U.S. patent application number 11/946687 was filed with the patent office on 2008-05-29 for method for releasing genetic material from solid phase.
This patent application is currently assigned to Canon U.S. Life Sciences, Inc.. Invention is credited to Michele R. Stone.
Application Number | 20080124777 11/946687 |
Document ID | / |
Family ID | 39468503 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124777 |
Kind Code |
A1 |
Stone; Michele R. |
May 29, 2008 |
METHOD FOR RELEASING GENETIC MATERIAL FROM SOLID PHASE
Abstract
The present invention relates to systems for releasing genetic
materials from a solid medium. The present invention also relates
to methods for releasing genetic materials from a solid medium. The
present invention further relates to methods for isolating genetic
material from a biological sample.
Inventors: |
Stone; Michele R.;
(Rockville, MD) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Canon U.S. Life Sciences,
Inc.
Rockville
MD
|
Family ID: |
39468503 |
Appl. No.: |
11/946687 |
Filed: |
November 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60867699 |
Nov 29, 2006 |
|
|
|
Current U.S.
Class: |
435/173.1 ;
181/141; 310/334; 435/283.1 |
Current CPC
Class: |
G01N 2001/4094 20130101;
G01N 1/405 20130101 |
Class at
Publication: |
435/173.1 ;
435/283.1; 310/334; 181/141 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12M 1/42 20060101 C12M001/42; H04R 1/00 20060101
H04R001/00 |
Claims
1. A genetic material releasing system comprising: a pressure wave
generating device, a chamber and a solid medium in the chamber,
wherein the solid medium is configured to adsorb or bind genetic
material from a sample, the pressure wave generating device is
configured to emit pressure waves, and the solid medium and the
chamber are configured so that when a portion of the pressure waves
impinges upon the solid medium, a portion of the adsorbed or bound
genetic material is released from the solid medium.
2. The genetic material releasing system of claim 1, wherein the
pressure wave generating device is a sound wave generating
device.
3. The genetic material releasing system of claim 2, wherein the
sound wave generating device is an acoustic energy emitting
device.
4. The genetic material releasing system of claim 3, wherein the
acoustic energy emitting device is an ultrasonic transducer, a
piezoelectric transducer, a magnorestrictive transducer or an
electrostatic transducer.
5. The genetic material releasing system of claim 3, wherein the
acoustic energy emitting device is inserted into a liquid in
contact with the solid medium.
6. The genetic material releasing system of claim 5, wherein the
acoustic energy emitting device is a sonicator.
7. The genetic material releasing system of claim 3, wherein the
chamber is inserted into a liquid in contact with the acoustic
energy emitting device.
8. The genetic material releasing system of claim 7, wherein the
acoustic energy emitting device is a transducer that focuses
acoustic energy.
9. The genetic material releasing system of claim 8, wherein the
acoustic energy emitting device is a dish-shaped transducer.
10. The genetic material releasing system of claim 3, wherein the
acoustic energy emitting device is in direct contact with the
chamber.
11. The genetic material releasing system of claim 1, wherein the
solid medium is selected from the group consisting of filter paper,
silica particles, silica gel particles, glass particles, glass
fibers, glass microfibers, glass fiber fleece, polymeric materials,
cellulosic materials, metallic beads, magnetic beads, metallic
particles and magnetic particles.
12. The genetic material releasing system of claim 11, wherein the
cellulosic material is a cellulose based substrate.
13. The genetic releasing system of claim 1 which further comprises
a mechanical energy device, wherein solid medium and the chamber
are configured so that when a portion of the mechanical energy
impinges upon the solid medium, a portion of the adsorbed or bound
genetic material is released from the solid medium.
14. A method of releasing genetic material from a solid medium
having the genetic material adsorbed or bound thereto, said method
comprising subjecting a solid medium having genetic material
adsorbed or bound thereto to a pressure wave to release at least a
portion of the genetic material from the solid medium.
15. The method of claim 14, wherein the pressure wave is a sound
wave.
16. The method of claim 15, wherein the sound wave is produced by
an acoustic energy emitting device.
17. The method of claim 16, wherein the acoustic energy emitting
device is an ultrasonic transducer, a piezoelectric transducer, a
magnorestrictive transducer or an electrostatic transducer.
18. The method of claim 16, wherein the solid medium is subjected
to the acoustic energy by inserting an acoustic energy emitting
device into a liquid in contact with the solid medium.
19. The method of claim 18, wherein the solid medium is subjected
to the acoustic energy by inserting a sonicator into a liquid in
contact with the solid medium.
20. The method of claim 16, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with an acoustic energy emitting
device.
21. The method of claim 20, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with an transducer that focuses
acoustic energy.
22. The method of claim 21, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with a dish-shaped transducer.
23. The method of claim 16, wherein the acoustic energy emitting
device is in direct contact with the chamber.
24. The method of claim 14, wherein the solid medium is selected
from the group consisting of filter paper, silica particles, silica
gel particles, glass particles, glass fibers, glass microfibers,
glass fiber fleece, polymeric materials, cellulosic materials,
metallic beads, magnetic beads, metallic particles and magnetic
particles.
25. The method of claim 24, wherein the cellulosic material is a
cellulose based substrate.
26. The method of claim 14, which further comprises subjecting the
solid medium to mechanical energy to release at least a portion of
the genetic material from the solid medium.
27. The method of claim 26, wherein the solid medium is subjected
to the mechanical energy and the pressure wave at the same
time.
28. A method of recovering genetic material from a biological
sample which comprises the steps of: (a) contacting a biological
sample comprising genetic material with a solid medium; (b)
retaining the genetic material with the solid medium; (c)
subjecting the solid medium with retained genetic material to a
pressure wave to release at least a portion of the genetic material
from the solid medium; and (d) recovering the released genetic
material.
29. The method of claim 28, wherein the pressure wave is a sound
wave.
30. The method of claim 29, wherein the sound wave is produced by
an acoustic energy emitting device.
31. The method of claim 30, wherein the acoustic energy emitting
device is an ultrasonic transducer, a piezoelectric transducer, a
magnorestrictive transducer or an electrostatic transducer.
32. The method of claim 30, wherein the solid medium is subjected
to the acoustic energy by inserting an acoustic energy emitting
device into a liquid in contact with the solid medium.
33. The method of claim 32, wherein the solid medium is subjected
to the acoustic energy by inserting a sonicator into a liquid in
contact with the solid medium.
34. The method of claim 30, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with an acoustic energy emitting
device.
35. The method of claim 34, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with a transducer that focuses
acoustic energy.
36. The method of claim 35, wherein the solid medium is subjected
to the acoustic energy by inserting a chamber holding the solid
medium into a liquid in contact with a dish-shaped transducer.
37. The method of claim 30, wherein the acoustic energy emitting
device is in direct contact with the chamber.
38. The method of claim 30, wherein the acoustic energy emitting
device is positioned sufficiently close to the chamber such that
the acoustic energy emitted from the acoustic energy emitting
device causes the release of at least a portion of genetic material
from the solid medium.
39. The method of claim 28, wherein the solid medium is selected
from the group consisting of filter paper, silica particles, silica
gel particles, glass particles, glass fibers, glass microfibers,
glass fiber fleece, polymeric materials, cellulosic materials,
metallic beads, magnetic beads, metallic particles and magnetic
particles.
40. The method of claim 39, wherein the cellulosic material is a
cellulose based substrate.
41. The method of claim 28, wherein the biological sample comprises
cells containing the genetic material and the cells are lysed
before, while or after contacting the biological sample with the
solid medium.
42. The method of claim 41, wherein the solid medium is selected
from the group consisting of filter paper, silica particles, silica
gel particles, glass particles, glass fibers, glass microfibers,
glass fiber fleece, polymeric materials, cellulosic materials,
metallic beads, magnetic beads, metallic particles and magnetic
particles.
43. The method of claim 42, wherein the cellulosic material is a
cellulose based substrate.
44. The method of claim 28, wherein the biological sample comprises
cells containing the genetic material and the solid medium is
capable of lysing the cells.
45. The method of claim 44, wherein the solid medium is selected
from the group consisting of filter paper, silica particles, silica
gel particles, glass particles, glass fibers, glass microfibers,
glass fiber fleece, polymeric materials, cellulosic materials,
metallic beads, magnetic beads, metallic particles and magnetic
particles.
46. The method of claim 45, wherein the cellulosic material is a
cellulose based substrate.
47. The method of claim 28, which further comprises subjecting the
solid medium to mechanical energy.
48. The method of claim 47, wherein the solid medium is subjected
to the mechanical energy and the pressure wave at the same
time.
49. A nucleic acid releasing device comprising: an energy emitting
device; a chamber; and an adsorption substrate, wherein the
absorption substrate is within the chamber, wherein the absorption
substrate is configured to adsorb nucleic acid from a sample, and
wherein the energy emitting device is configured to emit energy,
wherein the adsorption substrate and chamber are configured so that
when a portion of the energy impinges upon the adsorption
substrate, a portion of the adsorbed nucleic acid is released from
the adsorption substrate.
50. The nucleic acid releasing device according to claim 49,
wherein the energy is contained in pressure waves.
51. The nucleic acid releasing device according to claim 50,
wherein the pressure waves are sound waves.
52. The nucleic acid releasing device according to claim 51,
wherein the sound waves are produced by an acoustic energy emitting
device.
53. The nucleic acid releasing device according to claim 52,
wherein the acoustic energy emitting device is an ultrasonic
transducer, a piezoelectric transducer, a magnorestrictive
transducer or an electrostatic transducer.
54. The nucleic acid releasing device according to claim 52,
wherein the acoustic energy emitting device is inserted into a
liquid in contact with the adsorption substrate.
55. The nucleic acid releasing device according to claim 54,
wherein the acoustic energy emitting device is a sonicator.
56. The nucleic acid releasing device according to claim 52,
wherein the chamber is inserted into a liquid in contact with the
acoustic energy emitting device.
57. The nucleic acid releasing device according to claim 56,
wherein the acoustic energy emitting device is a transducer that
focuses acoustic energy.
58. The nucleic acid releasing device according to claim 57,
wherein the acoustic energy emitting device is a dish-shaped
transducer.
59. The nucleic acid releasing device according to claim 52,
wherein the acoustic energy emitting device is in direct contact
with the chamber.
60. The nucleic acid releasing device according to claim 52,
wherein the acoustic energy emitting device is positioned
sufficiently close to the chamber such that the acoustic energy
emitted from the acoustic energy emitting device causes the release
of at least a portion of genetic material from the solid
medium.
61. The nucleic acid releasing device according to claim 49,
wherein the energy is contained in thermal energy.
62. The genetic releasing system of claim 49 which further
comprises a mechanical energy device, wherein the solid medium and
the chamber are configured so that when a portion of the mechanical
energy impinges upon the solid medium, a portion of the adsorbed or
bound nucleic acid is released from the solid medium.
Description
[0001] This application claims the benefit of Provisional Patent
Application No. 60/867,699, filed on Nov. 29, 2006, which is
incorporated herein by this reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to systems for releasing
genetic materials from a solid medium. The present invention also
relates to methods for releasing genetic materials from a solid
medium. The present invention further relates to methods for
isolating genetic material from a biological sample.
[0004] 2. Description of the Related Art
[0005] Genetic material in blood samples, tissue samples and other
fluids is used for the purposes of monitoring and diagnosing
genetic diseases, blood-borne parasitic diseases such as malaria,
and other diseases and disorders. Genetic material further can be
used for determining paternity and monitoring other unusual cell
populations in blood and other fluids. Analysis of genetic material
can be achieved through numerous techniques and utilizes various
materials. Generally, these techniques and methods involve the
initial collection of the genetic material, storage of the genetic
material and then subsequent analysis of the genetic material.
[0006] Human genomic DNA is purified by a variety of methods
(Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel
et al. (1992), Current Protocols in Molecular Biology, John Wiley
& Sons, New York, including periodic updates). Consequently,
many commercial kit manufacturers provide products for such
techniques, for example: AmpReady.TM. (Promega, Madison, Wis.),
DNeasy.TM. (Qiagen, Valencia, Calif.), and Split Second.TM. (Roche
Molecular Biochemicals, Indianapolis, Ind.). These products rely on
the use of specialized matrices or buffer systems for the rapid
isolation of the genomic DNA molecule.
[0007] More recently, microporous filter-based techniques have
surfaced as tools for the purification of genomic DNA as well as a
whole multitude of nucleic acids. The advantages of filter-based
matrices are that they can be fashioned into many formats that
include tubes, spin tubes, sheets, and microwell plates.
Microporous filter membranes as purification support matrices have
other advantages within the art. They provide a compact, easy to
manipulate system allowing for the capture of the desired molecule
and the removal of unwanted components in a fluid phase at higher
throughput and faster processing times than possible with column
chromatography. This is due to the fast diffusion rates possible on
filter membranes.
[0008] Nucleic acid molecules have been captured on filter
membranes, generally either through simple adsorption or through a
chemical reaction between complementary reactive groups present on
the filter membrane or on a filter-bound ligand resulting in the
formation of a covalent bond between the ligand and the desired
nucleic acid.
[0009] Porous filter membrane materials used for non-covalent
nucleic acid immobilization have included materials such as nylon,
nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and
glass microfiber. A number of methods and reagents have also been
developed to also allow the direct coupling of nucleic acids onto
solid supports, such as oligonucleotides and primers (e.g., Coull
et al. (1986), Tetrahedron Lett 27:3991-3994; Connolly (1987),
Nucleic Acids Res 15:3131-3139, 1987; Connolly and Rider (1985),
Nucleic Acids Res 12:4485-4502; Yang et al. (1998), Proc Natl Acad
Sci USA 95:5462-5467). UV cross-linking of DNA (Church et al.
(1984), Proc Natl Acad Sci USA 81:1991-1995), RNA (Khandjian et al.
(1986), Anal Biochem 159:227-232) to nylon membranes, The
Generation Capture Column Kit (Qiagen, Valencia, Calif.) QIAamp DNA
Blood Mini Kit, QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.),
ChargeSwitch.RTM. technology (Invitrogen, Corp., Carlsbad, Calif.),
MagaZorb.RTM. isolation kits (Cortex Biochem, Inc., San Leandro,
Calif.) and NucliSENS.RTM. Isolation Kit (bioMerieux, Inc., Durham,
N.H.) have also been reported.
[0010] Many chemical methods have been utilized for the
immobilization of molecules such as nucleic acids on filter
membranes. For example, activated paper (TransBind.TM., Schleicher
& Schuell Ltd., Keene, N.H.), carbodimidazole-activated
hydrogel-coated PVDF membrane (Immobilin-IAV.TM., Millipore Corp.,
Bedford, Mass.), MAP paper (Amersham, Littlechalfont Bucks, Wis.),
activated nylon (BioDyne.TM., Pall Corp., (Glen Cove, N.Y.), DVS-
and cyanogen bromide-activated nitrocellulose. Membranes bound with
specific ligands are also known such as the SAM2.TM. Biotin Capture
Membrane (Promega) which binds biotinylated molecules based on
their affinity to streptavidin or MAC affinity membrane system
(protein A/G) (Amicon, Bedford, Mass.). A primary disadvantage of
covalent attachment of biomolecules onto activated membranes is
that the covalently bound molecules can not be retrieved from the
filter membrane.
[0011] More recently, glass microfiber has been shown to
specifically bind nucleic acids from a variety of nucleic acid
containing sources very effectively (e.g., Itoh et al. (1997),
Nucleic Acids Res 25:1315-1316; Andersson et al (1996),
BioTechniques 20:1022-1027; U.S. Pat. No. 5,910,246). Under the
correct salt and buffering conditions, nucleic acids will bind to
glass or silica with high specificity. U.S. Pat. No. 5,234,809
describes a method in which nucleic acids are bound to a solid
medium in the form of silica particles, in the presence of a
chaotropic agent such as a guanidinium salt, and thereby separated
from the remainder of the sample. International published
application No. WO 91/12079 describes a method whereby nucleic acid
is trapped on the surface of a solid medium by precipitation.
Generally speaking, alcohols and salts are used as precipitants.
U.S. Pat. No. 6,617,105 describes a method for isolating nucleic
acids from cells in which cells are non-specifically or
specifically bound to a solid medium, such as glass, silica, latex
or polymeric materials, the cells are lysed allowing the DNA to be
bound to the same solid phase which is then recovered. A similar
process using a porous matrix is described in U.S. Pat. No.
5,653,141.
[0012] Nucleic acids or genetic material can be immobilized to a
cellulosic-based dry solid support or filter (FTA.RTM. filter;
FTA.RTM. cellulosic filter material; Whatman, plc). See, for
example, U.S. Pat. Nos. 5,496,562, 5,756,126, 5,807,527, 6,322,983
and 6,627,226. The solid support described is conditioned with a
chemical composition that is capable of carrying out several
functions: (i) lyse intact cellular material upon contact,
releasing genetic material, (ii) enable and allow for the
conditions that facilitate genetic material immobilization to the
solid support (probably by a combination of mechanical and
chaotropic), (iii) maintain the immobilized genetic material in a
stable state without damage due to degradation, endonuclease
activity, UV interference, and microbial attack, and (iv) maintain
the genetic material as a support-bound molecule that is not
removed from the solid support during any down stream processing
(e.g., Del Rio et al. (1995), BioTechniques 20:970-974).
[0013] The usefulness of the so called FTA.RTM. cellulosic filter
material described in the above patents has been illustrated for
several nucleic acid techniques such as bacterial ribotyping
(Rogers and Burgoyne (1997), Anal Biochem 247: 223-227), detection
of single base differences in viral and human DNA (Ibrahim et al.
(1998), Anal Chem 70: 2013-2017), DNA databasing (Ledray et al.
(1997), J Emergency Nursing 23:156-158), automated processing for
STR electrophoresis (Belgrader and Marino (1996), L.R.A. 9:3-7;
Belgrader et al. (1995), BioTechniques 19:427-432), and
oligonucleotide ligation assay for diagnostics (Baron et al.
(1996), Nature Biotech 14:1279-1282).
[0014] As illustrated above, various materials and solid media have
been and continue to be utilized to provide a base for performing
any desired analysis of the genetic material. Those materials
include, for example, FTA.RTM. filter paper or FTA.RTM.-coated
materials. In particular, FTA.RTM.-coated materials have been
successfully utilized for preparing all types of genetic material
for subsequent genetic analysis. Genetic material prepared using
FTA.RTM.-coated materials and FTA.RTM. techniques yields highly
purified material bound to the cellulosic base filter for the
duration of various subsequent applications and amplification
reactions. FTA.RTM.-coated base filter materials include, but are
not limited to Whatman cellulosic BFC-180, 31-ET, glass
microfibers, and other similar filter materials known to those of
skill in the art.
[0015] It is known that high molecular weight genetic material does
not release well from any media. For example, it has been shown
that nucleic acid or genetic material applied to, and immobilized
to, FTA.RTM. filters cannot be simply removed, or eluted from the
solid support once bound (Del Rio et al. (1995), BioTechniques
20:970-974). This is a major disadvantage for applications where
several downstream processes are required from the same sample,
such a STR profiling and genotyping. This disadvantage has recently
been confirmed. Specifically, it has been shown that not all
commercial methods are capable of extracting sufficient DNA for use
in a whole genome amplification step prior to a quantitative PCR
(Q-PCR) reaction (Sjoholm et al. (2007), Clin Chem 53:1401-1407).
These commercial methods are extremely cumbersome and many hours
are required to obtain enough material for use in a Q-PCR
reaction.
[0016] The difficulty in removing genetic material from FTA.RTM.
filters has been well recognized in the art, and several techniques
have been developed for removing genetic material from FTA.RTM.
filters. One technique includes the use of chemical methods, such
as the use of special buffer compositions (U.S. Pat. No.
6,410,725). This technique, as well as other techniques that rely
on the use of chemical methods to release the genetic material,
require additional reagents and steps, thus increasing the
complexity of the isolation of genetic material. Other techniques
include photolysis (U.S. Pat. No. 6,972,329), heat (U.S. Pat. No.
6,645,717) and treatment of the genetic material on the paper for
detection (U.S. Pat. No. 6,746,841).
[0017] Although the above methods speed up the nucleic acid
separation process, a need still exists for methods which are quick
and simple to perform, which have higher efficiency, and in
particular which are readily amenable to isolating nucleic acids
from cells for use in microfluidic environments, such as
microfluidic PCR methods.
SUMMARY OF THE INVENTION
[0018] The present invention relates to systems for releasing
genetic materials from a solid medium. The present invention also
relates to methods for releasing genetic materials from a solid
medium. The present invention further relates to methods for
isolating genetic material from a biological sample.
[0019] Thus, in one aspect, the present invention provides a
genetic material releasing system comprising a pressure wave
emitting device, a chamber and a solid medium in the chamber. The
solid medium is configured to adsorb or bind genetic material from
a sample added to the chamber. In one embodiment, the adsorption or
binding is non-specific. The pressure wave emitting device is
configured to emit pressure waves. The solid medium and the chamber
are configured so that when a portion of the pressure waves impinge
upon the solid medium, a portion of the adsorbed or bound genetic
material is released from the solid medium. In one embodiment, the
pressure waves are sound waves and the pressure wave emitting
device is an acoustic energy emitting device. In one embodiment,
the solid medium is any solid material that is capable of adsorbing
or binding genetic material. In another embodiment, the solid
medium is solid material that includes a coating which is capable
of adsorbing or binding genetic material. In a further embodiment,
the solid medium is selected from the group consisting of FTA.RTM.
paper or FTA.RTM.-coated materials, silica particles, silica gel
particles, glass particles, glass fibers, glass microfibers, glass
fiber fleece, cellulosic materials, such as a cellulose based
substrates, metallic beads, magnetic beads, metallic particles and
magnetic particles. In a further embodiment, the genetic material
system further comprises a mechanical energy device. The solid
medium and the chamber are configured so that when a portion of the
mechanical energy impinges upon the solid medium, a portion of the
adsorbed or bound genetic material is released from the solid
medium.
[0020] In a second aspect, the present invention provides a method
for releasing genetic material from a solid medium which has
adsorbed or bound genetic material. In accordance with this aspect,
the method comprises subjecting a solid medium having genetic
material adsorbed or bound thereto to pressure waves to release at
least a portion of the genetic material from the solid medium. In
one embodiment, the pressure waves are sound waves and the sound
waves are produced by an acoustic energy emitting device. In one
embodiment, the solid medium is any solid material that is capable
of adsorbing or binding genetic material. In another embodiment,
the solid medium is solid material that includes a coating which is
capable of adsorbing or binding genetic material. In a further
embodiment, the solid medium is selected from the group consisting
of FTA.RTM. paper or FTA.RTM.-coated materials, silica particles,
silica gel particles, glass particles, glass fibers, glass
microfibers, glass fiber fleece, cellulosic materials, such as a
cellulose based substrates, metallic beads, magnetic beads,
metallic particles and magnetic particles. In a further embodiment,
the method for releasing genetic material from a solid medium
further comprises subjecting the solid medium to mechanical energy
to release at least a portion of the genetic material from the
solid medium. The released genetic material can be recovered and
stored for future use. Alternatively, the released genetic material
can be separated from the solid medium and directly processed for
nucleic acid analysis such as PCR reactions. The method is
particularly suited for releasing genetic material from a solid
medium that can be used in microfluidic PCR techniques for nucleic
acid analysis and detection. In addition to causing the release of
the genetic material from the solid medium, one additional
advantage of the use of acoustic energy in accordance with the
present invention is the fragmentation of the DNA by the acoustic
energy which makes the DNA better suited for analysis.
[0021] In a third aspect, the present invention provides a method
for recovering genetic material from a biological sample. In
accordance with this aspect, the method comprises contacting a
biological sample comprising genetic material with a solid medium,
retaining the genetic material with the solid medium, subjecting
the solid medium with retained genetic material to pressure waves
to release at least a portion of the genetic material from the
solid medium and recovering the released genetic material. The
genetic material is retained with the solid medium by adsorption or
binding.
[0022] In one embodiment, the pressure waves are sound waves and
sound waves are produced by an acoustic energy emitting device. In
one embodiment, the genetic material is free in the biological
sample. In another embodiment, the biological sample comprises
cells containing the genetic material and the cells are lysed
before contacting the biological sample with the solid medium. In
an additional embodiment, the biological sample comprises cells
containing the genetic material and the cells are lysed while
contacting the biological sample with the solid medium. In a
further embodiment, the biological sample comprises cells
containing the genetic material and the solid medium is capable of
lysing the cells. In a still further embodiment, the biological
sample comprises cells containing the genetic material and the
cells are lysed after contacting the biological sample with the
solid medium. In one embodiment, the solid medium is washed to
remove non-genetic material prior to releasing the genetic material
from the solid medium. In one embodiment, the solid medium is any
solid material that is capable of adsorbing or binding genetic
material. In another embodiment, the solid medium is solid material
that includes a coating which is capable of adsorbing or binding
genetic material. In a further embodiment, the solid medium is
selected from the group consisting of FTA.RTM. paper or
FTA.RTM.-coated materials, silica particles, silica gel particles,
glass particles, glass fibers, glass microfibers, glass fiber
fleece, cellulosic materials, such as a cellulose based substrates,
metallic beads, magnetic beads, metallic particles and magnetic
particles. In a further embodiment, the method for releasing
genetic material from a solid medium further comprises subjecting
the solid medium to mechanical energy to release at least a portion
of the genetic material from the solid medium.
[0023] The released genetic material can be recovered, e.g.,
separated from the solid medium, and stored for future use.
Alternatively, the released genetic material can be recovered,
e.g., separated from the solid medium, and directly processed for
nucleic acid analysis such as PCR reactions. The method is
particularly suited for releasing genetic material from a solid
medium that can be used in microfluidic PCR techniques for nucleic
acid analysis and detection. In addition to causing the release of
the genetic material from the solid medium, one additional
advantage of the use of acoustic energy in accordance with the
present invention is the fragmentation of the DNA by the acoustic
energy which makes the DNA better suited for analysis.
[0024] In a fourth aspect, the present invention provides for a
nucleic acid releasing device which comprises an energy emitting
device, a chamber and an adsorption substrate, wherein the
absorption substrate is within the chamber, wherein the absorption
substrate is configured to adsorb nucleic acid from a sample, and
wherein the energy emitting device is configured to emit energy,
wherein the adsorption substrate and chamber are configured so that
when a portion of the energy impinges upon the adsorption
substrate, a portion of the adsorbed nucleic acid is released from
the adsorption substrate. In one embodiment, the energy emitting
device is a pressure wave emitting device. In another embodiment,
the pressure wave emitting device is an acoustic energy emitting
device. In yet another aspect of this embodiment, the energy is
contained in thermal energy. In a further embodiment, the nucleic
acid releasing device further comprises a mechanical energy device.
The absorption substrate and the chamber are configured so that
when a portion of the mechanical energy impinges upon the solid
medium, a portion of the adsorbed or bound genetic material is
released from the solid medium.
[0025] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The accompanying drawings, which are incorporated herein and
form part of the specification, illustrate various embodiments of
the present invention.
[0027] FIG. 1 is an electron micrograph showing DNA entrapped
within the FTA.RTM. matrix (magnification.times.10,000).
[0028] FIG. 2A is an illustration of one embodiment of the present
invention in which genetic material is bound to beads and the
sample is subjected to sonication to release the genetic material
from the beads.
[0029] FIG. 2B is an illustration of another embodiment of the
present invention in which genetic material is bound to cellulosic
substrate and the sample is subjected to sonication to release the
genetic material from the cellulosic substrate.
[0030] FIG. 3A illustrates an adaptive focused acoustic system that
can be used to send acoustic energy wave packets into a sample
container that may contain genetic material bound to a solid
phase.
[0031] FIG. 3B illustrates the transducer shown in FIG. 3A.
[0032] FIG. 3C illustrates the transducer shown in FIG. 3B with
beam of energy.
[0033] FIG. 4 is an illustration of a non-contact acoustic
system.
[0034] FIG. 5A shows a comparison of different extraction methods
for FTA.RTM. paper for gram positive microorganism.
[0035] FIG. 5B shows a comparison of different extraction methods
for FTA.RTM. paper for gram negative microorganism.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT
[0036] The present invention has several embodiments and relies on
patents, patent applications and other references for details known
to those of the art. Therefore, when a patent, patent application,
or other reference is cited or repeated herein, it should be
understood that it is incorporated by reference in its entirety for
all purposes as well as for the proposition that is recited.
[0037] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, biochemistry, and
immunology, which are within the skill of the art. Such
conventional techniques include polymer array synthesis,
hybridization, ligation, and detection of hybridization using a
label. Specific illustrations of suitable techniques can be had by
reference to the example herein below. However, other equivalent
conventional procedures can, of course, also be used. Such
conventional techniques and descriptions can be found in standard
laboratory manuals such as Genome Analysis: A Laboratory Manual
Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells:
A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular
Cloning: A Laboratory Manual (all from Cold Spring Harbor
Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.)
Freeman, N.Y., Gait, Oligonucleotide Synthesis: A Practical
Approach, 1984, IRL Press, London, Nelson and Cox (2000),
Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub.,
New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W.H.
Freeman Pub., New York, N.Y., all of which are herein incorporated
in their entirety by reference for all purposes.
[0038] In one aspect, the present invention provides a genetic
material releasing system comprising a pressure wave emitting
device, a chamber and a solid medium in the chamber. The solid
medium is configured to adsorb or bind genetic material from a
sample added to the chamber. The pressure wave emitting device is
configured to emit pressure waves in a manner that causes the
release of at least a portion of the genetic material from the
solid medium. In one embodiment, the pressure waves are sound waves
and the pressure wave emitting device is an acoustic energy
emitting device.
[0039] As used herein, "genetic material" means any nucleic acid,
including DNA and RNA. Thus, genetic material may include a gene, a
part of a gene, a group of genes, a fragment of many genes, a
molecule of DNA or RNA, molecules of DNA or RNA, a fragment of a
DNA or RNA molecule, or fragments of many DNA or RNA molecules.
Genetic material can refer to anything from a small fragment of DNA
or RNA to the entire genome of an organism.
[0040] As used herein, the term "chamber" refers to any device to
which a sample can be added and treated in accordance with the
present invention.
[0041] The "solid medium" or "solid substrate" or "solid phase" or
"solid matrix" is not critical and can be any solid material
generally used by those skilled in the art. A "solid material" or
"solid phase material" or "solid phase," as used herein, refers to
any material which is insoluble, or can be made insoluble by a
subsequent reaction. Any known solid support may be used. Examples
of commonly used solid phase materials include, but are not limited
to, matrices, particles, micro beads and macro beads free in
solution, made of any known material, e.g., cellulose,
nitrocellulose, nylon, glass, polyacrylates, mixed polymers,
polystyrene, silane polypropylene, silica gel, metal, such as
non-magnetic and magnetic beads and particles. See, for example,
U.S. Pat. Nos. 4,358,535, 4,797,355, 5,237,016, 5,652,141,
6,645,717, 6,617,105, 6,627,226, 7,214,780 and 7,294,489. In some
embodiments, the solid substrate may include a magnetic bead, a
matrix, a particle, a polymeric bead, a chromatographic resin,
filter paper, a membrane or a hydrogel.
[0042] The solid material may be capable of adsorbing or binding
the genetic material directly or it may be coated with a material
that is capable of adsorbing or binding the genetic material. As
used herein, adsorption or binding refers to the immobilization of
genetic material on solid phases through ionic interactions,
hydrophobic interactions, covalent interactions, chelation and the
like. The interactions may be direct or they may be indirect, such
as through one or more linkers, as is well known to the skilled
artisan. Among the advantages of solid phase systems is that they
can be washed with relative ease to remove cellular components
other than the bound genetic material.
[0043] One solid medium that has found significant use for
collecting and storing DNA samples is FTA.RTM. solid substrates.
These solid substrates also contain chemicals that lyse cells,
denature proteins and protect the genetic material from enzymatic
or other degradation. FIG. 1 is an electron micrograph showing DNA
entrapped within the FTA.RTM. matrix (magnification.times.10,000).
As discussed above, it is well known that it is difficult to remove
the genetic material from FTA.RTM. solid substrates.
[0044] The pressure wave emitting device may be any device the is
capable of generating pressure waves within the chamber such that
the impingement of the pressure waves on the solid medium causes a
release of at least a portion of the genetic material. Suitable
pressure wave emitting devices are well known to the skilled
artisan. Examples of pressure wave emitting devices include, but
are not limited to, a transducer that is a vibrating type or a
transducer that is an oscillating device. These devices are
activated to generate pressure waves in the chamber.
[0045] In one embodiment, the pressure wave emitting device is an
acoustic energy emitting device. The acoustic energy emitting
device produces acoustic energy this is used to release genetic
material from a solid support. Any device that generates sound
waves can be used as a source of acoustic energy. Such devices
include, but are not limited to, ultrasonic transducers,
piezoelectric transducers, magnorestrictive transducers and
electrostatic transducers. Suitable devices are well known in the
art including such commercially available devices as Sonicator 4000
(Misonix, Inc., Farmingdale, N.Y., USA), Microson.RTM. Sonicator
Microprobe or Micro Cup Horn (Kimble/Kontes, Vineland, N.J., USA)
and Covaris.TM. Adaptive Focused Acoustics (Nexus Biosystems,
Poway, Calif., USA). Other suitable devices are described in U.S.
Pat. Nos. 6,881,541 and 6,878,540 and in U.S. Patent Application
Publication No. 2007/0170812. One advantage of using acoustic
energy to release the genetic material from the solid support is
that not only is the genetic material released, but the genetic
material is also sheared to generate fragments of genetic
material.
[0046] FIGS. 2-4 show illustrations of the acoustic energy devices
referenced above. Specifically, FIGS. 2A and 2B illustrate an
acoustic energy device which includes a probe which is inserted
into a liquid in contact with the solid medium. Contact sonicators,
i.e., sonicators placed in the sample containing chamber, are
examples of such an acoustic energy device. FIG. 3A illustrates an
acoustic energy emitting device 100 which includes a transducer 102
that focuses acoustic energy into a chamber 101 that is inserted
into a liquid 103 in contact with the device. In the non-limiting
embodiment shown in FIG. 3A, the acoustic energy emitting device is
a dish-shaped transducer 102 that focuses the acoustic energy to a
focal zone 104 in the chamber 101. FIG. 3B is a further
illustration of transducer 102. FIG. 3C illustrates transducer 102
with beam of energy 105. FIG. 4 illustrates a non-contact acoustic
energy emitting device 400 in which the device is not in contact
with the sample but is in direct contact with the chamber 401. The
system can be used to transmit acoustic energy through the sample
chamber. In another aspect, the acoustic energy emitting device is
positioned sufficiently close to the chamber such that the acoustic
energy emitted from the acoustic energy emitting device causes the
release of at least a portion of genetic material from the solid
medium.
[0047] In a further embodiment, genetic material releasing system
further comprises a mechanical energy device. The mechanical energy
device is configured in a manner so that the mechanical energy
causes the release of at least a portion of the genetic material
from the solid medium. Any suitable mechanical energy device can be
used and such devices are well known to the skilled artisan.
Examples of mechanical energy devices include, but are not limited
to, devices which cause vibration, vortexing and homogenization.
For example, Pro Scientific offers a variety of homogenizers (e.g.
Pro 200-Pro 400), VWR Scientific offers a variety of vortexers
(e.g. MV-1 Mini Vortexer), and BioSpec Products, Inc. offers a
handheld ultrasonic homogenizer (Sonozap), all of which can be used
to provide the energy necessary to liberate DNA from a solid
medium.
[0048] In a second aspect, the present invention provides a method
for releasing genetic material from a solid medium which has
adsorbed or bound genetic material. In accordance with this aspect,
the method comprises subjecting a solid medium having genetic
material adsorbed or bound thereto to pressure waves to release at
least a portion of the genetic material from the solid medium. The
genetic material and solid medium are as described herein. The
pressure waves are generated using a pressure wave emitting device
as described herein. In one embodiment, the pressure wave emitting
device is an acoustic energy emitting device, as described herein.
The pressure waves are generated in a chamber containing the solid
medium with the genetic material for a time sufficient to release
at least a portion of the genetic material from the solid medium.
The length of time is empirically determined on the basis of the
amount of genetic material that may be needed for downstream
processing. This determination is well within the skill in the
art.
[0049] FIGS. 2A and 2B illustrate embodiments of this method of the
present invention. A chamber is provided which contains a solid
medium having genetic material adsorbed or bound thereto. The DNA
is bound to a solid medium in a conventional manner. In the
illustrated examples, the solid medium may be beads (FIG. 2A) which
may be non-magnetic or magnetic or FTA.RTM. paper (FIG. 2B). The
solid medium having the adsorbed or bound DNA is exposed to
ultrasonic waves to physically disrupt the interaction of the DNA
with the solid medium. FIGS. 2A and 2B show the use of a micro tip
probe sonicator to produce ultrasonic energy in a liquid via a
probe introduced directly into the liquid. The sample is sonicated
and the sonication results in the release of at least a part of the
genetic material from the solid medium.
[0050] In a further embodiment, the method further comprises
subjecting the sample to mechanical energy to cause the release of
at least a portion of the genetic material associated with the
solid medium. The mechanical energy, as described herein, is
generated in a chamber containing the solid medium with the genetic
material for a time sufficient to release at least a portion of the
genetic material from the solid medium. The length of time is
empirically determined on the basis of the amount of genetic
material that may be need for downstream processing. This
determination is well within the skill in the art. The use of
mechanical energy in conjunction with the use of pressure waves,
particularly acoustic energy, enables using a lower amount of
pressure waves or acoustic energy in releasing the genetic
material.
[0051] In other embodiments, the sample comprises cells and the
method further comprises first lysing the cells before subjecting
the sample to the pressure waves. In some embodiments, the lysis is
performed by chemical lysis. Typical chemical lysing agents fall
into several categories, such as enzymes, and detergents. Lysosyme
is an enzyme that hydrolytically attacks the cell walls of many
bacteria; trypsin is a protease enzyme that breaks the cell
membrane of most eukaryotic cells. Other proteases with specificity
for certain peptide sequences can be employed and are preferred if
the target moiety is liable to certain proteases. Proteinase K is
often used because it also digests nuclear proteins and host cell
enzymes that may interfere with polymerase chain reaction (PCR).
For eucaryotic cells, detergents such as Triton X-100 or sodium
dodecyl sulfate solubilize the cell membrane and release
intracellular contents. Commercial cell lysis products can be used
to lyse cells in the cellular sample. Such commercial cell lysis
products include, but are not limited to, Poppers Cell Lysis
Reagents (Pierce, Rockville, Ill., USA), Wizard.RTM. Genomic DNA
Purification Kit (Promega Corp., Madison, Wis., USA), lysis
solutions from Qiagen, Inc. (Valencia, Calif., USA), and Cell Lysis
Solution (Spectrum Chemical and Laboratory Products, Gardena,
Calif., USA). Alternatively, acoustic energy, such as described
herein and used to release the genetic material from the solid
phase, can be used to lyse cells in a cellular sample.
[0052] The lysis may occur prior to contacting the sample with the
solid medium. In this embodiment, the sample could be treated to
remove contaminants as is well known to the skilled artisan.
Alternatively, the solid medium may contain components that lyse
intact cellular material upon contact thereby releasing the genetic
material which is then adsorbed or bound to the solid medium. One
example of such a solid medium is FTA.RTM. paper or FTA.RTM.-coated
materials.
[0053] The released genetic material can be recovered, e.g.,
separated from the solid medium, and stored for future use.
Alternatively, the released genetic material can be recovered,
e.g., separated from the solid medium, and directly processed for
nucleic acid analysis such as PCR reactions.
[0054] Experimental data has been generated to determine the
efficiency of sonication versus the methods recommended by the
manufacturer of FTA.RTM. paper. In the experiment detailed below,
Whatman FTA.RTM. paper is the solid substrate used to bind DNA
after cells lyse upon contact with the paper. The general procedure
for DNA elution from the paper is incubating the paper in TE buffer
for 10-15 minutes, vortexing a few times, and then centrifuging the
sample for 1 minute at 13,000 rpm. To increase the efficiency of
elution of DNA from the FTA.RTM. paper the manufacturer has also
recommended incubating the paper for 5 minutes in a pH 13 solution
and neutralizing for 15 minutes with a second buffer, then
centrifuge for 1 minute at 13,000 rpm. Both methods were tested for
efficiency and compared with sonicating the FTA.RTM. paper in the
presence of water or TE buffer in accordance with one embodiment of
the present invention.
[0055] As shown in FIGS. 5A and 5B, the sonication results are
significantly better than that of altering pH or TE elution. FIG.
5A shows a comparison of different extractions methods for FTA.RTM.
paper for gram positive microorganism. The extraction methods
examined included TE (10 mM Tris-HCl, 1 mM EDTA; pH 7.5)
extraction, high pH (i.e., pH13), sonication (3.times.5 sec pulses
at 20 kH) and sonication with high pH. In FIG. 5A, it is clear that
treatment with high pH is deleterious to the DNA, and that
sonication is clearly more efficient than either TE elution or high
pH elution.
[0056] FIG. 5B shows a comparison of different extraction methods
for FTA.RTM. paper for gram negative microorganism. The extraction
methods examined included TE extraction, high pH (i.e., pH13),
sonication and sonication with high pH. In FIG. 5B, sonication is
about an order of magnitude more efficient at eluting DNA when
compared with the TE elutions, and is slightly better than the pH
13 elutions in most cases.
[0057] The data in FIGS. 5A and 5B reflect the improved efficiency
of eluting DNA from FTA.RTM. paper when sonication is used as the
elution method.
[0058] In a third aspect, the present invention provides a method
for recovering genetic material from a biological sample. In
accordance with this aspect, the method comprises contacting a
biological sample comprising genetic material with a solid medium,
retaining the genetic material with the solid medium, subjecting
the solid medium with retained genetic material to pressure waves
to release at least a portion of the genetic material from the
solid medium and recovering the released genetic material. The
solid medium is as described herein. In one embodiment, the genetic
material is free in the biological sample. In another embodiment
the biological sample comprises cells. The biological sample
comprising cells may be a blood sample, a urine sample, a saliva
sample, a sputum sample, a cerebrospinal fluid sample, a body fluid
sample, a tissue sample, or the like. The genetic material is
retained with the solid medium by adsorption or binding. The
biological sample is contacted with the solid medium, such as the
solid media described herein, using conventional techniques well
known to the skilled artisan.
[0059] The pressure waves are generated using a pressure wave
emitting device as described herein. In one embodiment, the
pressure wave emitting device is an acoustic energy emitting
device, as described herein. The pressure waves are generated in a
chamber containing the solid medium with the genetic material for a
time sufficient to release at least a portion of the genetic
material from the solid medium as described herein.
[0060] In another embodiment, the biological sample comprising
cells containing the genetic material is first treated to lyse the
cells before contacting the biological sample with the solid medium
as described herein. In an additional embodiment, the biological
sample comprises cells containing the genetic material and the
cells are lysed while contacting the biological sample with the
solid medium as described herein. In a further embodiment, the
biological sample comprises cells containing the genetic material
and the solid medium is capable of lysing the cells. In a still
further embodiment, the biological sample comprises cells
containing the genetic material and the cells are lysed after
contacting the biological sample with the solid medium. In one
embodiment, the solid medium is washed to remove non-genetic
material prior to releasing the genetic material from the solid
medium.
[0061] In a further embodiment, the method for releasing genetic
material from a solid medium further comprises subjecting the solid
medium to mechanical energy as described herein to release at least
a portion of the genetic material from the solid medium.
[0062] The released genetic material can be recovered, e.g.,
separated from the solid medium, and stored for future use or used
in downstream applications as described herein or as well known to
the skilled artisan.
[0063] In a fourth aspect, the present invention provides for a
nucleic acid releasing device comprises an energy emitting device,
a chamber and an adsorption substrate, wherein the absorption
substrate is within the chamber, wherein the absorption substrate
is configured to adsorb nucleic acid from a sample, and wherein the
energy emitting device is configured to emit energy, wherein the
adsorption substrate and chamber are configured so that when a
portion of the energy impinges upon the adsorption substrate, a
portion of the adsorbed nucleic acid is released from the
adsorption substrate. The nucleic acid may be DNA or RNA. The
adsorption substrates are well known to the skilled artisan and
include those solid media described herein which adsorb nucleic
acids. In one embodiment, the energy emitting device is a pressure
wave emitting device as described herein. In another embodiment,
the pressure wave emitting device is an acoustic energy emitting
device as described herein. In yet another aspect of this
embodiment, the energy is contained in thermal energy, such as, for
example, heat produced by pressure waves within the chamber. In a
further embodiment, the nucleic acid releasing device further
comprises a mechanical energy device as described herein. The
absorption substrate and the chamber are configured so that when a
portion of the mechanical energy impinges upon the solid medium, a
portion of the adsorbed or bound genetic material is released from
the solid medium as described herein.
[0064] There are several advantages to the method of the present
invention versus conventional methods of eluting DNA from a solid
substrate. For example, the DNA is liberated from a solid material
in seconds when acoustic energy is applied, versus minutes to hours
when other methods are utilized. In addition, DNA is eluted more
efficiently when acoustic energy is used versus traditional
methods. Recovery of the DNA can be greater than an order of
magnitude higher when using acoustic energy versus conventional
elution methods. Further, the DNA is also in more manageable
fragments for down stream applications when acoustic energy is
applied to liberate DNA from a solid matrix.
[0065] The method is particularly suited for releasing genetic
material from a solid medium that can be used in microfluidic PCR
techniques for nucleic acid analysis and detection. In addition to
causing the release of the genetic material from the solid medium,
one additional advantage of the use of acoustic energy in
accordance with the present invention is the fragmentation of the
DNA by the acoustic energy which makes the DNA better suited for
analysis.
[0066] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. For example, if the range 10-15 is disclosed, then
11, 12, 13, and 14 are also disclosed. All methods described herein
can be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.
[0067] It will be appreciated that the methods and compositions of
the instant invention can be incorporated in the form of a variety
of embodiments, only a few of which are disclosed herein.
Variations of those embodiments may become apparent to those of
ordinary skill in the art upon reading the foregoing description.
The inventors expect skilled artisans to employ such variations as
appropriate, and the inventors intend for the invention to be
practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
* * * * *