phase comprises an
immobilized nucleic acid molecule.
49. The method of claim 18, wherein said phase comprises
silica.
50. The method of claim 18, wherein said phase comprises an
anion-exchange resin.
51. The method of claim 18, wherein said phase comprises a
pressure-sensitive gel.
52. The method of claim 18, wherein said phase comprises a
pressure-stable medium.
53. The method of claim 52, wherein said medium is a non-porous
resin comprising 1 to 50 .mu.m beads having a positively charged
surface.
54. The method of claim 18, further comprising concentrating the
nucleic acids between two membranes by electrophoresis, wherein one
of said membranes is substantially impermeable to nucleic acids and
the second membrane has increased permeability to nucleic acids
under applied electrical potential.
55. The method of claim 54, wherein said concentration is carried
out at said modified pressure.
56. The method of claim 18, further comprising trapping the
spatially separated nucleic acids in a filter by
electrophoresis.
57. The method of claim 18, further comprising transporting the
spatially separated nucleic acids to an analytical device.
58. The method of claim 57, wherein said analytical device is a
matrix-assisted laser desorption and ionization (MALDI) mass
spectrometer.
59. A device for carrying out the method of claim 18, the device
comprising: a pressure modulation apparatus; and a pressurizable
cell containing said phase, wherein said cell is adapted to fit
within said apparatus.
60. A device for pressurizing a sample, the device comprising: a
sample compartment; and a pressure-transmitting apparatus to
transmit pressure from a pressurizing medium outside of said device
to the sample compartment, without allowing fluid flow between the
medium and the sample compartment.
61. The device of claim 60, further comprising a chamber having an
orifice, wherein said sample compartment and pressure-transmitting
apparatus are configured within said orifice.
62. The device of claim 60, wherein said pressure-transmitting
device comprises a shape-memory alloy device.
63. The device of claim 60, wherein said pressure-transmitting
apparatus comprises a magnetostrictive device.
64. The device of claim 61, wherein said chamber comprises a
cylinder and said pressure-transmitting apparatus comprises a
piston.
65. The device of claim 64, wherein said cylinder comprises a
plastic tube having a sealed end and an open end, and said piston
comprises a rubber piston.
66. The device of claim 60, wherein said chamber comprises a well
in a microtiter plate.
67. A method for permeabilizing cells, the method comprising:
charging the sample compartment of the device of claim 60 with
cells at an initial pressure; introducing the device into a
pressure modulation apparatus; and momentarily increasing the
pressure to at least 10,000 psi to permeabilize the cells.
68. The method of claim 67, wherein said sample compartment is also
charged with a gas.
69. The method of claim 67, further comprising applying a voltage
across the sample compartment to spatially separate at least some
components of the permeabilized cells from other components of the
cells.
70. The method of claim 67, further comprising freezing the
cells.
71. An improved ion-exchange chromatography method, the improvement
comprising using hyperbaric pressure to modulate binding affinities
associated with an ion-exchange material.
72. A method for the isolation of molecules from cells, the method
comprising: exposing the cells to an elevated pressure of at least
500 psi in a pressure chamber to form lysed cells; and separating
the molecules from the cells within the pressure chamber.
73. The method of claim 72, further comprising cycling the pressure
between the elevated pressure and ambient pressure at least
twice.
74. The method of claim 72, wherein the molecules are extracted by
elution with a flowing solvent, electrophoresis, electroosmosis,
selective absorption to an absorptive medium, filtration,
differential sedimentation, volatilization, distillation, gas
chromatography, or precipitation.
75. The method of claim 72, wherein the pressure is raised to its
final value in less than 1 second.
76. The method of claim 72, wherein the pressure is raised to its
final value in less than 0.1 second.
77. The method of claim 72, wherein the molecules are extracted
while the cells are at said elevated pressure.
78. The method of claim 72, further comprising returning the cells
to ambient pressure.
79. The method of claim 72, further comprising purifying the
molecules, at least partially, within the pressure chamber.
80. The method of claim 79, wherein the molecules are purified by
elution with a flowing solvent, electrophoresis, electroosmosis,
selective absorption to an absorptive medium, filtration,
differential sedimentation, volatilization, distillation, gas
chromatography, or precipitation.
81. The method of claim 79, wherein the purifying step requires a
change in pressure of at least 500 psi.
82. The method of claim 72, wherein the cells are selected from the
group consisting of yeast, bacteria, fungi, animal cells, plant
cells, insect cells, and protozoan cells.
83. The method of claim 78, wherein the cells are returned to
ambient pressure in 1 second or less.
84. The method of claim 78, wherein the cells are returned to
ambient pressure in 0.1 second or less.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/962,280, filed on Oct. 31, 1997, which is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention is in the general field of methods and devices
for isolating and purifying compounds from mixtures.
BACKGROUND OF THE INVENTION
[0003] Many methods for separating biomolecules from mixtures such
as cell lysates or synthetic preparations are based on a procedure
in which the sample is loaded onto a column packed with a solid
phase.
[0004] In the case of nucleic acids, for example, the solid phase
can include an anion-exchange medium or resin. The negatively
charged, anionic phosphate backbone of a nucleic acid can bind to
and is thereby effectively immobilized by the resin. The resin can
be washed with a low salt solution (e.g., 0.2 M sodium chloride),
which flushes away the neutral, cationic, and less highly charged
anionic components of the original mixture without substantially
disrupting the binding of the nucleic acid molecules to the solid
phase.
[0005] A high salt buffer solution (e.g., a buffer containing 1 M
sodium chloride) is then used to elute the nucleic acid molecules
away from the solid phase. The high salt concentration, however,
can interfere with mass spectroscopy, electrophoresis, and many
downstream enzymatic processes commonly employed in the laboratory
or clinic, for example, for diagnostics, forensics, or genomic
analysis. It is therefore necessary, in many cases, to remove at
least some of the salt from the nucleic acid in an additional,
frequently time-consuming step. Desalting can be accomplished by
any of several procedures, including ethanol precipitation,
dialysis, and purification from glass or silica beads or resin. In
some cases it may also be necessary to add nuclease inhibitors to
the wash and buffer solutions to prevent degradation of the nucleic
acid.
SUMMARY OF THE INVENTION
[0006] The invention is based on the discovery that hyperbaric,
hydrostatic pressure reversibly alters the partitioning of
biomolecules between certain adsorbed and solvated phases relative
to partitioning at ambient pressure. The new methods and devices
disclosed herein make use of this discovery for highly selective
and efficient, low salt isolation and purification of nucleic acids
from a broad range of sample types, including forensic samples,
blood and other body fluids, and cultured cells.
[0007] In one embodiment, the invention features a
pressure-modulation apparatus. The apparatus includes an electrode
array system having at least two (i.e., two, three, four, or more)
electrodes; and a conduit interconnecting the electrodes. The
conduit contains an electrically conductive fluid in contact with a
phase positioned in a pressure chamber. The phase can be, for
example, a binding medium or stationary phase. It can be a gel
(e.g., a pressure-sensitive gel), a resin (e.g., an ion-exchange
resin, a hydrophobic resin, a reversed phase resin, or a size
exclusion resin), a plastic, a glass, hydroxyapatite, an
immobilized oligonucleotide, a silica, an ion-exchange material,
silicon or other metal, an alumina, a zeolite, a cellulose, a
particle, a microparticle, a nanoparticle, a coating on a
substrate, a soluble polymer, a micelle, a liposome, a porous solid
medium, a membrane, a pressure-stable medium (e.g., DEAE-coated
glass, quartz, thermoplastic polymer, gel, or a non-porous resin
made up of 1 to 50 Am beads with positively charged surface), or a
phase of a phase-separated liquid. The electrodes can have a
protective coating (e.g., of polyacrylamide gel.
[0008] The apparatus can also include at least one (i.e., one, two,
three, four, or more) reservoir in communication with the conduit
to contain materials transported by the conduit. The reservoir can
also be positioned in the pressure chamber. The conduit can be, for
example, an electrically non-conducting tube. The apparatus can
also include a pressure-transmitting apparatus (e.g., an
electrically mediated pressure actuator, such as an
electrostrictive apparatus, magnetostrictive apparatus, or a
shape-memory alloy device) that can transmit pressure to or from
the pressure chamber. If there are three electrodes or more, the
electrodes can be configured in a straight line or can
alternatively define two or more (i.e., two, three, four, or more)
axes. The conduit can include an electrophoretic or electroosmotic
capillary. The electrode array system can be configured on a
microchip.
[0009] The invention also features a method for purifying nucleic
acids from a sample. The method includes the steps of contacting
the sample with the phase of the aforementioned apparatuses at an
initial pressure (i.e., where the phase is a phase that
non-specifically binds to nucleic acids with greater affinity than
it does to non-nucleic acid components of the sample); transporting
(e.g., electrophoretically or electroosmotically) at least some of
the non-nucleic acid components (e.g., towards one electrode, or
away from the nucleic acids); modifying the pressure to a level
sufficient to disrupt the binding of the nucleic acids to the
phase; and transporting (e.g., electrophoretically or
electroosmotically) the nucleic acids (e.g., towards a second
electrode, or away from the phase).
[0010] In another embodiment, the invention features another method
for isolating and purifying nucleic acids from a sample. The method
includes the steps of applying the sample to a phase at an initial
pressure (i.e., where the phase non-specifically binds to nucleic
acids with greater affinity than it does to non-nucleic acid
components of the sample); spatially separating (e.g., by
electrophoresis, electroosmosis, or fluid flow) at least some of
the non-nucleic acid components from the phase and the nucleic
acids; modifying the pressure to a level sufficient to disrupt the
binding of at least some of the nucleic acids to the phase; and
spatially separating the nucleic acids from the phase at the
modified pressure. The "applying" and first "spatially separating"
steps, at least, are carried out within a single reaction vessel
(e.g., a pressure modulation apparatus, or a pressurized
vessel).
[0011] The first "spatially separating" step can include
transporting the non-nucleic acid components into a reservoir. The
reservoir can optionally include binding materials such as
ion-exchange materials, desalting (mixed ion-exchange) resin,
nonspecific affinity resin, polystyrene resin, gamma-irradiated
polystyrene resin, a covalent attachment resin (e.g., an
aldehyde-rich surface, a carbodiimide-rich surface, an
o-methylisourea-rich surface, an amidine-rich surface, a
dicarbonyl-rich surface, a hydrazide-rich surface, or a thiol-rich
surface), a resin or combination of resins possessing different
binding functionalities, or a hydrophobic material; alternatively,
an anion-exchange material can be placed at one or more electrodes
of positive potential or a cation-exchange material can be placed
at one or more electrodes of negative potential.
[0012] The initial pressure can be, for example, ambient pressure
and the modified pressure can be an elevated pressure (e.g., 100 to
200,000 psi, 500 to 100,000 psi, 1,000 to 50,000 psi, or 2,000 to
25,000 psi).
[0013] In some instances, the sample can include cells; the method
would then also include subjecting the sample to a hyperbaric
pressure sufficient to lyse the cells. The cells can include both
external and nuclear membranes, and the hyperbaric pressure can be
sufficient to lyse both membranes, or alternatively, only to lyse
the external membrane, not the nuclear membranes.
[0014] The sample can also include nucleic acid-binding proteins
(e.g., nuclease enzymes); the method can thus also include
subjecting the sample to a hyperbaric pressure sufficient to
inactivate the nucleic acid-binding proteins.
[0015] The sample can include various sizes of nucleic acids; the
modified pressure level can, for example, be sufficient only to
disrupt the binding of relatively small nucleic acids to the phase.
To disrupt the binding of larger nucleic acids, and the method also
includes the steps of further modifying the pressure to a level
sufficient to disrupt the binding of the relatively larger nucleic
acids to the phase; and spatially separating the nucleic acids from
the phase at the further modified pressure. By this method, for
example, a 250 base pair nucleic acid can be separated from a 500
base pair nucleic acid, a 1000 base pair nucleic acid can be
separated from a 2000 base pair nucleic acid, or a 10,000 base pair
nucleic acid can be separated from a 20,000 base pair nucleic
acid.
[0016] The sample can be, for example, a biological fluid, whole
blood, serum, plasma, cultured cells, tumor biopsy tissue, plant
tissue, or living tissue (e.g., tissue in which most normally
processes associated with life are ongoing; can be from a living or
deceased organism).
[0017] The nucleic acids can be partially digested, and fragments
of a particular size distribution can be recovered (e.g., for use
in sequencing or hybridization analysis). The nucleic acids can
include RNA (e.g., total RNA, messenger RNA (mRNA), viral RNA,
ribosomal RNA (rRNA)) or DNA (e.g., chromosomal DNA, a vector, or
viral DNA).
[0018] The modified pressure can be sufficient to elute vector DNA
(e.g., typically around 5,000 to 20,000 base pairs, regardless of
source; it can include, e.g., digested chromosomal DNA) but not
high enough to elute chromosomal DNA (e.g., typically 50,000 base
pairs or more). This method would require pressures in the range
of, for example, 15000 to 30000 psi, depending on the nature of the
phase, temperature, pH, ion concentration, etc.
[0019] Similarly, the modified pressure can be sufficient to elute
RNA but not high enough to elute chromosomal DNA (e.g., 10,000 to
30,000 psi, depending on phase and other conditions).
[0020] A dicarbonyl compound can also be added to the sample to
inactivate nucleic-acid binding proteins such as nucleases.
Pressure can, for example, accelerate the condensation of guanido
moieties, such as arginine residues within the proteins, with the
dicarbonyls.
[0021] In some cases, the nucleic acids can be concentrated (e.g.,
at an elevated pressure) between two membranes by electrophoresis,
wherein one of said membranes is substantially impermeable to
nucleic acids and the second membrane has increased permeability to
nucleic acids under applied electrical potential. In another case,
the nucleic acids can be trapped in a filter by
electrophoresis.
[0022] The nucleic acids can be transported to an analytical device
(e.g., a matrix-assisted laser desorption and ionization (MALDI)
mass spectrometer).
[0023] The invention also features a device for carrying out the
aforementioned methods. The device includes a pressure modulation
apparatus; and a pressurizable cell containing the phase. The cell
is adapted to fit within the apparatus.
[0024] In still another embodiment, the invention features a device
for pressurizing a sample. The device includes a sample
compartment; and a pressure-transmitting apparatus to transmit
pressure from a pressurizing medium outside of the device to the
sample compartment, without allowing fluid flow between the medium
and the sample compartment.
[0025] The device can also include a chamber having an orifice,
wherein the sample compartment and the pressure-transmitting
apparatus are configured within the orifice. The
pressure-transmitting device can include, for example, a
shape-memory alloy device, or a magnetostrictive device. The
chamber can be in the form of a cylinder (e.g., a plastic tube with
one sealed end and one open end) and the pressure-transmitting
apparatus can be a piston (e.g., a rubber piston, or a syringe
plunger). The chamber can alternatively be a well in a microtiter
plate. The invention also features a method for permeabilizing (or
lysing) cells. The method includes the steps of charging the sample
compartment of the preceding device with cells at an initial
pressure; introducing the device into a pressure modulation
apparatus; and momentarily increasing the pressure to at least
10,000 psi to permeabilize the cells. The cells can be, for
example, yeast, bacteria, animal, or plant; the initial pressure
can be less than, equal to, or greater than atmospheric pressure;
the permeabilized cells can be removed and electrophoresed, or
purified electrically (e.g., electrophoretic washing or washing
with fluid driven by electroosmotic flow); a detergent can be added
to the cells prior to or after pressure treatment. The sample
compartment can also be charged with a gas (e.g., air).
Additionally, a voltage can be applied across the sample
compartment to spatially separate at least some components of the
permeabilized cells from other components of the cells. The cells
can additionally be frozen.
[0026] Yet another embodiment of the invention is the use of
hyperbaric pressure to modulate binding affinities associated with
an ion-exchange material (e.g., an anion-exchange resin, or a
cation-exchange resin) for use in ion-exchange chromatography. This
can include traditional or capillary chromatography, and the
chromatographic substrates can include nucleic acids, proteins,
carbohydrates, or other small molecules. The method can be also be
integrated with lysis or electrophoresis methods.
[0027] Another embodiment of the invention is a method for the
isolation of molecules from cells. The method includes the steps of
exposing the cells to an elevated pressure of at least 500 psi
(e.g., 1,000, 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, or
100,000 psi, or even higher) in a pressure chamber to form lysed
cells; and separating the molecules from the cells within the
pressure chamber. The method can be carried out in an integrated
device (e.g., a consumable, single-use cartridge). The pressure can
be pulsed or cycled between the elevated pressure and ambient
pressure at least twice (e.g., two, three, four, or more times).
The cells can be, for example, yeast, bacteria, fungi, animal
cells, plant cells, insect cells, or protozoan cells.
[0028] The molecules can be extracted by elution with a flowing
solvent, electrophoresis, electroosmosis, selective absorption to
an absorptive medium, filtration, differential sedimentation,
volatilization, distillation, gas chromatography, or precipitation.
The molecules can be extracted while the cells are at the elevated
pressure. The pressure can be raised to its final value in less
than 1 second (e.g., less than 0.1 second). The method can also
include the step of returning the cells to ambient pressure, for
example, in 1 second or less (e.g., 0.1 second or less). The
molecules can be purified, at least partially, within the
integrated device.
[0029] The molecules can also be purified by elution with a flowing
solvent, electrophoresis, electroosmosis, selective absorption to
an absorptive medium, filtration, differential sedimentation,
volatilization, distillation, gas chromatography, or precipitation.
The purifying step can, for example, requires a change in pressure
of at least 500 psi.
[0030] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described below.
All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present application, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0031] An important advantage of the new methods is the potential
for use of a single solvent for both isolation and purification of
nucleic acids. A single solvent can be used for 1) loading the
nucleic acid-containing sample onto the immobilized solid phase, 2)
washing non-nucleic acid impurities away from the immobilized
nucleic acid, and 3) dissociating the nucleic acid from the solid
phase. Additionally, if the sample includes cells, the cells can be
lysed by hyperbaric pressure in the same solvent as is used for
loading, washing, and dissociating.
[0032] A single solvent method can be more cost-efficient, can
generate less waste, and is generally simpler to implement.
Furthermore, the solvent can be the same buffer that is used for a
downstream reaction. For example, prepackaged buffers, such as
those containing magnesium salts and other cofactors for use in the
polymerase chain reaction (PCR), can be used as the loading,
washing, and elution buffer in the new methods.
[0033] The use of a single, low-salt solution enables
electrophoresis of biomolecules through an ion-exchange matrix, at
a pressure that allows the desired molecules to adhere to the solid
phase, while undesired molecules are removed by electrophoresis to
a waste reservoir. The pressure can then be modulated to release
the desired molecules, which can be collected by further
application of an electric field. This method is compatible with
miniaturized "biochip" devices which can utilize methods of
high-volume manufacturing.
[0034] Another advantage of the new methods is the use of solvents
that minimize damage to biomolecular constituents. Because pressure
can be used to assist the lysis of the cells (if any) in the
sample, there is no need for harsh lysis solutions (e.g.,
phenol/chloroform, guanidinium salts, chaotropic salts) that are
often used in vast excess and must subsequently be removed. Since
pressure can also be used to reduce the affinity of the
biomolecules for the solid phase, high-salt elution solvents are
not necessary.
[0035] Pressure can also be used to selectively lyse, for example,
the cell wall or external membrane without lysing the nuclear
membrane. This can be useful for isolation of vector DNA, for
example, from the cytoplasm, while leaving chromosomal DNA (i.e.,
in the nucleus) behind.
[0036] Yet another advantage ensues from the obviation of the need
for high-salt elution solvents: the need for desalting procedures
is avoided. Desalting is generally necessary if, for example, the
purified nucleic acids are to be used in further reactions or
processes such as PCR, transfection, transformation,
electroporation, electrophoresis, mass spectroscopy, quantification
with fluorescent dyes, in vitro translation, stringent
hybridization, sequencing, genetic engineering, ligation,
restriction digestion, genomic mapping, clinical diagnostics, or
hybridization with other molecules. In the present methods, the
eluted biomolecule-containing solution does not need to be
desalted. The new methods also do not require precipitation in
organic solvents, or binding of the nucleic acids to silicon or
glass beads for desalting.
[0037] The use of low salt buffers also allows the new methods to
be compatible with electrophoretic or electroosmotic transfer of
materials. Salt can cause excessive heat generation in these
processes. Electrophoretic devices are generally inexpensive, can
be incorporated into other devices, and can allow isolation of, for
example, less fragmented nucleic acids (e.g., compared to flow
techniques). Electrophoresis can also be used to concentrate
nucleic acid samples (i.e., electroconcentration).
[0038] Still another advantage of the present methods is that the
methods avoid the need for addition of nuclease inhibitors. The
majority of proteins are believed to be denatured at pressures
lower than 100,000 psi at ambient temperature and neutral pH,
whereas nucleic acids can withstand substantially higher pressures.
Altering pH or temperature can further enhance protein
denaturation. Thus, a pressure pulse of, for example, 120,000 psi
at pH 4 and 25.degree. C. can effectively inactivate nuclease
activity without adversely affecting the desired nucleic acids.
[0039] Moreover, it is known that arginine residues of proteins
react with 1,2 and 1,3-dicarbonyl compounds such as phenyl glyoxal,
2,3-butanedione and 1,2-cyclohexanedione, to form condensation
products that can be stabilized by borate ions (Creighton, T E,
"Proteins: Structures and Molecular Properties, 1993, W.H. Freeman
and Company: New York, pp 12-13 and references therein). By using a
dicarbonyl compound that is attached to a solid support, nucleic
acid binding proteins such as nucleases and histones may be
retained in the purification process. A charged molecule bearing a
dicarbonyl moiety is useful since excess reagent can be removed by
electrophoresis. The condensation of arginine with a dicarbonyl
compound can be accelerated by pressure.
[0040] Centrifugation is generally avoided in the processing of the
samples for the new methods. This is an advantage in that
centrifugation can generate shearing forces and pressure drops that
may irreparably damage the integrity of many biomolecules, thereby
decreasing the yield and quality of the isolation. Moreover, the
new methods eliminate much of the handling and pipetting of the
biomolecule-containing solutions. As a result, much longer mRNA
strands, for example, which can be shorn by routine handling and
pipetting, can be isolated intact, thereby facilitating formation
of more reliable cDNA libraries, even from mRNA molecules present
in low concentration or low copy number. The new methods can give
yields of greater than 95% with high purity and speed.
[0041] Because all of the steps in the new methods can be carried
out in a single solvent, no additional time is required for
manipulation of solvents prior to each step. Moreover, the effects
of pressure are manifest very rapidly; pressure is transferred
through the sample at the speed of sound. As a result, the new
methods require only the time that it takes to spatially separate
the sample constituents; the need to wait for the nucleic acid to
precipitate in alcohol, for example, is avoided.
[0042] Furthermore, the new methods can be scaled up or down over a
large range of sample sizes, from the isolation of the genomic DNA
from a single hair follicle to the purification of a plasmid from a
megaprep of bacteria. Sample volumes as small as 1 femtoliter or as
large as 5 liters (e.g., for commercial nucleic acid preparation)
can be accommodated by the new methods. Small-scale nucleic acid
isolations can be completed within seconds; large-scale isolations
may take a few minutes.
[0043] Moreover, essentially the same methods can be used for the
isolation of small nucleic acids (e.g., less than 50 bp) or large
nucleic acids (e.g., larger than 1,000,000 bp). The small molecules
elute at lower pressures and lower salt concentrations, and can
therefore be independently isolated from samples containing both
large and small nucleic acids.
[0044] The new methods are also suitable for isolating nucleic acid
from a broad range of samples, including, but not limited to,
blood, urine, semen, mucal scrapings, sweat, hair, bone, pus,
saliva, fecal matter, biopsy tissue, amniotic fluid, synovial
fluid, plasma, prokaryotic (e.g., bacteria) or eukaryotic cultures
(e.g., plant tissue, yeast, tumor cells), viruses, viroids, and
blood-stained materials. Pressure can also enhance dissociation of
proteins from nucleic acids.
[0045] Hyperbaric pressure can cause nucleic acids to adopt compact
configurations which confer added resistance to shearing, nicking,
and enzymatic degradation, thus yielding a purified nucleic acid of
improved quality.
[0046] The use of hyperbaric pressure also improves electrophoretic
and electroosmotic processes by suppression of gas bubble
formation, which can block the transmission of electric fields.
[0047] The new methods are also amenable to automation. The new
methods require little human intervention; no additional pipetting,
decanting, centrifugation, precipitation, or resuspension of the
nucleic acid is generally required. The methods are also highly
efficient, and are thus both cost-effective and suitable for
high-throughput screening processes (e.g., genetic screening, drug
screening). Since the new methods rely on physical processes,
little customization is required for different applications (i.e.,
sample specimens).
[0048] In an example of high-throughput methods, a multi-column
array is used. Such an array can include ninety-six miniature
columns built into a microtiter plate-type device, each column
packed with DEAE cellulose retained by a frit and having a volume
of a few hundred microliters. In another version, the array can
include patches of a NUCLEPORE.RTM.-type (Corning Separations
Division, Acton, Mass.) track etch membrane, derivatized to include
charged groups. Each individual pore would effectively be a
"column" of ion-exchange material, with a volume of about a
femtoliter each. Several thousand of these columns can be present
in each patch. The separation material and the wall of the column
can be made of the same substance. In still another version,
separation columns can be microfabricated on chips measuring only a
few microns in lateral dimension. Such columns can either contain a
filling material or use the walls of the device as a separation
material.
[0049] Typical procedures for RNA purification require lysis using
chaotropic agents (e.g., guanidinium salts, sodium dodecyl sulfate,
sarcosyl, urea, phenol, or chloroform), which disrupt the plasma
membrane and subcellular organelles, and inactivate ribonucleases,
or using a gentler solution that only solubilizes the plasma
membrane (e.g., hypotonic nonidet P-40 lysis buffer). The latter
reagents also require addition of a nuclease inhibitor. Organic
solvent extraction or silica membrane absorption methods are then
be used to extract the RNA from the cell lysate. Using the new
pressure-based methods, however, cell lysis and RNA purification
can be combined in a single procedure. This offers the advantages
of reduced human intervention, better control of contamination with
RNase, and a rapid processing speed, which also reduced the
potential for RNA degradation.
[0050] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 is a drawing of a resin-filled cartridge for use in a
pressure-modulation apparatus.
[0052] FIG. 2 is a drawing of a five electrode chip for use in a
pressure-modulation apparatus.
[0053] FIG. 3 is a drawing of an eight electrode chip for use in a
pressure-modulation apparatus.
[0054] FIGS. 4A to 4C are views of a chip that includes a diaphragm
for relaying pressure.
[0055] FIGS. 5A to 5C are views of a chip that includes a
hydrophobic valve for relaying pressure.
[0056] FIGS. 6A to 6C are views of a chip that includes a
compressible piston for relaying pressure.
[0057] FIG. 7 is a graph of percent recovery of nucleic acids as a
function of sodium chloride concentration at constant pressure for
three sizes of DNA: 50 bp ( . . . ), 4.6 kb ( - - - ), and 48.4 kb
(--).
[0058] FIG. 8 is a graph of percent recovery of nucleic acids as a
function of pressure at constant sodium chloride concentration for
three sizes of DNA: 50 bp ( . . . ), 4.6 kb ( - - - ), and 48.4 kb
(--).
[0059] FIG. 9 is a view of a sample cell for pressurization.
[0060] FIGS. 10A and 10B are side and top views, respectively, of a
high pressure purification cartridge.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Methods and devices are described for the highly selective
and efficient, low ionic strength isolation and purification of
biomolecules (e.g., nucleic acids, proteins, carbohydrates, and
small molecules) from many types of samples. The invention is based
on the observation that pressure can be used to effect the
dissociation of nucleic acids and other biomolecules from solid
phases to which they normally bind tightly (e.g., anion-exchange
resins), even at low salt concentrations.
[0062] General Procedure
[0063] In general, a solution containing a biomolecule to be
purified is introduced onto a solid phase at low pressure (e.g.,
ambient pressure). The solid phase, to which the biomolecule
present in the solution should now be bound, is washed with a
buffered second solution, in which the desired biomolecule will
remain bound to the solid phase at elevated pressure, whereas the
undesirable contaminants (e.g., proteins and lipids) will be
removed from the solid phase. When the washing has been completed,
the pressure is increased further to a level sufficient to cause
the desired biomolecule to be freed from the solid phase. While
this elevated pressure is maintained, fresh low salt buffer can be
used to wash the liberated biomolecule away from the solid phase
and into a collection vessel. These procedures can be fully
automated. The recovered biomolecule is free from high salt and can
be used in downstream enzymatic reactions.
[0064] Biomolecules that can be purified by this procedure include
nucleic acids (e.g., chromosomal DNA, viral DNA, plasmid DNA,
mitochondrial DNA, a DNA vector, an oligonucleotide, mRNA,
mitochondrial RNA, viral RNA, or mixtures of nucleic acids),
proteins (e.g., enzymes, antibodies, structural proteins,
metalloproteins, hormones, glycoproteins, mucins), and
carbohydrates and other small molecules (e.g., sugars, dyes,
synthetic drugs, cofactors, amino acids). The solid phase can be
made from any substance that selectively binds the desired
biomolecule at ambient pressure and has reduced affinity at
elevated pressure, such as an anion-exchange column, an oligo-dT
column, or an electrode coated with absorptive polymers.
[0065] In addition to binding to the desired biomolecule, the solid
phase can have other functions. For example, the solid phase can
absorb the biological samples (e.g., a sponge-type polymer); it can
assist in the lysis of the cells, for example, by mixing the solid
phase material with proteases (e.g., pepsin or trypsin), lipases,
or glycosidases (e.g., lysozyme) to digest proteins, lipids, and
polysaccharides, respectively; or it can include DNase for RNA
purifications, or RNase for DNA purification. Some solid phases can
bind nucleic acids, but only weakly interact with other negatively
charged molecules such as some proteins or lipids, or vice
versa.
[0066] The same solution or different solutions can be used to load
the biomolecule sample onto the solid phase, elute the impurities
away, and elute the biomolecule away from the solid phase.
Nonetheless, it is generally most desirable to use a single buffer,
both for ease of operation and to reduce waste. Whether the
solution acts as a wash buffer or as an elution buffer depends on
the pressure. At pressures greater than about 25,000 psi, for
example, large nucleic acids (e.g., more than 5,000 bp) can be
eluted in low salt buffers. In addition, at 25,000 psi, small
nucleic acids such as those used in the Sanger sequencing method
can be eluted at still lower salt concentrations. At ambient
pressure, however, it is necessary to use an eluent having a much
higher concentration of salt. High salt eluents can interfere with
downstream reactions, especially enzymatic reactions used in the
manipulation of nucleic acids (e.g., for sequencing or
amplification), and are therefore ideally avoided.
[0067] As described above, for a low salt buffer to be an effective
elution solvent, the pressure in the vicinity of the solid phase
must be greatly increased, often to several thousand times ambient
pressure. Suitable pressure-modulation apparatuses for generating
the requisite pressure are described in PCT Appln. No. US/96/03232,
PCT Appln. No. US/97/11198, and U.S. Ser. No. 08/903,615, which are
hereby incorporated by reference. A chip or a cartridge containing
the solid phase can be inserted into this apparatus, for example,
and the purification can be carried out within the apparatus. The
apparatus can be made in various configurations to accommodate the
full range of sample sizes.
[0068] Other properties important to separation can also be altered
by pressure. These include the denaturation and refolding of
proteins and the association of nucleic acids into double-stranded
forms (or dissociation into single-stranded forms), both of which
can affect the filtration, sedimentation velocity or equilibrium,
radius of gyration, exclusion volume, electrophoretic mobility,
and/or chemical reactivity of biomolecules. Any of the separation
techniques described herein can be used to equivalent effect by
selection of appropriate conditions.
[0069] All of the steps of biomolecule purification, including
lysis, binding, elution, and isolation, can be automated.
Additionally, the pressure can be scaled up to allow elution of
progressively larger biomolecules, thereby facilitating the
isolation of specific sizes of molecules. A pressure gradient
(i.e., either stepped or continuous) can also be set up within the
devices. A pressure gradient (e.g., step function) can be used, for
example, to fractionate samples. Fractionation can be used to
purify specific fragments from a partially degraded sample or a
highly diverse sample (e.g., a cDNA library).
[0070] Pressure can alter the effective hydrodynamic radius of
gyration of a macromolecule such as a nucleic acid or a protein. In
general, such a change can alter the elution position (i.e.,
V.sub.e, the exclusion volume) of the macromolecule on a
size-exclusion medium (e.g., silica, a rigid plastic such as
polystyrene, or a porous hydrogel such as SEPHADEX.TM. and
SEPHAROSE.TM. (Pharmacia) resins). The molecule does not need to
bind to the resin; rather, the molecule's ability to enter a pore
can be affected by the hydrostatic pressure at which the separation
is conducted. The ability to select a pore size such that a given
molecule is included at one pressure and excluded at another can
allow improved separations. In particular, molecules that co-elute
at one pressure can be separated at another.
[0071] Nucleic Acids
[0072] Examples of applications of the present method include
purification of nucleic acids from blood, cell culture (genomics or
infectious disease) or tissue (e.g., tumor biopsy) for clinical or
research purposes, purification of microbial DNA for genetic or
biotechnology research, desalting of DNA, forensic analysis (e.g.,
purification of DNA from hair, blood, semen, or tissue found at the
scene of a crime), and purification of PCR products.
[0073] The isolation and purification techniques of the invention
can be applied to both natural and artificial nucleic acids.
Artificial nucleic acids are typically based on ribose or
deoxyribose, or geometrical analogs thereof. Linkages other than
the natural phosphodiester bonds can be employed in artificial
nucleic acids, including thiophosphate and amide bonds.
[0074] Among RNA molecules, the most common classes include mRNA
(messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), viral
RNA, and RNA that has been copied from DNA in vitro.
[0075] For most applications, chromosomal DNA is linearized from a
cyclic form, if present as such, and can also broken into smaller
pieces for ease of manipulation. The breaking may be done
non-specifically (e.g., using the exonuclease DNase I, or by
sonication); or by specific cutting with an enzyme (a "restriction
endonuclease") or by chemical means; helicases, topoisomerases,
kinases, and other nucleic acid-specific enzymes can also be used,
for example, to alter migration or absorption properties of the
nucleic acids. If no other alterations have been made, such DNA may
still be generally referred to as chromosomal.
[0076] A plasmid is an independently replicating DNA found in
bacteria, generally circular, and is often used for transmission of
genes in biotechnology. Plasmid DNA may also be cut into small
pieces, usually by restriction enzymes. The fragments produced by
restriction enzymes are often named by their apparent molecular
weights, in "base pairs" or "bases" of DNA. For example, a "4.6 kB"
DNA fragment is about 4,600 bases long, whether it is double
stranded or single stranded.
[0077] Most proteins (e.g., nucleases) are inhibited by pressure. A
120,000 psi pulse, for example, can irreversibly denature the
nucleases in a sample. It is important, especially in attempted
isolations of RNA, to denature nucleases such as RNAse to prevent
degradation of the desired nucleic acids during the isolation
process. Pressure can be used here in place of chemical inhibitors.
In some cases, nuclease denaturation and cell lysis can be
accomplished simultaneously.
[0078] Ribonuclease A is subject to cold-denaturation at elevated
pressures. Addition of a reducing agent can subsequently facilitate
irreversible denaturation by reducing the disulfide bonds of the
nuclease. Thus, for example, 10 mM .beta.-mercaptoethanol can be
added to a sample, the sample can be cooled to -20.degree. C., and
the pressure can be increased to 60,000 psi to irreversibly
denature the nuclease.
[0079] Additionally, activation of RNases may be desirable for
certain applications, such as for the extraction of genomic DNA. In
this case, conditions that activate or enhance RNase activity can
be obtained through a combination of temperature, pH, or pressure
(e.g., using pressures high enough to accelerate enzymatic
activity, but still lower than the pressures required to denature
the enzyme).
[0080] For example, at 220 MPa (about 32,000 psi), 100% of Lambda
DNA (.lambda.DNA) was eluted off a DEAE column with 50 mM Tris-HCl
buffer, pH 8.5. At atmospheric pressure (0.1 MPa, or 14 psi),
higher salt buffers (e.g., 1 M sodium chloride Tris-HCl buffer) are
generally required to elute the DNA away from the DEAE resin.
Moreover, plasmid DNA dissociated at lower salt concentrations and
lower pressures than .lambda.DNA. Thus, low molecular weight DNA
molecules can be dissociated at lower salt concentrations and lower
pressures than higher molecular weight molecules. For sample
analysis and other applications, it can be useful to separate
nucleic acid fragments by size.
[0081] Eukaryotic cells can express cloned genes (i.e., transient
and stable heterologous expression), using eukaryotic expression
vectors purified by the new methods. To analyze and identify the
function of cloned eukaryotic genes, for example, eukaryotic
expression plasmids carrying the gene of interest can be obtained
in a form suitable for introduction into mammalian cells. It is
often necessary to generate a large panel of mutants for
structure-function studies of a particular eukaryotic gene.
Therefore, the new methods provide a method for rapid and facile
analysis.
[0082] Isolation of DNA by the new methods can be used for numerous
applications including, but not limited to, protein expression and
protein structure function studies in eukaryotic cells, Southern
blot analysis, in vitro transcription, ligation, and
transformation, heterologous protein expression in bacteria or
yeast, microinjection studies, PCR, DNA sequencing, viral DNA
detection, paternity testing by RFLP analysis, and genetic
screening by single-strand conformation polymorphism (SSCP) or
non-isotopic RNase cleavage assay (NIRCA.TM.; Ambion, Austin,
Tex.). Similarly, isolation of RNA provides a variety of
applications including, but not limited to, genetic analysis, cDNA
library construction, microinjection into oocytes, differential
display, Northern blot analysis, RNase protection assays, in vitro
translation, reverse transcriptase PCR (RT-PCR), and detection of
viral RNA (e.g. HIV, hepatitis C, hepatitis A, and HTLV-1) in human
blood. Isolation of nucleic acids produced in vivo generally
requires the lysis of the host cells in which the nucleic acids are
contained. Any cell lysis method can be employed in conjunction
with the new methods, provided that it produces a yield and quality
of nucleic acid sufficient for an intended subsequent use. Lysis
can be carried out inside or outside of a pressurized or
pressurizable apparatus.
[0083] Cells differ in their resistance to lysis. For example, many
animal cells can be lysed through contact with even small amounts
of detergents or organic solvents. Animal cells have little or no
containing structure except a plasma membrane and its embedded
proteins. Dissolution of the cellular membrane allows all of the
cellular contents to diffuse into the lysing solution.
[0084] Animal cells can also be lysed by changing the osmolarity of
the solution (e.g., by lowering it from its normal level of about
300 mOsm to about 0 to 10 mOsm. Osmotic lysis is especially
effective on cells which are normally exposed to a fixed, normal
osmolarity, such as human tissue.
[0085] Animal cells can also be lysed by chemical, enzymatic (e.g.,
proteases can be used in conjunction with chemicals to lyse
membranes when purifying nucleic acids), or mechanical methods. For
example, mammalian tissues can be lysed by strong mechanical shear
in solution, such as by grinding and dispersing with a Dounce
homogenizer or a kitchen blender.
[0086] Viruses can generally be lysed under conditions similar to
those described above for the lysis of animal cells. Some viruses
can be lysed under even milder conditions, such as a change in
ionic conditions (e.g., by removal of polyvalent cations) or
temperature.
[0087] Bacterial cells often have strongly crosslinked cell walls
in addition to plasma membranes. Consequently, they can be more
difficult to lyse than animal cells. The cell walls are generally
resistant to most detergents and chemicals, and also stabilize the
plasma membrane against rupture by change in osmolarity. However,
bacteria can be grown in the presence of antibiotics (or under
specific metabolic conditions) that prevent the formation of cell
walls, facilitating subsequent lysis by the means described above
in connection with animal and viral cells.
[0088] Yeast cells, most plant cells, and some insect cells have
still more durable cell walls. More powerful methods are required
for lysis of such cells. These methods, which can also be used for
lysis of the less robust cell types described above, include sudden
depressurization from high pressure to atmospheric pressure, often
by flow from a pressurized cell through a fine needle (i.e., French
press). An alternative for rupturing the durable cell walls is the
grinding of cells with glass beads (or other durable particles),
often in a violently reciprocating shaker (e.g., in a "Nossal
shaker"). When prevention of enzymatic activity after cell lysis is
particularly critical, cells or tissues can be frozen, for example
in liquid nitrogen, and then ground in the frozen state
("cryogrinding").
[0089] One method for purifying DNA relies on the absorption of DNA
by silica at high concentrations of chaotropic salt, especially
sodium iodide. Typically, DNA is absorbed to the silica surface at
this high salt concentration, and impurities (and excess iodide)
are washed away in the presence of methanol/water. On reduction of
the methanol concentration, the DNA is released. This process can
be pressure-sensitive, and thus either some of the expensive sodium
iodide can be replaced by application of the DNA to the silica
resin at high pressure, or the elution itself can be facilitated by
pressure modulation.
[0090] Hyperbaric Cell Lysis and Extraction
[0091] For the purification of non-secreted biomolecules from cells
(e.g., from cell cultures or tissue), the cells must be lysed, or
at least permeabilized, prior to the introduction of the sample
onto the solid phase. There are many known methods for cell lysis,
as described above in connection with the isolation of nucleic
acids, including chemical methods (e.g., phenol/chloroform
extraction, treatment with guanidinium salts, chaotropic salts,
detergents such as sodium dodecyl sulfate, or enzymes such as
proteinase K) and physical methods (e.g., boiling, French pressing,
Douncing, vortexing in the presence of glass beads, or
sonication--see, e.g., Bollag et al., "Protein Methods," 2nd Ed.,
1996, pp. 27-56). Often these methods can be sensitive to
variations in time and temperature.
[0092] Another suitable method is the use of hyperbaric pressure to
cause cell lysis. Hyperbaric lysis can be carried out in the same
solvent as will be used as the loading buffer for later introducing
the sample onto the solid phase, or can be carried out in a
different solvent. Still another suitable method is the use of a
chemical agent (e.g., a detergent) in combination with pressure.
For example, a small amount of a chaotropic salt can be used to
prime the cells for lysis; after the cells have been treated with
the chaotropic agent, they can be lysed at lower pressure.
[0093] More gentle lysis procedures can be especially preferable
for isolating single-strand (e.g., RNA) or high molecular weight
nucleic acid molecules. Single strand nucleic acids are more easily
shorn; high molecular weight nucleic acids are statistically more
likely to be damaged during lysis. Furthermore, pressure-based
lysis can be used to preferentially fractionate cellular material
(e.g., lysis of external cell wall/membrane while maintaining the
nuclear membrane for the isolation of extranuclear constituents) By
modulating hydrostatic pressure, a tissue or cell suspension can be
rapidly freezed and thawed while maintaining a low temperature.
This technique can be advantageous in that the action of nuclease
enzymes can be inhibited at low temperature; nucleases can also be
denatured by pressure at low temperature.
[0094] In a typical procedure, the sample is cooled to -20.degree.
C. to freeze the sample, then the pressure is cycled between 60,000
psi (i.e., to melt the sample) and 14 psi (i.e., to re-freeze the
sample) repeatedly until the sample is sufficiently disrupted.
Alternatively, the temperature can be modulated by cyclic
application of pressure to a sample or a gas-containing
pressurizing medium in proximity to the sample. The compression and
decompression of the gas either in the sample compartment or in the
pressurizing medium modulate the temperature via the Joule-Thompson
effect.
[0095] Molecules can be extracted from the cell with pressure,
allowing direct application of the extracts to the various solid
supports. Pressure-based extraction methods are easily integrated
with the hyperbaric methods described herein. Exposure of cells to
high pressure can cause lysis or permeabilization of cells without
any further treatment.
[0096] For example, bacteria in a solution in a test tube can be
exposed to a pressure of 60,000 psi. While the cells are held at
such a pressure, molecules can diffuse out of the cells. Even
relatively large molecules such as nucleic acid plasmids can
diffuse under these conditions. Application of an electric field
across a pressurized sample can further increase the extraction
efficiency.
[0097] Repeated cycling between ambient and elevated pressures can
cause rupture of cells. For example, yeast cells exposed to 30,000
psi for 240 cycles were fragmented, releasing at least some of
their intracellular contents. This mechanism of fragmentation is
different from that used in a French-type pressure cell, where a
pressurized solution containing cells is released through a fine
orifice. There, the fragmentation generally requires both a sudden
pressure drop and high rate of shear in the solution. The new
methods require no shear, and are thus gentler to the cells. In at
least some cases, the new methods also do not require sudden
depressurization.
[0098] Because the sample integrity can be maintained during
molecule extraction, the new pressure-based extraction methods
allow hundreds, thousands, or even more samples to be processed in
parallel. The samples can be in the wells of microtiter plates, for
example. For smaller volumes, the cells can be present in droplets
on bibulous media. Imprinted boundaries of hydrophobic materials
can also be used to separate the droplets. The bibulous material
can also serve as the separation medium, binding the desired
molecules for later elution. The permeabilizing (or lysis) and
separation steps can each be carried out at a high, but isostatic,
pressure. Thus, multilayer arrays of bibulous and separation media
can be made, having multiple sample spots. Moreover, because the
pressure is isostatic, fluids can be processed through such arrays
at high pressure without requiring that the array, for example, or
machinery handling it within the hyperbaric volume, have sufficient
integrity to withstand the pressure differentials. Thus, for
example, cells contained in a moist DEAE grid on a paper strip can
be pressure-lysed in situ and then "blotted" (wet layer applied
"above", dry layer "below") to transfer the molecules of interest
to one layer, while the cell debris and extraneous molecules are
left behind or transferred to other layers. With suitable
equipment, selective extraction and purification of molecules from
cells can be substantially automated.
[0099] Cells can be lysed in a device intended to carry absorbed
biomolecules into a pressure chamber, either before or after
application of pressure. The cell debris can be retained by
filtration on a medium (e.g., filter paper), while the soluble
components (i.e., including the biomolecules) are carried to an
absorptive material by pressure-induced flow of buffer. The
biomolecules can be later released from the resin by changes in
pressure. If further purification is desired, the output of
pressure-released biomolecules from the retention medium can be
absorbed by another medium (e.g., another resin).
[0100] Provision can be made for control of such parameters as
shear forces and enzymatic attack on the nucleic acids to be
isolated. If, for example, the biomolecule of interest is a small,
double-stranded DNA molecule (e.g., a plasmid, a cosmid, or viral
DNA) from a bacterium, then it may not be necessary to completely
lyse the bacteria. Lysis of the plasma membrane, accompanied by
physical and/or chemical treatment (e.g., heating in the presence
of protease K) to inactivate DNA-degrading enzymes, can be used to
leave the contaminating bacterial chromosomal DNA inside the
bacterial cell wall for ease of isolation. Cartridges
[0101] One design for an isolation device is shown in FIG. 1. This
device is a cartridge 10 made of metal (e.g., titanium, stainless
steel, or aluminum), plastic (e.g., a thermoplastic such as
polypropylene or polytetrafluoroethylene), glass, quartz, stone
(e.g., sapphire), or a ceramic, adapted to fit into a
pressure-modulation apparatus such as that described in PCT Appln.
No. US/96/03232.
[0102] The cartridge is generally formed in the shape of a tubular
column, although other designs can be used. Regardless of the
shape, the cartridge usually has two openings 12 and 14, one 12 to
allow fluid to enter and another 14 to allow the fluid to exit.
Between the two openings, but within a channel 16 common to the
openings, a solid phase material 18 is packed. The solid phase can
be any of a multitude of nucleic acid-binding materials, including
silica gel, glass, anion-exchange resin (e.g., DEAE), tethered
specific binding molecules. Binding groups, bound to the resin by
suitable chemical or physical linkage, can include nucleotides or
nucleic acids, tethered proteins or peptides, polymers, DNA-binding
molecules (e.g., ethidium, acridinium), or other small molecules
(e.g., sugars, benzodiazepines, drugs). The solid phase should
ideally be able to withstand the hyperbaric pressures utilized in
the new methods without permanent deformation or malfunction. Thus,
solid phases that can withstand higher pressures can be preferable
(e.g., DEAE-coated glass can advantageously be used in place of
certain silica-based resins for some applications).
[0103] The cartridge can be designed such that the openings are in
direct fluid contact with the reaction chamber of the
pressure-modulation apparatus, or can be designed as a closed
system with valves and pistons that can open and close to regulate
the pressure and the fluid flow within the cartridge. The valves
and pistons in this embodiment can be controlled either
electronically or mechanically.
[0104] Cartridges designed for use with samples derived from lysed
whole cells can optionally include a filter or membrane 19, having
a pore size suitable for removal of any remaining cell debris prior
to introduction of the sample onto the solid phase. This filter may
be larger in cross-sectional area than the resin chamber to prevent
pressure gradients.
[0105] The volume of the cartridges can vary widely. For example,
the cartridge can have an internal volume that can range from a
femtoliter (fl) up to 10 ml or more (e.g., 1 .mu.l to 1 ml). A fl
is the approximate volume of a 10 .mu.m diameter capillary
penetrating a 100 .mu.m thick (4 mil) membrane. The volume of the
separation medium will depend upon the intended use. Typically, the
solid phase occupies about half of the internal volume of the
cartridge, although some cartridges can be filled to nearly to
their full capacity while others may be filled just one tenth of
the way. In some cases, the cartridges can be reused. The volume
applied to the column to load it is arbitrary, and the relevant
column parameter for separation is the binding capacity of the
column.
[0106] In an example of one mode of operation of the cartridges,
the sample is typically dissolved or suspended in a low-salt buffer
solution and introduced at opening 12. The cartridge 10 is placed
in the pressure-modulation apparatus. A low pressure flow of buffer
solution is used to force the sample through the membrane 19 and
through the solid phase 18. Nucleic acids in the sample bind to the
solid phase; the flow-through continues through the solid phase and
emerges from opening 14. In some cases, the flow-through is taken
up by a sample output tube leading to an input on a detection
device (e.g., a UV-vis spectrophotometer). The low pressure flow of
the buffer solution is continued until the detection device shows
that no additional residues are washed away. The flow-through is
discarded.
[0107] The pressure is then increased to 500 to 100,000 psi,
causing the nucleic acid to be released from the solid phase. More
of the buffer solution is introduced through opening 12, and the
nucleic acid-containing flow-through that emerges from opening 14
is collected. This flow-through can also be fed into a detection
device and analyzed, and the flow continued until the nucleic acid
detected in the flow-through falls below a set threshold level.
[0108] The cartridges can also include multiple compartments. For
example, the individual compartments can contain different solid
phase materials (e.g., ion-exchange resin, silica gel, tethered
oligonucleotides). Reactions can be carried out within the
cartridges.
[0109] For instance, a cartridge of the present invention can be
used as a PCR reaction vessel, if placed within a thermal cycling
apparatus after the solid phase has been washed to remove
non-nucleic acid impurities and the nucleic acid has been eluted
from the solid phase into, for example, a second compartment in the
cartridge.
[0110] A multi-compartment cartridge can also be used to
concentrate nucleic acids. In such a cartridge, fluids can be moved
hydrodynamically or electrically, or both. In one example, DNA from
a large sample can be concentrated hydrodynamically onto a resin,
small molecule impurities can be washed away, then the DNA can be
electrophoresed into a downstream cartridge. This process is herein
termed electroconcentration.
[0111] In another two-part cartridge, nucleic acids are eluted from
a first compartment (e.g., containing an anion-exchange resin),
using pressure, and concentrated in a second compartment (e.g.,
containing silica gel) that requires different conditions for
elution. Thus, concentrated nucleic acids can be isolated from eve
dilute samples containing many impurities. Alternatively, the
eluted sample can be automatically transferred to another device
(e.g., a disk, a pad, a bead, or a detection device).
[0112] Silica and glass are commonly used in isolation of nucleic
acids, particularly double-stranded DNA (dsDNA). In a high
concentration of a chaotropic salt, such as NaI (sodium iodide),
DNA binds to glass surfaces. After other impurities are washed away
by a solvent which retains the DNA on the glass, which solution can
be the chaotropic salt solution, or an aqueous solution containing
a nonsolvent for DNA, such as an alcohol, then the DNA can be
released and eluted by exposure of the column to dilute buffer.
There are several steps in this procedure in which high pressure
might be used to simplify the procedure.
[0113] Cartridges containing multiple, layered resins are also
within the scope of the claims. A layer of cation-exchange resin,
for instance, will capture any positively charged proteins which
might bind to the DNA. Hydrophobic (e.g., reverse-phase) resins can
bind to the lipids in the sample.
[0114] Devices Using Electrophoresis or Electroosmosis
[0115] Alternative designs for isolation devices are depicted in
FIGS. 2 and 3. These devices are in the form of a chip, with an
electrode array aligned along at least two axes. The individual
electrodes are coated with a solid phase material. In some cases,
all of the electrodes are coated with the same material; in other
cases, the coatings differ from electrode to electrode or form a
coating gradient along a capillary connecting two or more
electrodes. The chips can optionally be interfaced with an
analytical device such as a mass spectrometer or a capillary
electrophoresis device.
[0116] Although the design of the chips can vary widely, the
operation of the chips is similar irrespective of the design. In
one of the simplest designs (FIG. 2), electrodes 20, 30, 50, 70,
and 80 are electrically connected to contact points 22, 32, 52, 72,
and 82, respectively. A sample containing, for example, chromosomal
DNA to be isolated is introduced at electrode 20 at ambient
pressure. In addition to the nucleic acids to be isolated, the
sample can include salt (e.g., 50 to 350 mM sodium chloride) and
various impurities. Electrode 20 is coated with a material that
absorbs the sample (e.g., an ion-exchange resin such as DEAE).
[0117] The chip 25 is placed within the sample chamber of a
pressure-modulation apparatus (e.g., the apparatus described in
U.S. Ser. No. 08/903,615) adapted to supply a switchable electrical
voltage at the contact points 22, 32, 52, 72, and 82. A voltage
potential is supplied between electrodes 20 and 30 (i.e., electrode
20 is the anode and electrode 30 is the cathode) while the system
is at ambient pressure. The potential causes the sample to flow
through capillary 40, which is filled with a size-exclusion
filtration material (e.g., 0.5%-2% agarose) that retains large
cellular debris but allows nucleic acids, proteins, lipids, and
other small cellular components to pass through.
[0118] The flow-through then passes through to electrode 50, which
is coated with an anion-exchange resin. Nucleic acids in the
molecule are trapped at electrode 50, while other components in the
flow-through continue through the aqueous solution in capillary 60,
and ultimately, to electrode 30. Electrode 30 includes a material
(e.g., polyacrylamide) that traps the impurities that reach it. The
voltage potential between electrodes 20 and 30 is then
discontinued.
[0119] The pressure in the system is increased to a moderately
elevated level (e.g., 500 to 10,000 psi). A voltage potential is
set up between electrodes 50 (anode) and 80 (cathode). The moderate
pressure causes the smallest nucleic acids (e.g., less than 5,000
bp) to dissociate from the anion-exchange resin at electrode 50,
and the potential causes the nucleic acid to migrate through the
liquid phase in capillary 90 and finally into electrode 80, which
includes a reservoir. The potential is discontinued.
[0120] The pressure in the system is increased to a more elevated
level (e.g., 12,000 to 100,000 psi). A voltage potential is set up
between electrodes 50 (anode) and 70 (cathode). The high pressure
causes the remaining nucleic acids to dissociate from the
anion-exchange resin at electrode 50, and the potential causes the
nucleic acid to migrate through the liquid phase in capillary 75
and finally into electrode 70, which includes a reservoir. The
potential is discontinued, the pressure is lowered to ambient
pressure, the chip is removed from the pressure-modulation
apparatus, and the large nucleic acid fraction, including the
chromosomal DNA, can be removed from electrode 70.
[0121] In another design (FIG. 3), electrodes 100, 110, 120, 130,
140, 170, 200, and 220 are electrically connected to contact points
102, 112, 122, 132, 142, 172, 202, and 222, respectively. A whole
blood sample is introduced at electrode 100 at ambient pressure.
Electrode 100 is coated with a wicking material that absorbs the
sample.
[0122] The chip 190 is placed within the sample chamber of a
pressure-modulation apparatus adapted to supply a switchable
electrical voltage at the contact points 102, 112, 122, 132, 142,
172, 202, and 222. A voltage potential is supplied between
electrodes 100 (anode) and 120 (cathode) while the system is at
ambient pressure. The potential causes the sample to flow through
liquid-filled capillaries 105 and 125 and electrode 110. At the
junction between electrode 110 and capillary 125, a filter 114
prevents white blood cells from passing. An example of a suitable
filter is the HEMAFIL.phi. Nucleopore membrane (Corning Separations
Division, Acton, Mass.), a polymeric microporous track-etch
polycarbonate having a pore size of 4.7-5.0 .mu.m. Thus, the white
blood cells become trapped at electrode 110, while red blood cells
continue to migrate to electrode 120.
[0123] The pressure is increased momentarily (e.g., to 80,000 psi,
120,000 psi, or higher), lysing the cells at electrodes 110 and
120, and irreversibly inactivating any nucleases present in the
cell lysates. The pressure is then restored to ambient pressure. A
potential (e.g., 100-200 V, or a constant current, e.g., 20-40 mA,
or a constant power, e.g., 500 watts) is then provided between
electrodes 110 (anode) and 220 (cathode). Capillary 115 contains a
size-exclusion material or an ion-exchange material, for example,
that can retain large cellular debris but allow nucleic acids,
proteins, lipids, and other small cellular components to pass
through.
[0124] The flow-through then passes through to electrode 140, which
is coated with oligo-dT, and through to electrode 170, which is
coated with an anion-exchange resin. RNA in the white blood cell
lysate is trapped at electrode 140 and DNA in the white blood cell
lysate is trapped at electrode 170, while the remaining components
in the flow-through continue through the aqueous solution in
capillary 176 and ultimately to electrode 220. Electrode 220
includes a reservoir that traps the impurities that reach it. The
potential is then discontinued.
[0125] A potential is supplied between electrodes 140 (anode) and
130 (cathode). The potential causes the RNA to dissociate from the
solid phase at electrode 140, migrate through the liquid phase in
capillary 144, and finally to electrode 130, where a reservoir
traps the RNA. The potential is discontinued.
[0126] The pressure in the system is then increased to an elevated
level (e.g., 20,000 to 100,000 psi), and a potential is supplied
between electrodes 170 (anode) and 200 (cathode). The pressure
causes the DNA to dissociate from the solid phase at electrode 170,
and the potential causes the nucleic acid to migrate through the
liquid phase in capillary 174, and finally to electrode 200, where
a reservoir traps the DNA. The potential is discontinued, the chip
is removed from the pressure-modulation apparatus, the purified
white blood cell RNA can be removed from electrode 130, and the
purified white blood cell DNA can be removed from electrode
200.
[0127] There may be multiple versions of this device or consumables
for use with the device that are optimized for various applications
and sample sizes. For instance, a miniature version can be highly
parallel and/or interface into a downstream biochip. Examples of
sample sizes include 1 fl, 1 pl, 1 nl, 1 .mu.l, 1 ml, 10 ml, and
intermediate sizes.
[0128] The chips can be made from any suitable material which can
be planar in form and worked by conventional processes. Base
materials include plastics, such as polypropylene or
polytetrafluoroethylene (PTFE); inorganic oxides, such as silica,
glass and ceramics; metals; and semiconducting materials, such as
silicon. The contact points and electrodes are made of conductive
materials, including metals (such as gold, silver, copper, aluminum
or iron), semiconductors, conductive polymers, and aqueous
solutions, optionally stabilized by fabrics, gels, and the
like.
[0129] The chips can be designed such that the fluids in the
capillaries are in direct fluid contact with the reaction chamber
of the pressure-modulation apparatus. More preferably, the chips
can be designed as a closed system with a diaphragm (FIG. 4), a
piston (FIG. 5), or a hydrophobic valve (FIG. 6), which relays the
pressure from the reaction chamber to the capillaries and
electrodes.
[0130] As shown in FIGS. 4A to 4C, the chip 240 can include a
recessed area 242, in which the electrode array is situated. A
flexible, elastic membrane 244 spans the recessed area 242, to form
a diaphragm. One or both surfaces of the membrane can be flexible.
The membrane transmits external pressure to the electrode array,
while simultaneously providing a hermetic seal that prevents fluids
from being transferred.
[0131] The chip 250 shown in FIGS. 5A to 5C also includes a
recessed area 252, in which the electrode array is situated. A
solid lid 254 is placed over the recessed area. A channel 256 is
drilled through one side of the chip, leading into the recessed
area 252. The wall of the channel 256 is precoated with a
hydrophobic material, such that water and other fluids are unable
to traverse the length of the channel 256 under ambient conditions.
As the pressure is increased, however, the fluids overcome the
hydrophobic interactions and pass through the channel 256, thereby
modulating the pressure within the recessed area 252.
[0132] In a third design, shown in FIGS. 6A to 6C, a chip 260
includes a recessed area 262, in which the electrode array is
situated. A compressible, elastomeric piston 264 is mounted in the
recessed area 262. When the pressure in the reaction chamber is
increased, the piston 264 becomes compressed, thereby increasing
the pressure at the electrode array without allowing fluid transfer
between the reaction chamber and the electrode array.
[0133] In addition, it is possible to retain fluid in narrow
capillaries, such as capillaries having diameters in the range of
10 to 1000 microns, without needing an external restraint, provided
that the surface of the capillary has a polarity (surface energy)
which permits the fluid to wet the capillary surface. Processes may
be performed in such capillarity-filled capillaries without an
external cover, if the space above the open capillary is saturated
with the vapor phase of the fluid. Alternatively, any sufficiently
non-wettable (hydrophobic or solvophobic) surface can be used to
close the upper surface of the capillaries, and thereby allow
stacking of chips without having the fluid in the capillary spread
beyond the capillary by wetting the film. For aqueous solutions, a
sheet of polypropylene or PTFE, or a coating on the back of the
next chip in the stack, could serve the purpose.
[0134] Disposable Two-syringe Device
[0135] In an example of still another embodiment of the invention,
the sample is placed into a first (loading) syringe having a DEAE
resin cartridge attached at the narrow end. This system must have a
very small resin chamber so that high pressures can be generated
and the materials must be able to withstand the high pressures. The
plunger is slowly depressed, so as not to create a significant
pressure gradient. As the sample is loaded onto the resin, the
waste is discarded. Low salt (e.g., 10 to 300 mM) buffer is placed
in the syringe. The buffer can contain magnesium and other
cofactors necessary for downstream enzymatic techniques. A measured
quantity (e.g., 100 .mu.l to 10 ml) of the buffer is used to wash
the resin to remove non-DNA contaminants.
[0136] A second (collection) syringe is added to the first. The
resistance of the plunger of the second syringe is adjusted such
that the pressure needed to move the loading syringe causes the
dissociation of the nucleic acid from the resin. The resisting
force exerted by the collection syringe can be adjusted by means of
low-angle threads in the syringe and piston. The angle of the
threads can be adjusted to change the pressure. For applications
where consistency of yield and purity are crucial, the pressurizing
step can be carried out by, or with the aid of, a machine that
maintains a consistent pressure and flow rate, such as an
expression chamber with a check valve.
[0137] Another version of this system would use a device which
applied an equal force to two opposing pistons and (with much less
force) moved the two syringes simultaneously to achieve a flow. In
another version, two pistons supply force, with a small pressure
differential between them. This system can be immersed in a
pressurizing medium (such as water) so as to avoid the use of
pressure resistant materials and small resin capacity in the
disposable component.
[0138] Sample Cell for Pressurization
[0139] FIG. 9 is a view of a cylindrical chamber for pressurization
in a pressure modulation apparatus. The chamber 300 includes a
rigid closure 310, a sample cell 320, and a piston 330. The piston
communicates the pressure outside the chamber 300 to the sample
cell 320. The sample cell 320 also includes rigid end caps 340,
flexible walls 350, and a sample compartment 360. The rigid end
caps 340 prevent extrusion of the flexible walls 350 into the
clearance gap 370 between the piston 330 and cylinder walls 380.
The flexible walls 350 allow deformation of the sample cell 320 to
allow compression of the sample in the sample compartment 360.
[0140] The following illustrative examples are not intended to
limit the scope of the invention.
EXAMPLE 1
[0141] DNA Isolation and Purification in an Anion-exchange
Cartridge:
[0142] DNA samples were separated using a Qiagen DEAE
anion-exchange resin (Qiagen, Inc., Santa Clarita, Calif.) at
ambient and elevated pressures. The DEAE resin was packed into a 9
mm.times.4 mm I.D. (5 mm O.D.) stainless steel `half-length column`
capped with titanium frits with a 2 .mu.m pore size (Valco
Instrument Company, Inc., Houston, Tex.). Two half-length columns,
one containing resin and the other acting as a spacer and devoid of
resin, were placed into a column holder. The column holder was a
metal tube with an inner diameter of 5 mm and syringe fittings at
the ends to allow fluid to flow through the columns.
[0143] Pressure elution of DNA was performed using a pressure flow
apparatus as described in PCT Appln. No. US/96/03232, controlled by
a microcomputer with LABVIEW.TM. software (National Instruments,
Austin, Tex.). The columns were inserted into a pressure chamber
adapted to receive the columns. Liquid was injected and removed
from the chamber using a series of pneumatic valves and pistons as
described in the '232 application, allowing for elution of DNA from
the column while maintaining elevated pressure within the
column.
[0144] The DEAE column was initially washed with 1 ml high salt
elution buffer (1.25 M sodium chloride; 50 mM Tris-HCl, pH 8.5; 15%
ethanol) and equilibrated with 1 ml equilibration buffer (750 mM
sodium chloride; 50 mM MOPS, pH 7.0; 15% ethanol; 0.15% Triton
X-100). Approximately 300 .mu.l of 21 .mu.g/ml DNA in loading
buffer (1 M potassium acetate; 33 mM NaCl; 33 mM Tris-HCl, pH 5; 8
mM EDTA), was injected into the packed column over five minutes, in
four 1 minute intervals. 1 ml of MO washing buffer (containing 1 M
NaCl; 50 mM MOPS, pH 7.0; 15% ethanol) was then injected through
the holder to remove any remaining contaminants, followed by 200
.mu.l of elution buffer to displace the MO washing buffer prior to
elution either at atmospheric or elevated pressures. Elution
buffers used during the DNA elution step contained 50 mM Tris-HCl,
pH 8.5, and various concentrations of sodium chloride.
[0145] Four consecutive 300 .mu.l elution fractions were collected
during each experiment. Each fraction was collected over a three
minute interval, in which a 100 .mu.l pressure wash step with the
elution buffer was performed each minute. Experiments at
atmospheric pressure were performed with identical elution steps,
using a syringe to deliver the elution salt solutions through the
column holder.
[0146] DNA in the collected samples was quantified using OliGreen
DNA binding dye (Molecular Probes, Eugene, Oreg.). To reduce the
background signal and increase sensitivity, the salt concentration
in the DNA assay solutions to be assayed was first diluted 20-200
fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and then
complexed with the OliGreen dye (20,000:1 DNA:dye in volume).
Fluorescence emission intensity (.lambda..sub.em=520 nm,
.lambda..sub.ex=480 nm) of the DNA/OliGreen solution was measured
with an ISS PCI spectrofluorometer (ISS, Inc., Champaign, Ill.),
without background subtractions. Quantitation of DNA was made by
comparing the measured intensity with calibration curves obtained
from known DNA concentrations. Recovery of DNA in the elution
fractions was calculated by dividing total DNA in the elution
fractions with the total DNA bound in the column, (i.e., total DNA
loaded minus DNA in flow-through and MO washing solutions).
[0147] At elevated pressures, .lambda.DNA (Worthington Biochemical
Company, Freehold, N.J.) was released from the DEAE resin with a
buffer of lower salt concentration, as shown in Table 1. The
percentages given in the table are percent recoveries, and numbers
in parentheses after the percentages are the number of times the
corresponding experiments were carried out. The error range was
calculated to be approximately 5% based on the duplicated data.
1TABLE 1 .lambda.DNA Purification with Low Salt Buffer at Various
Hyperbaric Pressures [NaCl] (M) in Tris Pressure (MPa) Buffer 0.1
90 170 220 0.10 trace (1) trace (1) 40% (1) 0.25 trace (2) 20% (3)
100% (1) 0.40 25% (1) 0.50 trace (2) 15% (1) 70% (2) 60% (1) 0.75
trace (2) 15% (1) 100% (2) trace (1) 1.00 100% (1) 100% (1)
[0148] Table 1 shows a correlation: the higher the pressure, the
lower the salt concentration needed for dissociation. At 0.1 MPa
(i.e., atmospheric pressure), 1 M sodium chloride was required for
more than just a trace amount (i.e., less than 10%) of DNA to be
eluted. At 90 MPa (about 12,500 psi), the DNA showed a slightly
increased tendency to dissociate; at 170 MPa (about 24,000 psi),
70% of the .lambda.DNA was dissociated with 0.50 M NaCl. 100% of
the .lambda.DNA dissociated with 0.25 M NaCl at 220 MPa (about
32,000 psi). Interestingly, less of the DNA eluted at this pressure
when the salt concentration was raised, possibly due to a phase
change in the silica resin that is aided by electrostatic shielding
in the high salt environment. A similar effect is seen at lower
salt concentrations and higher pressures.
[0149] A follow-up experiment included three different sizes of
DNA. Human cell extract high molecular weight DNA K562 (number of
bp is unknown; Pharmacia Biotech, Inc., Piscataway, N.J.) and
.lambda.DNA (.about.48.4 kb) behaved similarly. Both yielded a 25%
recovery with 0.40 M NaCl Tris-HCl buffer at 170 MPa. On the other
hand, 100% recovery of the plasmid pKK223-3 (about 4.6 kb DNA) was
observed under the same conditions.
[0150] To test the effect of salt concentration for three different
sized nucleic acids (i.e., 50 bp, 4.6 kb, and 48.4 kb), the
pressure was held at 23,600 as the concentration of sodium chloride
was increased from 0 to 1 M. The nucleic acids were detected as
they eluted from the cartridge. The results are shown in the graph
in FIG. 7. Most of the smallest nucleic acid, 50 bp, was eluted by
100 mM sodium chloride (i.e., as indicated by the dotted line). The
4.6 kb fragment was eluted at 250 mM, as shown by the dashed line.
The solid line indicates that 500 mM sodium chloride was required
to elute the largest nucleic acid, 48.4 kb. Thus, the nucleic acids
can be separated on the basis of size by varying the salt
concentration.
[0151] The effect of pressure was also studied, using the same
three nucleic acid fragments. In this experiment, the sodium
chloride concentration was held constant at 250 mM, as the pressure
was increased from 14 to 40,000 psi. The nucleic acids were
detected as they eluted from the cartridge. The results are shown
in the graph in FIG. 8. Most of the smallest nucleic acid, 50 bp,
was eluted at around 7,000 psi, as indicated by the dotted line.
The 4.6 kb fragment was eluted at about 20,000 psi, as shown by the
dashed line. The solid line indicates that approximately 32,000 psi
was necessary to elute the largest nucleic acid, 48.4 kb. Thus, the
nucleic acids can be separated on the basis of size by varying the
elution pressure.
[0152] To test the specificity of the resin for nucleic acids,
bovine serum albumin (BSA) was applied to the DEAE column. Serum
albumins are multivalent and highly absorptive, and are the most
abundant proteins in mammalian blood. It is therefore highly
desirable that any DNA purification procedure for isolating DNA
from blood be capable of separating BSA from DNA. Indeed, all of
the protein was recovered in the flow through and MO washing
solutions.
[0153] Agarose gels were used to check the integrity of DNA in the
eluent solutions. Where sufficient DNA was recovered for analysis,
the DNA molecules were found to be intact. In the remaining cases,
there was not enough DNA to test on a gel. The DNA in the elution
solutions was also quantified, using PicoGreen, a dye specific for
double-stranded DNA, PicoGreen (Molecular Probes, Eugene, Oreg.).
The dye indicated that the majority of DNA (i.e., about 90% of the
DNA) was still double-stranded after applying pressure with a high
concentration of salt.
EXAMPLE 2
[0154] Restriction Digestion of Eluate without Desalting:
[0155] pCMV-SV40T plasmid was isolated from 1.5 ml of an overnight
culture of an JM109 E. coli strain by alkaline lysis (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory Press: Plainview, N.Y., 1989, pp. 1.25-1.26). 1.5
ml of an overnight bacterial culture was placed in a
microcentrifuge tube and spun for 30 seconds at 12,000 g. The
medium was removed and the cells were resuspended with vigorous
vortexing in 100 .mu.l of a solution containing 50 mM glucose, 25
mM Tris-HCl (pH 8.0), and 10 mM EDTA (pH 8.0). 200 .mu.l of a
freshly made solution of 0.2 N NaOH (containing 1% sodium dodecyl
sulfate, SDS) was added, and the tube was inverted five times. 150
.mu.l of ice cold solution III (prepared by mixing 60 ml of 5 M
potassium acetate, 11.5 ml of glacial acetic acid, and 28.5 ml of
water) was added, then the sample was gently vortexed and incubated
on ice for 4 minutes. The sample was centrifuged at 12,000 g for 5
minutes. The cleared, neutralized supernatant was transferred to a
fresh tube and water was added to a final volume of 700 .mu.l. 300
.mu.l of the sample was purified using the Qiagen.TM. #12129
plasmid kit (Santa Clarita, Calif.) without the final isopropanol
precipitation. Another 300 .mu.l of the sample was loaded onto a
cartridge and processed as described in Example 1. The plasmid was
eluted with 400 mM NaCl at 23,6000 psi. 45 .mu.l of purified
plasmid solution was mixed with 55 .mu.l of buffer (containing 18
mM Tris-HCl pH 8.0, 18 mM MgCl.sub.2, 1.8 mM dithiothreitol, and
180 .mu.g/ml of BSA). A restriction digest reaction was initiated
by adding 0.5 .mu.l (40 units) of BamHI enzyme (Promega, Madison,
Wis.) and incubated for 1 hour at 37.degree. C.
[0156] The results were analyzed by agarose gel electrophoresis.
The gel was stained using SYBR1 (Molecular Probes; Eugene, Oreg.).
No digestion was seen with the Qiagen.TM. purified DNA, whereas the
pressure eluted DNA showed two bands, indicating digestion of the
plasmid at the two BamHI sites. This result demonstrated that, in
contrast to a traditional nucleic acid elution procedure, DNA
eluted under high pressure can be cleaved by a restriction enzyme
without a precipitation or desalting step, other than a 1:1
dilution into reaction buffer.
EXAMPLE 3
[0157] Protein Expression in Mammalian Cells Utilizing Plasmid DNA
Isolated and Purified by Hyperbaric Pressure
[0158] The vector pCMV-SV40TAg, which encodes the large tumor
antigen (TAg) of the SV40 virus, was transformed into a bacterial
strain, JM109, and isolated by standard alkaline lysis procedure
(Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press; Plainview, N.Y., 1989) essentially
as described in Example 2. 1.0 ml of the sample was purified using
the Qiagen #12129 plasmid kit (Santa Clarita, Calif.). Another 1.0
ml of the sample was aliquoted into three equal portions and
processed batchwise as described in Example 1. The plasmid was
eluted with 500 mM NaCl at 29,000 psi. The solution containing the
purified pCMV-SV40TAg plasmid was ethanol precipitated and
resuspended in sterile water.
[0159] A transient protein expression assay was performed to
compare the purity and quality of the plasmid prepared using
hyperbaric pressure with that of the plasmid prepared with the
Qiagen kit. To assay the level of TAg protein expression, a monkey
kidney cell line, BSC 40, was transiently transfected with either
the pressure purified or the Qiagen purified pCMV-SV40TAg plasmid
using a standard calcium phosphate transfection procedure (Ausubel
et al., Current Protocols in Molecular Biology, John Wiley &
Sons; New York, 1987 pp. 9.1.1 to 9.1.4). Western blot analysis was
performed according to standard procedures (ibid., pp. 10.2.1 and
10.8.1). The results of this experiment indicated that pCMV-SV40TAg
DNA prepared using hyperbaric pressure was three- to five-fold more
efficient at directing TAg protein expression compared with the
same plasmid prepared using the Qiagen kit. These results
demonstrate the quality and stability of plasmid DNA following
isolation by hyperbaric pressure.
EXAMPLE 4
[0160] Isolation of total RNA on a DEAE Anion-Exchange
Cartridge:
[0161] BSC40 cells that stably express TAg were lysed by the method
of Chomczynski et al. (Anal. Biochem., 162:156-159, 1987). 1 ml RNA
STAT-60.TM. (Tel-test, Inc., Friendswood, Tex.) was added directly
to the cells. After incubating at room temperature for 5 minutes,
the cells were scraped from the plate, homogenized by pipetting,
and transferred to a sterile microcentrifuge tube. After addition
of 0.2 ml chloroform, the solution was mixed vigorously for 15
seconds and the upper aqueous phase was separated by
centrifugation. Following precipitation of the RNA with
isopropanol, the RNA was pelleted by centrifugation in a
microcentrifuge, and resuspended in 50 .mu.l of sterile RNAse-free
water. 10 .mu.l of the RNA sample was then mixed with 500 .mu.l of
equilibration buffer (containing 750 mM sodium chloride; 50 mM
MOPS, pH 7.0; 15% ethanol; and 0.15% Triton X-100).
[0162] Qiagen DEAE anion-exchange resin was packed in the
"half-column" as described in Example 1, and washed with 1 ml
equilibration buffer. 300 .mu.l RNA sample was injected onto the
DEAE column over 3 minutes. Then, 1 ml MO buffer (containing 1 M
NaCl; 50 mM MOPS, pH 7.0; 15% ethanol) and 200 .mu.l elution buffer
(containing 250 mM sodium chloride and 50 mM Tris, pH 8.5) was
applied to wash the column. Four consecutive 100 .mu.l and three
300 .mu.l elution fractions were collected at 23,600 psi. After
taking the DEAE column out of the pressure flow apparatus, the
column was washed with 1 ml of high salt buffer (containing 1.25 M
sodium chloride; 50 mM Tris-HC1, pH 8.5; and 15% ethanol).
[0163] RNA in the collected samples was quantified using OliGreen
DNA binding dye. To reduce background signals, RNA assay solutions
were diluted 125-fold with TE buffer (containing 10 mM Tris-HCl and
1 mM EDTA, pH 7.5), which contained 1200-fold diluted OliGreen.
Fluorescence emission intensity (.lambda..sub.ex=485 nm,
.lambda..sub.em=580 nm) was measured with a FLUOROCOUNT.TM.
microplate fluorometer (Packard Instrument Company, Meriden,
Conn.), without background subtractions. The quantity of RNA was
estimated by comparing the measured intensity with calibration
curves obtained from known .lambda.DNA concentrations. The recovery
of RNA in the elution fractions was calculated by dividing total
RNA in the elution fractions by the total RNA bound in the
column.
[0164] It was found that at 23,600 psi, more than 60% of RNA was
released from the DEAE resin in the first 4 fractions. The other
three fractions contained about 40%; thus 100% recovery was
achieved. The high salt wash solution was analyzed to verify this
result; indeed, no RNA was detected in the subsequent high salt
wash solutions. To compare the integrity and purity of RNA isolated
using hyperbaric pressure with that of the original RNA sample
preparation, both samples were analyzed on a 0.8% agarose gel. The
results indicated that there was no significant degradation of the
28S and 18S rRNA following isolation on the DEAE anion-exchange
cartridge with hyperbaric pressure. These results suggested that
total RNA was effectively isolated using the high pressure
procedure without shearing or degradation by RNases. The estimated
yield of RNA, based on the gel electrophoresis, was consistent with
the estimation made by fluorescence assay. Thus, it is shown that
high pressure elutes RNA from the column at lower salt
concentration while preserving physical integrity.
EXAMPLE 5
[0165] Purification of Messenger RNA (mRNA) from a Eukaryotic
Sample:
[0166] Two cartridges containing a solid phase are arranged in
serial order, such that eluent from the first cartridge enters the
second cartridge. The first cartridge is packed with an activated
acidic DEAE activated anion exchange resin and the second is packed
with a resin containing covalently linked polythymidine (poly-dT)
resin.
[0167] An mRNA standard (positive control) is purified by standard
methods using the POLY(A)PURE.TM. Kit (Ambion, Austin, Tex.). A
salt concentration is found (i.e., by multiple trials) at which
poly-da mRNA is poorly bound to poly-dT resin at atmospheric
pressure, but is more tightly bound at high pressure. Samples are
loaded onto the cartridge containing the poly-dT resin, in a buffer
containing 100 mM NaCl and 10 mM Tris-HCl, pH 7.2, at atmospheric
pressure. The samples are then eluted with 300 .mu.l of buffer
containing 10 mM Tris-HCl (pH 8.0) and NaCl concentrations of 0 to
100 mM in increments of 10 mM. The salt is removed from the sample
by washing twice in 10 mM Tris-HCl (pH 8.0) using a
Macrocon-100.TM. spin-filter (Millipore). The experiment is
repeated at 29,000 psi for each sample that has a different salt
concentration. A buffer which gives poor binding at atmospheric
pressure, but improved binding at high pressure is selected and
referred to as "solution A".
[0168] NIH 3T3 cells, grown in culture, are lysed by standard
procedures (Chomczynski et al., supra) and total RNA is isolated on
the DEAE column as described in Example 4. The cell debris is
removed by centrifugation. The sample is applied to the double
anion-exchange/poly-dT column. The column is washed with 300 .mu.l
of solution A. The column is then washed at 25,000 psi with 300
.mu.l of solution A, thereby directly transferring the mRNA from
the anion-exchange resin to the poly-dT resin. The pressure is then
lowered to atmospheric pressure and the resin is washed with 300
.mu.l of either solution A or distilled water to recover the mRNA.
The sample is analyzed for purity by reverse
transcriptase=polymerase chain reaction (RT-PCR) for specific
target transcripts, agarose gel electrophoresis, UV spectroscopy,
and a protein binding dye assay. To determine the quality and
integrity of the mRNA isolated utilizing this procedure,
.beta.-actin mRNA, for example, can be amplified using .beta.-actin
primers in RT-PCR (Promega, Madison, Wis.). The resulting DNA
products can then be analyzed on a 1.0% agarose gel and compared
with cDNA product resulting from the RT-PCR procedure using the
positive mRNA control. These results indicate that mRNA is
effectively isolated on a poly-dT cartridge, transferred to a
poly-dA cartridge using 25,000 psi, and effectively recovered for
subsequent analysis.
EXAMPLE 6
[0169] Detection of p53 Mutations in Human Malignancies Using
Hyperbaric Pressure Purified RNA:
[0170] Total RNA obtained from tumor samples is prepared by a
combination of the method of Chomczynski et al. (supra) and the
hyberbaric DEAE column purification as described in Example 4. The
homogenized human sample is lysed with 8 M urea and 50 mM Tris-Cl
buffer (pH 8.0). 500 .mu.l urea solution is then loaded onto the
activated DEAE resin, which is packed in a cartridge at atmospheric
pressure. Total RNA will elute off the column at 29000 psi. The
mRNA of interest (i.e., p53) is amplified by reverse
transcriptase-polymerase chain reaction (RT-PCR) using p53-specific
primers (Promega, Madison, Wis.). The resulting DNA product is then
sequenced with the dideoxy chain termination method using a
Sequenase 2.0 kit (United States Biochemicals), then analyzed for
mutations by comparing to the p53 consensus sequences. This
therefore indicates that hyperbaric pressure RNA isolation is an
effective and simplified procedure to obtain RNA molecules from
cells.
EXAMPLE 7
[0171] Isolation of RNA from Human Whole Blood:
[0172] 300 ml of the whole blood with anticoagulant, e.g., heparin,
is loaded onto a DEAE anion-exchange cartridge over three minutes
as described in Example 1. The column is then pressurized to 60,000
psi to cause cell lysis and nucleic acid molecules bind with the
DEAE resin. Subsequently, RNA is eluted at 29,000 psi as described
in Example 4, collected in consecutive fractions, precipitated with
isopropanol, and resuspended in 30 ml of RNase-free water.
[0173] As a control, total RNA is extracted by the method of
Chomczynski (Biotechniques, 15:532-536, 1993). 300 .mu.l of whole
blood is mixed with 1.0 ml of red blood cell lysis solution (RBCS;
containing 40 mM ammonium chloride, 10 mM potassium hydroxide, 7.5
mM potassium acetate, 2.5 mM sodium bicarbonate, 0.125 mM EDTA, and
0.1% glacial acetic acid). After 10 minutes at 4.degree. C., the
residual red blood cells are pelleted by centrifugation (30 seconds
at 12,000 g). An additional 1.0 ml RBCS is added to the pellet and
mixed thoroughly. The centrifugation step described above is
repeated. The supernatant is removed and the leukocyte pellet is
resuspended in 350 .mu.l of leukocyte lysis solution (LLS: 4 M
guanidinium isothiocyanate. 0.1 M .beta.-mercaptoethanol, 10 mM
sodium citrate, pH 7.0, 0.5 M lauryl sarcosine, and 2.0% Triton
X-100). The tube is vortexed vigorously prior to addition of 350
.mu.l 64% ethanol.
[0174] The quality and integrity of total RNA eluted from the
column is analyzed on a native 1.0% agarose gel stained with
ethidium bromide. Results show clear bands of the 28S and 185 rRNA.
Further, the RNA sample is examined for the existence of
.beta.-actin mRNA using RT-PCR as described in Example 5. The
results show a distinctive signal originating form .beta.-actin in
the purified blood sample.
EXAMPLE 8
[0175] Pressure Effects on Ion-exchange Electrophoresis
[0176] 5 .mu.l 384 .mu.M rhodamine-labeled 21-mer
deoxyoligonucleotide was mixed with 100 .mu.l of Qiagen silica
ion-exchange resin in 25 mM TBE buffer. The resin was placed in a
cartridge composed of acrylic that had four reservoirs for holding
resin or liquid. An electrode was molded into the bottom for each
reservoir and contacted the cartridge cap by means of a wire glued
to the outside of the cartridge. The cartridge was filled with
borate buffer and a cap with an o-ring was placed on top to form a
seal and act as a piston. The cartridge was designed such that it
could be plugged into four electrical leads in the cap of a
pressurizing apparatus. The pressurizing medium was silicone
oil.
[0177] As a control, 1.2 mA of electric current was applied at
5,000 psi of pressure for 15 minutes. No effect was observed.
However, when the same current was applied at 25,000 psi of
pressure for 15 minutes, the labeled oligonucleotide was observed
to travel from the chamber with the negative electrode to the
chamber contacting the positive electrode, resulting in white resin
at the former electrode. The electrode polarities were then
reversed and the color was seen to shift to the other side,
indicating that pressure was able to modulate the affinity of the
ion-exchange resin. This demonstrates that nucleic acid molecules
can be transported electrophoretically in an ion-exchange medium in
the presence of low salt buffer at hyperbaric pressure to
concentrate the sample.
EXAMPLE 9
[0178] Purification of Nucleic Acids from Cells or Viruses by
Hyberbaric Permeabilization and Electrophoresis.
[0179] A 5 ml culture of E. Coli cells containing a pACYC plasmid
is grown to an optical density of 0.6 at 600 nm in Luria broth
supplemented with 100 .mu.g/ml of ampicillin (LB/amp). One
milliliter of the culture was centrifuged at 10,000 g for 10
minutes to pellet the cells. The supernatant was discarded and the
cells were resuspended in 1 ml of distilled water. 90 .mu.l of the
resuspended cells and 2 .mu.l of 384 .mu.M rhodamine conjugated
21-mer oligonucleotide were loaded into a high-pressure
electrophoresis cartridge. as described in Example 8. A 1% agarose
gel was formed in another chamber of the cartridge. The cartridge
was pressurized to 30,000 psi and a 35 V electric field was applied
for 15 minutes. The labeled oligonucleotide was observed to have
moved into the gel, indicating that the electrical process was
adequate for the movement of DNA. The agarose plug was removed with
a needle and placed into the well of an agarose slab gel. The slab
gel was run with four control lanes: one containing a pure plasmid,
another containing untreated cells, one containing cells which had
been subjected to electrophoresis in the cartridge at atmospheric
pressure and the last contained cells that have been pressurized to
30,000 psi for 15 minutes without electrophoresis. The results
showed that high pressure can release plasmid DNA by permeabilizing
the cell walls and membranes. This result indicates the possibility
that plasmid DNA can be purified in a single-step process. Separate
steps are not required for lysis, neutralization, and
purification.
EXAMPLE 10
Cell Lysis and RNA Purification by Pressure Pulsing and/or Constant
Pressure
[0180] Murine NIH 3T3 cells were grown on tissue culture dishes
according to standard methods. Cells in the tissue culture plate
were washed twice with 8 ml of phosphate buffered saline (PBS),
lifted, and resuspended in 500 ml of PBS. 50 .mu.l of the cell
mixture was placed in hollow capsules, which were inserted into
pressure chambers filled with silica melting point oil (Sigma
Chemicals, St. Louis, Mo.). The capsules containing the cell
solution were pressurized and depressurized sixty times. In each
cycle, the capsules were pressurized to 30,000 psi for 1.25 seconds
and returned to atmospheric pressure for 1.25 seconds. In a second
experiment, the capsules were kept at a constant 60,000 psi for 10
minutes.
[0181] To determine the extent of cell lysis, a portion of the cell
lysis solution was removed from the capsule and observed in a phase
contrast optical Olympus microscope. Compared to the unpressurized
control cells, the pressurized cell solution was found to contain
fragmented cells and cellular debris despite the existence of a
large number of intact cells. 20 .mu.l aliquot from each sample
were mixed with 200 .mu.l of OliGreen solution (diluted 1:1,000)
(Molecular Probes, Eugene, Oreg.). Fluorescence emission
intensities at 530 nm were detected with an excitation wavelength
of 485 nm. The results indicated a 10-fold increase in fluorescence
intensity for the cells lysed by both pulsing and constant
pressurization. Nucleic acid products in the cell solution were
also examined using 1% agarose gel electrophoresis. The results
showed that the majority of the nucleic acid in the supernatant of
the pressurized cell solutions was RNA, as determined by the
presence of 28S and 18S rRNA. This hypothesis was confirmed by a
QIAamp purification test, assuming that DNA binds to QIAamp
membrane and RNA does not.
[0182] As a positive control, total RNA released from NIH 3T3 cells
by either pressure pulsing or pressing was purified using RNeasy
kit #74103 (Qiagen, Santa Clarita, Calif.). RNA products isolated
from the hyperbaric pressure purification or the RNeasy kit were
analyzed by agarose gel electrophoresis. The results indicated that
the outer membranes of cells are destroyed by hyperbaric pressure
pulsing and/or pressing. RNA released by pressure lysis is similar
to RNA released by the conventional method. Thus, RNA molecules
released in the pressurization process allow the cooperation of
cell lysis and RNA purification in a single step purification
process.
EXAMPLE 11
Cell Lysis and Genomic DNA Purification by Hyperbaric Pressure
Pulsing and/or Constant Pressure Pressing
[0183] In addition to the disruption of cells by applying high
pressure pulsing and/or constant pressure pressing as described in
Example 10, additional agents (e.g., proteinase K, detergents) can
be supplemented to aid in the release of genomic DNA from
DNA/protein complexes in the nuclei. Neutral or positively charged
detergents are tested, as these compounds are compatible with the
downstream high pressure purification. Such detergents include
NP-40 and cetyltrimethylammonium chloride (CTMA). Initially, DEAE
resin is activated with 2% NP-40, 100 mM sodium acetate buffer, pH
4.5 and equilibrated with a buffer of 50 mM Tris-Cl, 400 mM NaCl,
2% NP-40, pH 8.5. Then, murine NIH 3T3 cells were washed with 8 ml
PBS twice, lifted in 1 ml PBS and loaded onto the DEAE column as
described in Example 1. High pressure pulsing and/or constant
pressing is applied to the cartridge, as described in Example 10.
Elution of the genomic DNA is achieved by electroelution at 35,000
psi. As a quantity and quality control, 200 .mu.l lifted NIH 3T3
cells were mixed with 200 .mu.l sucrose buffer (i.e., 1.28 M
sucrose, 40 mM Tris-Cl, 20 mM MgCl.sub.2, 4% triton X-100, pH 7.4).
After the mixture was centrifuged at 2,000 rpm for 15 minutes and
the supernatant was discarded, 400 .mu.l of general lysis buffer
(i.e., 0.8 M guanidine HCl, 30 mM Tris-HCl, 30 mM EDTA, 5%
Tween-20, 0.5% Triton X-100, pH 8.0) was added, vortexed briefly,
and followed by a proteinase K digestion reaction at 55.degree. C.
for one hour. After the digestion, the lysis solution was
centrifuged at 14,000 rpm for 10 minutes. 200 .mu.l supernatant was
then collected and nucleic acid purified using protocol described
in QIAamp Tissue kit #29304. Purified nucleic acid was finally
eluted in 100 =82 l distilled water. The nucleic acid content of
this solution was analyzed using the standard OliGreen assay. If
the fluorescence intensity of the control is found to be similar to
that of the cells that have been pressurized, this example would
suggest that the two types of pressurization treatments are as
efficient as the conventional method.
[0184] These pressurization lysis procedures were also applied in
the lysis of yeast (S. cerevisiae). First, yeast culture cells were
grown either over night or for 3 hours. 1 ml of cell cultures were
washed twice with 1 ml of TN buffer (20 mM Tris-HCl, pH 7.4, 100 mM
NaCl), pelleted, and resuspended in 1 ml of TN buffer with 1 mM
EDTA (TNE buffer). 50 .mu.l aliquots of the yeast in the TNE
solution were placed in hollow capsules and pressurized either to
30,000 psi, with 2.5 second pressurization and depressurization
steps repeated 240 times, or using 60,000 psi constant pressure for
10 minutes. To determine the amount of nucleic acid released by
pressurization, OliGreen assays were performed. The results showed
that there were two- to four-fold increases in fluorescence for the
pressurized yeast as compared to the untreated cells. To improve
the lysis efficiency, glass beads (300.mu.) were added to the yeast
TNE solution and the mixture was vortexed for 2 minutes, prior to
the hyperbaric pressure lysis steps. However, no significant
changes in the yield of nucleic acids were observed. As in the
lysis method used for the NIH 3T3 cells mentioned above, addition
of detergent molecules is required to break the nuclei and release
the genomic DNA. As a positive control, yeast cells were also lysed
using standard lyticase and proteinase K enzymatic lysis procedures
(Qiagen Genome DNA Purification Manual, Santa Clarita, Calif.). The
nucleic acids were then purified using QIAamp Tissue kit #29304
(Qiagen, Santa Clarita, Calif.). The level of nucleic acids
obtained was analyzed using agarose gel electrophoresis and
OliGreen binding assay. The results indicated that both the control
method and the pressure lysis procedure yielded a similar amount of
nucleic acid.
EXAMPLE 12
[0185] DNA DNase I Fragment Purification in an Anion-exchange
Cartridge
[0186] To obtain random digested, various-length DNA fragments
(e.g., from genomic DNA), a method based on anion-exchange
chromatography is carried out. A rapid DNA fragmentation is started
with a purified biological sample and followed by repeated
hyperbaric pressure anion exchange and in combination of DNase I
digestion. Thus, human blood is lysed and purified in an
anion-exchange cartridge as described in Example 7, and genomic DNA
elutes off from the column at 45,000 psi in a buffer that has 100
mM NaCl, 50 mM Tris-Cl, pH 7.4. This solution is then mixed with
DNase I (Pharmacia, Piscataway, N.J.) and incubated at 37.degree.
C. for various lengths of time. To inactive the DNase I, the
digestion solution is either heated at 90.degree. C. for 3 minutes
or EDTA is added to a final concentration of 25 mM. Alternatively,
the DNA digestion solution is loaded onto a new DEAE cartridge for
a second step purification without inactivating DNase I. The heat-
or EDTA-treated solution is then loaded onto a new DEAE cartridge
(can be the same or simplified as used in genome DNA purification).
The digested DNA fragments are then eluted with 100 .mu.l of a
buffer containing 100 mM NaCl, 50 mM Tris-Cl, pH 7.0 at 40,000 psi.
The size distribution of the resulting DNA fraction can be analyzed
by agarose gel electrophoresis. This sample preparation method can
be incorporated in DNA hybridization chips, allowing a single step
sample preparation and downstream hybridization analysis. 1%
agarose gel electrophoresis is used to evaluate the size
distributions of the DNA fragments.
EXAMPLE 13
A Cartridge for Integrated High Pressure-mediated Cell Lysis and
Nucleic Acid Purification
[0187] The designed cartridge is composed by four essential
compartments as illustrated in FIGS. 10A and 10B. The starting
materials (e.g., cell solutions or cell lysis solutions) are loaded
into compartment 390. Compartment 395 (including a first half 460
and a second half 470) is filled with an ion-exchange resin such as
DEAE on solid support for nucleic acid purification. This
compartment is also accessible to four electrodes, 410, 420, 430,
and 440, which are protected by polyacrylamide gel. Electrodes 410
and 420 are used during pre-elution purification under relative low
pressure to remove contaminants (e.g., proteins, polysaccharides,
and lipids). Electrodes 430 and 440 are used when nucleic acid
products are extracted under high pressure. All of the electrodes
can be used in the lysis step as described in Examples 10 and 11.
Compartment 395 is separated by a plastic wall 450, which allows
nucleic acid molecules travel from the first half 460 to the second
half 470 such that resolution can be improved by introducing
chromatographic effects. For example, high resolution can be
achieved based on both the charge and the size of the nucleic
acids. Since larger DNA requires higher pressure to release from
the resin, relatively small DNA molecules will be eluted off at
first, at the appropriate pressure. The chromatographic effect
enhances the separation of different sizes of DNA. Compartment 400
contains layers of absorptive materials (e.g., cellulose-based
filter membranes, silica gels, CaO, and cotton). The adsorptive
material helps in absorbing extra fluids and thus relatively large
quantities of solution can be introduced into the cartridge so that
the yield of the product can be improved. Compartment 405 is
separated from compartments 395 and 400 by a nucleic acid permeable
membrane 480, and contains a low salt buffer (i.e., 50 mM NaCl, 50
mM Tris-HCl, pH 8.5). Although compartment 405 is physically
separated from compartment 390, the barrier 490 between the can be
punched through using a needle, and the nucleic acid product can be
collected with a syringe.
[0188] In the operation of the cartridge, biological samples, e.g.,
blood, cell culture and homogenized plant or tissue cultures, are
injected into compartment 390. Then, the cartridge is transferred
into a pressure modulation apparatus, in which the electrodes 410,
420, 430, and 440 are attached to voltage sources and voltage
changes are computer controlled. The pressure is increased to
60,000 psi. At this pressure, cell fluid saturates the resin and
the absorptive materials in compartments 395 and 400.
Simultaneously, the cells are lysed and the nucleic acids bind to
the resin. After holding at high pressure for a short period of
time, pressure is decreased to 10,000 psi. Electrodes 410 and 420
are turned on. At this pressure level, proteins and other
contaminant molecules to which the resin does not bind will travel
towards the electrodes and be trapped in the polyacrylamide gel
surrounding these electrodes. Once purification is complete, the
pressure in the chamber is increased, and nucleic acid product
collection begins. For example, the pressure is increased to 23,000
psi to collect RNA sample, 35,000 psi to collect plasmid, and
45,000 psi to collect genomic DNA. The nucleic acid products are
gathered in compartment 405 by turning on electrodes 430 and 440.
Once elution finishes, the pressure is decreased, and the cartridge
is removed from the pressure modulation apparatus. The nucleic acid
products are recovered by punching a needle into compartment 405
and collecting the nucleic acids with a syringe.
EXAMPLE 14
Cell Lysis Assisted by Pumping High Pressure Air
[0189] 50 .mu.l of an overnight yeast cell culture was loaded onto
a 500 ml column. A piston was inserted into the column, allowing
external pressure to be transmitted to the column. The column was
transferred to a pressure modulation apparatus and then pressurized
to 60,000 psi for 5 minutes, depressurized, kept at atmospheric
pressure for 1 minute and pressurized again. After two more
depressurization/pressurization cycles, the cell solution was
removed from the column at atmospheric pressure. A microscopic
examination was conducted, and the nucleic acid content was
estimated using fluorescence dye binding assay. The results
indicated that high pressure air pumping afforded a similar level
of cell lysis as that achieved through high pressure pulsing.
EXAMPLE 15
Separation of RNA from an RNA/DNA Mixture
[0190] Total RNA from Torula Yeast (Type IV) was purchased from
Sigma (St. Louis, Mo.). pKK223-3 (Pharmacia, Piscataway, N.J.) was
used as the DNA control. The RNA and DNA were mixed in an NTM
buffer solution, pH 7.0 (NTM: 175 mM NaCl, 35 mM Tris, 0.5 mM
MgCl.sub.2). The mixture was pre-bound to a Qiagen DEAE resin in a
stainless steel cartridge. 0.4 ml of the RNA/DNA solution was
injected into a cartridge containing activated DEAE resin, followed
by washing with 200 .mu.l of NTM buffer, pH 8.5. 1200 .mu.l elution
fractions were collected at 23,600 psi in NTM, pH 8.5. The
cartridge was washed with 1 ml of high salt buffer (1.25 M NaCl, 50
mM Tris-HCl, pH 8.5, 15% ethanol). The elutions were analyzed using
OliGreen fluorescence assay. The remaining elution fractions and
high salt buffer wash solutions were ethanol precipitated and
dissolved in 20 .mu.l double distilled water. These were checked by
agarose gel electrophoresis. The results indicated that the RNA was
completely separated from the DNA and RNA is purified. The DNA was
recovered in the high salt washing step with little RNA
contamination. Thus, RNA can be separated from an RNA/DNA mixture
by high pressure-mediated purification.
Other Embodiments
[0191] From the description above, one skilled in the art can
ascertain the essential characteristics of the invention and
without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
[0192] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof,
that the foregoing description is intended to illustrate and not to
limit the scope of the appended claims. Other aspects, advantages,
and modifications are within the scope of the following claims.
* * * * *