U.S. patent application number 15/858140 was filed with the patent office on 2018-05-17 for methods for separating nucleic acids with graphene coated magnetic beads.
The applicant listed for this patent is Life Magnetics, Inc.. Invention is credited to Kevin Hagedorn, Saravana Murthy.
Application Number | 20180135040 15/858140 |
Document ID | / |
Family ID | 62106709 |
Filed Date | 2018-05-17 |
United States Patent
Application |
20180135040 |
Kind Code |
A1 |
Hagedorn; Kevin ; et
al. |
May 17, 2018 |
METHODS FOR SEPARATING NUCLEIC ACIDS WITH GRAPHENE COATED MAGNETIC
BEADS
Abstract
Methods for separating and identifying nucleic acids utilize
carbon coated magnetic beads. The method teaches that multivalent
cations promote binding of single stranded nucleic acids to the
beads and that the single stranded nucleic acids can be released
with the addition of chelating agents that bind the multivalent
cations such as EDTA. The method further teaches that fragile
single stranded nucleic acids, such as RNA, can be stored on the
surface of the beads. Lastly, the method also teaches that by
iteratively adding complimentary DNA oligos, single stranded
nucleic acids can be quantified or individually isolated using the
carbon coated magnetic beads.
Inventors: |
Hagedorn; Kevin; (AnnArbor,
MI) ; Murthy; Saravana; (Gaitersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Magnetics, Inc. |
Ann Arbor |
MI |
US |
|
|
Family ID: |
62106709 |
Appl. No.: |
15/858140 |
Filed: |
December 29, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15434470 |
Feb 16, 2017 |
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15858140 |
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62295985 |
Feb 16, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1013 20130101;
C12Q 1/686 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12Q 1/686 20060101 C12Q001/686 |
Claims
1. A method for separating single stranded nucleic acids from
associated biological material, the method comprising: creating a
complex of a carbon coated material and single stranded nucleic
acids from a mixture containing single stranded nucleic acids,
double stranded nucleic acids, the carbon coated material, at least
one chaotropic salt and at least one alcohol having between one and
four carbon atoms, wherein the material is a chaotropic salt
present at a concentration from 0.01M to 3M and wherein the
alcohols are present as 5 vol % to 99 vol %; and after the complex
of carbon coated material and single stranded nucleic acids has
been created, removing the carbon coated material complexed with
single-stranded nucleic acid from the mixture.
2. The method of claim 1 wherein the carbon coating present on the
carbon coated material is at least one of graphene, pyrolytic
carbon or a mixture of graphene and pyrolytic carbon.
3. The method of claim 1, wherein chaotropic salt is a guanidinium
salt composed of a guanidinium cation and an anion selected from
the group consisting of chloride, bromide, fluoride, acetate,
sulfate, nitrate, carbonate, thiocyanate and mixtures thereon.
4. The method of claim 1, wherein the chaotropic salt is present at
a concentration between 0.01 M and 0.6M.
5. The method of claim 1, wherein the alcohol is present in an
amount between 10 vol % and 85 vol %.
6. A method for isolating nucleic acids having a defined sequence
from an aqueous solution, the method comprising: contacting a
composition containing single stranded nucleic acid and double
stranded nucleic acid with at least one nucleic acid probe, the
nucleic acid probe complimentary with amino acids present on at
least a portion of the nucleic acid in the aqueous solution, the
contacting step occurring for an interval sufficient for the at
least one nucleic acid probe to duplex with amino acid sequences
present on at least a portion of the nucleic acid present in the
solution to produce an admixture composed of duplexed nucleic acid
material and non-duplexed nucleic acid material; to the resulting
admixture containing duplexed nucleic acid material, adding
carbon-coated material, at least one chaotropic salt and
short-chain alcohol, the short-chain alcohol present in an amount
between 2 vol % and 99 vol %; allowing contact for an interval
sufficient to permit association between the carbon coated material
and at least a portion of the single stranded nucleic acids present
in the admixture; separating carbon-coated material complexed with
the single stranded nucleic acids from liquid supernatant, the
liquid supernatant containing complexed double stranded nucleic
acid.
7. The method of claim 6, wherein the carbon coated material is
configured as beads, the beads each having a core and an outer
surface wherein at least the core is magnetic and contains at least
30% by weight of a metal selected from the group consisting of Ni,
Fe, Co, or mixtures thereof.
8. The method of claim 6, wherein the chaotropic salt is added in
an amount sufficient to provide a concentration between 0.01M and
3M.
9. The method of claim 8, wherein chaotropic salt is selected from
the group consisting of a guanidinium salts, urea, thiourea,
lithium acetate, lithium perchlorate, magnesium chloride and
mixtures thereof.
10. The method of claim 9, wherein chaotropic salt is a guanidinium
salt composed of a guanidinium cation and an anion selected from
the group consisting of chloride, bromide, fluoride, acetate,
sulfate, nitrate, carbonate, thiocyanate and mixtures thereof.
11. The method of claim 10, wherein the concentration of the
chaotropic salt is between 0.01 and 8M.
12. The method of claim 6, wherein the short chain alcohol is
selected from the group consisting of methanol, ethanol, propanol,
isopronaol, phenol, and mixtures thereof.
13. The method of claim 6, wherein the removing step includes
subjecting the resulting complex of carbon-coated magnetic beads
and nucleic acid to at least one of a magnetic field, a centrifugal
force, precipitation, competitive binding, or mixtures thereof.
14. The method of claim 6, wherein the concentration of alcohols is
between 10% and 95%.
15. The method of claim 6, wherein the carbon coated material
comprises a substrate and an outer carbon coating, the carbon
coating having a thickness between 1 angstrom and 50 nm.
16. The method of claim 6, further comprising: after removing the
carbon coated magnetic beads from the supernatant, releasing
single-stranded nucleic acid from attachment to the carbon coated
material, wherein the releasing step occurs with addition of water
or suitable buffer solution into contact with the removed carbon
coated material.
17. The method of claim 16 wherein the removal step includes at
least one of the following: centrifugation, precipitation,
competitive binding, subjecting the composition to a magnetic
field.
18. The method of claim 6, wherein the carbon-coated material is
configured as individual beads, each bead having an interior core
and an outer surface, the beads composed of one of the following: a
magnetic metal containing at 30% by weight of a metal selected from
the group consisting of Ni, Fe, Co, or mixtures thereof, silica or
polymeric substrates, and wherein the carbon coating is present as
a carbon layer on at least a portion of the outer surface of the
bead, the carbon layer comprising at least one of graphene,
pyrolytic carbon or a mixture of graphene and pyrolytic carbon, and
wherein the multivalent cations are alkali earth metals or alkali
earth metal salts or mixtures thereof.
19. The method of claim 6, wherein the complementary nucleic acid
probe is a chain of nucleic acids selected such that the chain of
nucleic acids contains complimentary bases with corresponding
positions to the single stranded nucleic acid that is to be
isolated or purified.
20. The method of claim 6, wherein the complementary nucleic acid
probe may be DNA or a synthetic nucleic acid and wherein the method
further comprises the step of quantifying the sequence for study by
performing PCR on the supernatant which remains after removal of
carbon coated magnetic material and associated single stranded
nucleic acids.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 15/434,470, filed Feb. 16, 2017, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
62/295,985, each of which is incorporated herein in its entirety by
reference.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to nucleic acid purification and
isolation, magnetic bead biological separations, and carbon and its
allotropes
BACKGROUND
[0003] The need for effective techniques for isolating single
stranded nucleic acid components from associated biological
materials persists in a variety of research and development
endeavors. Heretofore, nucleic acid separation techniques can
include various chelating agents, solid support columns using
fibrous or silica matrices to bind nucleic acids and/or magnetic
beads to which nucleic acid can bind. The isolation methods
presently in use do not provide effective yield in all instances.
Thus, it would be desirable to provide a method and material that
can provide more effective and efficient nucleic acid
isolation.
SUMMARY
[0004] An improved method for separating single stranded nucleic
acids from a sample that includes the steps of: providing a mixture
containing nucleic acids; and providing carbon coated material;
providing a chaotropic salt such as guanidinium thiocyanate;
providing alcohols such as ethanol or organic solvents; and
creating a complex of the carbon coated material and nucleic acids.
In certain embodiments, the carbon-coated material employed can be
in the form of magnetic beads, silica gel or various polymeric
materials. The method disclosed herein can also include the steps
of removing the carbon coated material complexed with nucleic acids
from the resulting mixture by means of magnetic separation or
centrifugation. The method may further include the steps of washing
the carbon-coated material and nucleic acids complex with alcohol
based solutions. The method may also include the step of reversing
the complexation of the nucleic acids with the carbon-coated
material by the addition of water or aqueous liquids.
[0005] In certain embodiments, carbon coated magnetic beads may be
employed that can have a magnetic core coated surrounded by a
suitable carbon material. In other embodiments, the carbon coated
material may have a non-magnetic core such as silica coated with a
suitable carbon material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The various features, advantages and other uses of the
present apparatus will become more apparent by referring to the
following detailed drawings.
[0007] FIG. 1 is a schematic drawing for how single stranded
nucleic acids are isolated.
[0008] FIG. 2 is a flow chart of an embodiment of the process
disclosed herein and depicted in FIG. 1.
[0009] FIG. 3 is a schematic drawing for how a single stranded
nucleic acid in a sample may be isolated and may also be
quantified.
[0010] FIG. 4 is a flow chart for the process of how a single
stranded nucleic acid in a sample may be isolated and may also be
quantified.
[0011] FIG. 5 is a transmission electron micrographs of a carbon
coated magnetic beads which may be used in this process.
[0012] FIG. 6 is a flow chart of a process for separating and
producing a single stranded nucleic acid.
[0013] FIG. 7 is a gel electrophoresis image showing nucleic acids
extracted from MCF7 immortalized breast cancer cells using 125
.mu.l of: 2M, 3M, 4M, 5M, and 6M guanidinium thiocyanate with 500
.mu.l of 70% ethanol and 30% pH 8 Tris.
[0014] FIG. 8 is a gel electrophoresis image showing nucleic acids
extracted from MCF7 immortalized breast cancer cells using 125
.mu.l of 6M guanidinium thiocyanate without ethanol added and with
500 .mu.l of 70% ethanol and 30% pH 8 Tris.
[0015] FIG. 9 is a gel electrophoresis image comparing nucleic
acids extracted from MCF7 immortalized breast cancer cells using a
buffer composed of 125 .mu.l of 2M guanidinium thiocyanate and 500
.mu.l of 70% ethanol and carbon coated magnetic beads compared with
a nucleic acid isolation kit sold by Fisher Scientific.
[0016] FIG. 10 is a flow chart for the process of how sequence
specific analysis may be performed using chaotropic salts,
alcohols, and carbon coated materials.
DETAILED DESCRIPTION
[0017] An improved method of separating single stranded nucleic
acids from a mixture is disclosed herein. The method includes the
steps of providing a sample containing single stranded nucleic
acids and contacting the sample containing single-stranded nucleic
acid with a carbon-coated material, at least one chaotropic salt,
and at least one alcohol to effect separation.
[0018] An embodiment of the separation process as disclosed herein
is illustrated in FIG. 1 and described as a flow chart in FIG.
2.
[0019] As depicted FIG. 1, the process 100 begins with a collected
sample 12 containing nucleic acids and a suitable solvent, such as
would be found in in various microorganisms, plant and animal
specimens such as mammalian tissue, a non-limiting example of which
is human liver tissue. The collected sample 12 can be maintained in
a suitable vessel such as that depicted in FIG. 1 at reference
numeral 15. The process as disclosed herein can take placed in a
suitable reaction vessel or container or semi-permeable matrix. In
the embodiment depicted in FIG. 1, the reaction vessel is an
elongated tube.
[0020] To this sample 12 is added a chaotropic material 14 such as
a chaotropic salt solution and carbon-coated materials 16. This
creates a mixture of nucleic acids, chaotropic materials 14, and
carbon-coated materials 16 as at reference numeral 17. Without
being bound to any theory, it is believed that contact between the
nucleic acids present in the sample and the chaotropic material
results in separation of single stranded nucleic acid material 18a
and double stranded nucleic acid material 18b into the solution
with single and double stranded nucleic acid material collectively
referend to as reference numeral 18.
[0021] To this resulting mixture is added an alcohol as at
reference numeral 19. The alcohol of choice can be a short-chain
alcohol such as ethanol or the like. Once the nucleic acid material
18, chaotropic material 14, carbon-coated material 16, and solvent
are present together, the single-stranded nucleic acids 18a present
in the sample complex with the carbon-coated material 16 and create
a nucleic acids-carbon complex 16A as at reference numeral 19.
[0022] While the embodiment depicted in FIG. 1, depicts a process
in which the various material components are introduced in a
specific sequential order, it is considered within the purview of
this disclosure that the four materials may be provided in any
order such that the four materials are present together before the
separation step such as that denoted at reference numeral 21. Where
desired or required, the four components can be admixed to ensure
contact between the various components.
[0023] Once all four material components are in admixture together,
the admixed materials can remain in contact with one another for a
suitable period sufficient for the nucleic acids present in the
initial sample to complex with the carbon coated material. Where
desired or required, the contact interval can be between 1 second
and 5 minutes. In certain embodiments, the complexing period can be
between 10 seconds and 5 minutes.
[0024] The carbon-coated material can be configured in any suitable
size and shape that would facilitate complexing with the desired
nucleic acids. Non-limiting examples of such configurations include
beads in which each individual bead has an interior core and an
outer surface, the beads composed of one of the following: a
magnetic metal containing at between 20 and 40% by weight of a
metal selected from the group consisting of Ni, Fe, Co, or mixtures
thereof, silica or polymeric substrates, and wherein the carbon
coating is present as a carbon layer on at least a portion of the
outer surface of the bead, the carbon layer comprising at least one
of graphene, pyrolytic carbon or a mixture of graphene and
pyrolytic carbon, and wherein the multivalent cations are alkali
earth metals or alkali earth metal salts or mixtures thereof. Thus,
in certain embodiments, it is contemplated that magnetic particles
can be used as the carbon-coated material 16. In situations where
carbon coated materials have a magnetic substrate, it is
contemplated that exposure to a suitable magnetic field such as
that produced by magnet 20 will produce separation between the
nucleic acid carbon material complexes 16A and the liquid
components present resulting in the development of a supernatant
solution 26 and collected nucleic acids complexed with carbon
material 16A. This may be accomplished by placing a magnet 20 near
a specific location on the exterior of the reaction vessel or
container as at reference numeral 22 to collect the carbon coated
material and facilitate the process of separation as at reference
numeral 28 and illustrated in FIG. 1. This results in a supernatant
solution 26 that comprises double stranded nucleic acid, as well as
other materials that may be present in the initial sample as
depicted in reference numeral 23A. The complexes 16A can be
transferred to a separate vessel for additional processing as at
reference numeral 23B.
[0025] Where desired or required, the complexed carbon material 16A
can be washed one or more times to ensure that all the supernatant
liquid containing the cellular contaminants is removed. The washing
step may be performed by contacting the separated material with one
or more portions of a solution containing one or more of alcohols.
The washing step may be performed multiple times to ensure that all
the cellular contaminants present in the original supernatant have
been removed. After several washes with a material such as an
ethanol solution, the wash step can be completed by removing all of
the wash solution. Air drying or other drying means may be employed
to speed removal of the alcohol solution from the complex of the
carbon coated material and nucleic acids. Pure water or water with
less than 1M salt can then be added at reference numeral 30 in
order to facilitate removal of the nucleic acids from complex with
the carbon-containing material. In certain embodiments, it is
contemplated that 50 .mu.l or 100 .mu.l of pure water can be
employed in situations in which around 1 to 5 million cells are
used as the source sample, yielding a solution with about 10 to 100
.mu.g of single stranded nucleic acid (RNA) dissolved in the pure
water solution.
[0026] An embodiment of the separation process is illustrated in
the flow chart in FIG. 2 in which nucleic acids are separated from
a mixture using a chaotropic material such as a chaotropic salt
solution, a polar solvent such as ethanol, and a carbon coated
material.
[0027] As depicted in FIG. 2 at reference numeral 200, a sample
containing nucleic acids is contacted with a chaotropic material
such as a chaotropic salt to form an aqueous solution as at
reference numeral 210. Carbon-coated materials are introduced to
the aqueous solution at reference numeral 212. Alcohols are
introduced to the aqueous solution at reference numeral 214. It is
also considered to be with in the purview of this disclosure that
the four materials may be provided in any order such that the four
materials are present together prior to any separation operations
on the resulting solution. The process as disclosed herein can
occur in a suitable reaction vessel or container.
[0028] Once all four material components are in mixture together,
the materials can remain in contact with one another for a suitable
period sufficient for the nucleic acids present in the initial
sample to complex with the carbon coated material such as carbon
beads. Where desired or required, the contact interval can be
between 1 s and 5 min.
[0029] Once formed, the complexes of nucleic acids and
carbon-containing material are separated from the supernatant
liquid as at reference numeral 616. The resulting supernatant
liquid can contain the initial liquid components in addition to
extraneous cellular component material.
[0030] In certain embodiments, it is contemplated that magnetic
beads coated with carbon can be used as the carbon-coated material.
Separation can be accomplished by one or more mechanisms including,
but not limited to, decanting, filtration and various mechanical
separation methods. In situations where carbon coated beads had a
magnetic substrate, it is contemplated that the separation step
utilizing magnetic beads can be accomplished by placing a magnet
near the exterior of reaction vessel or container to collect the
carbon coated material and facilitate removal of the remaining
solution. If the substrate onto which the carbon is coated is not
magnetic, physical removal, centrifugation, or other methods may be
used to separate the carbon material.
[0031] Where desired or required, the complexed carbon material can
be washed one or more times to ensure that all the supernatant
liquid containing the cellular contaminants is removed as at
reference numeral 218. The washing step may be performed by
contacting the separated material with one or more portions of a
solution containing one or more of alcohols enumerated previously.
The washing step may be performed multiple times to ensure that all
the cellular contaminants present in the original supernatant have
been removed. After several washes with a material such as an
ethanol solution, the wash step can be completed by removing all of
the wash solution. Air drying or other drying means may be employed
to speed removal of the alcohol solution from the complex of the
carbon coated material and nucleic acids. Purified water or water
with less than 1M salt can then be added in order to facilitate
removal of the nucleic acids from complex with the
carbon-containing material at reference numeral 220. In certain
embodiments, it is contemplated that, 50 .mu.l or 100 .mu.l of this
water can be employed to release the single stranded nucleic
acids.
[0032] The method as disclosed herein can also be employed to
isolate and quantify a single stranded nucleic acid material with
specific sequencing. A non-limiting exemplary method is illustrated
in FIG. 3. A biological material to be analyzed is collected as at
reference numeral 312, which may be the result of process 100. The
sample in question can contain target single stranded nucleic acids
314 as well as double stranded nucleic acids 315 some of which are
nucleic acids that contain a desired sequence for quantification
together in a suitable solution 319.
[0033] Specific probes 316 can be added to the sample as
illustrated at reference numeral 318 and allowed to react with any
complementary nucleic acid material that is present in the sample
314 to form a duplex comprising the probe and the target. At step
317, a chaotropic salt, a suitable carbon-coated material 320, and
an organic solvent are added to the solution if not already
present. Once all four material components are in admixture
together, the materials can remain in contact with one another for
a suitable period sufficient for the single stranded nucleic acids
314 present in the initial sample to complex with the carbon coated
material 320. Where desired or required, the contact interval can
be between 1 second and 10 minutes, with intervals between 10
seconds and 5 minutes being employed in certain embodiment. The
duplexes which contain both a probe 316 and a double stranded
nucleic acid 315 target remain in solution 319.
[0034] In situations where carbon coated beads 320 have a magnetic
substrate, it is contemplated that separation of the supernatant
containing the duplex double stranded nucleic acids 315 and single
stranded nucleic acids 314 complexed with carbon material 320 may
be accomplished by placing a magnet 322 near the exterior of
reaction vessel or container 324 to collect the carbon coated
material as a pellet and facilitate the process of separation.
[0035] It is contemplated that the complementary nucleic acid
material may be modified with a protein which may be used to
isolate strands with specific sequencing. Once separated the duplex
double stranded nucleic acids 315 modified with complementary
proteins to those on the complementary nucleic acids may be added
to the mixture to concentrate or isolate the target sequences. By
way of non-limiting example, biotin may be attached to the
complementary nucleic acids and streptavidin may be attached to a
substrate or magnetic beads.
[0036] Separation of the supernatant liquid from contact with the
carbon coated beads 320 yields a solution of the duplex material
316 that is amenable to various analytic techniques including, but
not limited to florescent tag detection 336, simple PCR 338,
magnetic sensor detection 340 and the like.
[0037] It is further conceived that the method as disclosed herein
can be employed to perform sequence specific analysis and one
non-limiting example of such a procedure is further illustrated in
the flow chart diagram of FIG. 10 as at reference numeral 1000. A
suitable sample of single stranded nucleic acids can be collected
as at reference numeral 1010. It is contemplated that the single
stranded nucleic acid can be derived from any suitable source or by
any suitable method; one suitable process for separating and
producing single stranded nucleic acid process disclosed herein in
FIG. 6.
[0038] In the process as disclosed, at least one complimentary
nucleic acid probe can be added to the collected material as at
reference numeral 1012. It is contemplated that the nucleic acid
probe can be a suitable diagnostic material. Non-limiting examples
of such material include DNA, RNA, as well as synthetic nucleic
acids such as peptide nucleic acids. If desired or required, the
added material can be allowed to react with the single strand
nucleic acid for a suitable interval.
[0039] Once any optional reaction interval has elapsed, alcohols,
chaotropic materials such as chaotropic salts, and a carbon coated
material such as those described herein can be added to the sample
to initiate binding of the single stranded nucleic acids to the
carbon-coated material as at reference numeral 1014. In certain
embodiments, it is contemplated that this step will resemble the
process step outlined in with regard to FIG. 6. It is also
considered with in the purview of this disclosure that where little
or no protein or cellular contamination is present, the addition of
chaotropic materials such as chaotropic salts may be reduced or
eliminated entirely, It is contemplated that the complex of the
carbon coated material and single stranded nucleic acids that is
produced may also contains complimentary probes which were added at
reference numeral 1012 but do not find a target with which to
hybridize.
[0040] Where desired or required, the resulting material can be
permitted to form suitable complexes of the single stranded nucleic
acid to which complementary probes have bonded and
carbon-containing material as at reference numeral 1016. The
formation interval can be any suitable period between 10 seconds
and 24 hours in certain embodiments.
[0041] The resulting complex of nucleic acid and carbon coated
material can then be separated as at reference number 1018.
Separation can be accomplished by one or more mechanisms including,
but not limited to, decanting, filtration and various mechanical
separation methods. In situations where carbon coated beads have a
magnetic substrate, it is contemplated that the separation step
utilizing magnetic beads can be accomplished by placing a magnet
near the exterior of reaction vessel or container or semi-permeable
matrix to collect the carbon coated material and facilitate removal
of the remaining solution.
[0042] The resulting supernatant can be removed for further
analysis as desired or required as at reference numeral 1020
contains double stranded hybrids of the target and complimentary
nucleic acids, which may be quantified as at reference numeral
1022. Non-limiting examples of suitable quantification methods
include by PCR amplification reference numeral 1024, or by labeling
the synthetic nucleic acid with a detectable fluorescent or
magnetic tag as at reference numeral 1026. It is within the purview
of this disclosure that the synthetic nucleic acids may be labeled
before being added to the sample as at reference numeral 1012. It
is also contemplated that the probes can be labeled after the
supernatant is separated from the nucleic acid-carbon complex as at
reference numeral 1026.
[0043] The nucleic acids that are complexed with the carbon
material may be washed and eluted from the carbon coated material
as at reference numeral 1028. Where desired or required, the
complexed carbon material can be washed one or more times to ensure
that all the supernatant liquid containing the cellular
contaminants is removed as at reference numeral. The washing step
may be performed by contacting the separated material with one or
more portions of a solution containing one or more of alcohols
enumerated previously. The washing step may be performed multiple
times to ensure that all the cellular contaminants present in the
original supernatant have been removed. After several washes with a
material such as an ethanol solution, the wash step can be
completed is completed by removing all of the wash solution. Air
drying or other drying means may be employed to speed removal of
the alcohol solution from the complex of the carbon coated material
and nucleic acids. Pure water or water with less than 1M salt can
then be added in order to facilitate removal of the nucleic acids
from complex with the carbon-containing material. In certain
embodiments, it is contemplated that, in certain embodiments, 50
.mu.l or 100 .mu.l of pure water can be employed in situations in
which around 1 million cells are used as the source sample. The
resulting sample of single stranded nucleic acids eluted as at
reference numeral 1028 is suitable for use as a sample at 1010 and
the process may be repeated.
[0044] The carbon-coated material can be composed of a suitable
substrate having an outer surface with a suitable carbon material
covering at least a portion of the outer surface of the substrate.
The resulting carbon-nucleic acid complex may be separated from the
remaining materials by various separation techniques. The
techniques employed can be chosen based on factors such as the
nature of the of the substrate material employed.
[0045] As used herein the term "nucleic acid probe" or "antibody
probe" refers to a chain of nucleic acids selected so that it
contains bases that are complementary or have affinity with
corresponding positions in at least one section of the length of
the nucleic acid chain under study. The complimentary nucleic acid
probe may be longer than the target and have additional bases with
an arbitrary sequence. Anti-nucleic acid antibody may have
additional chemical modifications such as biotin tag. The
complimentary probe may be synthetic nucleic acids such as peptide
nucleic acid (PNA) or Morpholino and locked nucleic acid (LNA) or
glycol nucleic acid (GNA) or threose nucleic acid (TNA).
[0046] Also disclosed is a composition that comprises a carbon
coated substrate and single-stranded nucleic acid complexed
therewith.
[0047] The carbon coated material as disclosed herein is one that
will complex with single stranded nucleic acid material present in
a sample. The single stranded nucleic acid material can include
various naturally derived and/or synthetically prepared nucleic
acids such as RNA, including any added complimentary nucleic acid
probes if the target for these probes is not present in the sample
under study. Particles with the single-stranded nucleic acids bound
to the magnetic beads may be removed by application of a magnetic
field, centrifugation, precipitation and the like.
[0048] In certain embodiments, the carbon coated material will
include a substrate that is configured as a particulate material
such as beads, or other spheroid bodies. When the substrate is
configured as beads or other particulate geometries that are
nanoparticulate in size; having a diameter between 20 and 200 nm or
between 10 nm and 100,000 nm in certain embodiments.
[0049] In certain embodiments, the carbon material can include one
of more of the following: pyrolytic carbon, graphite, graphene. In
certain embodiments, the suitable material is a material with a
metal core that is surrounded by a carbon coating in which the
carbon creates a generally seamless conformal coating. A suitable
carbon coated material that can be used in the process as disclosed
herein can be one such as the bead configuration presented in FIG.
5. Referring to FIG. 5, there is depicted a transmission electron
micrograph in which a metal core composed of Fe.sub.65Co.sub.35 is
surrounded by a carbon coating. This carbon coating has multiple
layers, in which each layer is similar to graphene and there may be
coupling between the layers. The presented carbon coated material
structure is particularly well suited to the described process
because the carbon creates a conformal coating without a seam
around the metal core. Exposure of the metal core to water has
undesirable effects due to oxidation of the metal. Oxidation of the
metal may lead to undesirable properties such as binding DNA and
reduction of magnetic moment. Suitable magnetic material that can
be employed as the substrate is available commercially from
Metastable Materials, Inc., doing business as Life Magnetics.
[0050] Carbon coated magnetic materials may be prepared by the
laser manufacturing process disclosed in WO2015095398A1 which is
disclosed herein by reference. It is contemplated that carbon
coated materials can be prepared by positioning a laser is incident
on target in a solvent such that the pulsed laser produces laser
pulses having a pulse duration greater than 1 ps at a wavelength
between 200 nm and 1500 nm at a pulse repetition rate of at least
10 Hz and a fluence greater than 10 J/cm.sup.2. The laser beam may
be scanned across the surface of the target; i.e., the desired core
material (Silica, magnetic metal and the like). Non-limiting
examples of satiable magnetic metal material includes metals and
alloys containing iron, nickel, cobalt, or the like as well as
mixtures and alloys of the aforementioned. The solvent employed can
be an organic solvent which contains carbon and in certain
embodiments will be composed of xylenes, toluene, benzene, or
mixtures thereof. Where desired or required, the organic solvent
may further include any hydrocarbon containing benzene rings. In
certain embodiments, the magnetic beads employed will have a
saturation magnetization between 10 and 80 A m.sup.2/kg (emu/g); a
coercive force between 0.8 and 15.9 KA/m, and a sedimentation rate
between 2% and 15% in 30 minutes.
[0051] It is envisioned that the chaotropic material imparted may
be may be provided independently in certain embodiments or may be
introduced in combination with various other components depending
on the nature of the sample containing the single-stranded nucleic
acids. In certain embodiments, the chaotropic material composition
may further contain components on or more additional components.
These include, but are not limited to one or more of the following:
ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT),
2-mercaptoethanol (BME), Lithium Chloride (LiCl), Urea,
N-lauroylsarcosine, IGEPAL 630, NP-40, SDS (sodium dodecyl
sulfate), TCEP (tris(2-carboxyethyl) phosphine), Tributylphosphine
(in N-methyl-2-pyrrolidinone), glycogen or the like. Where desired
or required this compound can be present t a concentration of
between 0.001% and 1% by volume. Without being bound to any theory,
it is believed that the compound functions to inhibit protein
activity. The chaotropic composition can also include a suitable
buffer compound present at a concentration suitable to impart
buffering characteristics to the composition.
[0052] Non-limiting examples of suitable buffer compounds include
tris(hydroxymethyl)aminomethane (Tris), citric acid, sodium
citrate, (3-(N-morpholino) propanesulfonic acid) (MOPS),
2-(N-morpholino)ethanesufonic acid (MES) as well as mixtures of
these compounds. In certain embodiments, the buffer compound(s) may
be present at a concentration between 50 mM and 500 mM to buffer
the pH of the solution. The composition can also include detergents
such as Triton X-100, or sodium dodecyl sulfate (SDS) or Tween-20
or Tween-40 or N-lauroylsarcosine or IGEPAL 630 or NP-40 where
desired or required.
[0053] In one non-limiting example of a suitable chaotropic
material that can be employed one or more embodiments of the method
as disclosed herein may be a solution of chaotropic salts which
also contains between 1 to 100 mM EDTA, 1 to 100 mM of a buffer
composed of citric acid, sodium citrate, and/or Tris, 0.01 to 5
volume % Triton X-100, and 0.1 to 5 mM DTT. The pH of the solution
may be between 3 to 10. In one exemplary case, the chaotropic
material is a chaotropic salt solution of 4M guanidinium
thiocyanate, 20 mM citric acid buffer, and the pH is adjusted to
5.
[0054] It is further envisioned that, in certain embodiments,
additional wash steps may be necessary as would be the case for
biological substances having a high protein content such as blood.
The process as disclosed herein may include one or more additional
washing steps in which the wash solution employed may contain
suitable materials employed individually or in combination such as
reducing agents, ethanol, and chaotropic salts such as guanidinium
thiocyanate, to aid in the solubilization and removal of the
protein. For example, an additional wash step may be included which
uses a wash solution composed of 0.6M to 2M chaotropic salts such
as guanidinium thiocyanate, and 30% to 60% alcohol by volume. If
this additional wash step is included, it is contemplated that a
final washing step with a solution of ethanol without chaotropic
salts will still occur.
[0055] It is envisioned that the chaotropic material may be
chaotropic salts solutions other than guanidinium thiocyanate, such
as guanidinium chloride, guanidinium bromide, guanidinium fluoride,
guanidine acetate, guanidine sulfate, guanidine nitrate, guanidine
carbonate, or may be other chaotropic compounds such as guanidine
hydrochloride, urea, thiourea, lithium acetate, or lithium
perchlorate and these compounds may have different efficacy in the
process, it is expected different concentration ranges may be
required depending on the efficacy of the salt. The chaotropic
material guanidinium thiocyanate is used as a salt solution such
that the concentration of the guanidinium thiocyanate salt is 0.1
to 1M when the four materials are present together at reference
numeral 21. These other compounds may be expected to have similar
effects but at different concentrations, meaning that the
concentration of chaotropic salts may be 0.01M to 4M at reference
numeral 21 depending on the identity of the chaotropic salt, which
other components are present, and the pH of the solution.
[0056] In the process as disclosed, a short chain alcohol such as
ethanol can be added to the composition in an amount sufficient to
facilitate complexation of single stranded nucleic acids with
carbon materials such as carbon-coated magnetic beads. In certain
embodiments, the short-chain alcohol can be added such that the
final concentration of the chaotropic material such as guanidinium
thiocyanate is between 0.1M and 1M and the short-chain alcohol
composes between 40 vol % and 80 vol % of the resulting solution
employed at reference numeral 21 or reference numeral 615 in the
flow chart. It has been found that under these conditions, single
stranded nucleic acids complex with the carbon coated magnetic
beads selectively rather than complexing with double stranded
nucleic acids. It is also conceived that alcohols other than
ethanol may be used, and depending on the alcohol, the ideal
percentage of the total volume may be different. Alcohols such as
propanol, methanol, butanol, phenol, and the like may replace
ethanol. It is further conceived that a range of 20 vol % to 90 vol
% alcohols may be used.
[0057] In certain embodiments, it is contemplated that the mixture
which contains the sample containing nucleic acids, the
carbon-coated material, the chaotropic material, and the alcohols,
has carbon-coated beads at a concentration between 1.times.10.sup.5
nanoparticulate beads per ml and 1.times.10.sup.20 nanoparticulate
beads per ml.
[0058] It is contemplated that the process and material as
disclosed herein can be employed is to store RNA long term. RNA is
known to be fragile and to degrade upon exposure to various enzymes
and proteins extant in the environment. One non-limiting example of
such material is ribonuclease (RNase). It has been found that
single-strand nucleic acids such as RNA bind to the surface of the
carbon-coated magnetic beads as disclosed herein in a manner that
renders them resistant to interaction with enzymes such as RNase.
Thus, the bio-complex of a single-strand nucleic acid such as RNA
and the carbon coated magnetic beads as disclosed herein can
provide a stable long-term storage vector for fragile single-strand
nucleic acids.
[0059] It is contemplated that the stable stored single-strand
nucleic acid material can comprise 10-5000 ng of nucleic acids per
microliter and 50-500,000 ng of carbon coated material per
microliter, wherein the carbon coated material is preferably
magnetic or silica nanoparticulate for this embodiment. The
suspension for storage contains between 20% and 80% alcohols. The
single-strand nucleic acid, while on the surface of the
carbon-based material such as graphene, cannot be cleaved by
enzymes such as RNase and will not spontaneously degrade.
[0060] Aspects of the disclosed implementations also include a
method for fabricating the carbon coated magnetic beads that can be
employed in the method and process disclosed herein. One such
fabrication method is discussed in US Published Application Number
2015-0170807 to Hagedorn, K. and Malizia, H, the specification of
which is incorporated by reference herein. The referenced patent
discloses methods for preparing magnetic beads. The magnetic beads
prepared in carbon containing organic solvents such as xylenes or
toluene may create a carbon coating on the surface of the magnetic
beads suitable for use in the disclosed methods.
Example I
[0061] To further illustrate the nucleic acid separation method
described herein, the process was used to separate RNA from samples
of 1 million MCF7 breast cancer cells with various process
parameters and the RNA separated was quantified with gel
electrophoresis in FIG. 7. Samples of 1 million MCF7 breast cancer
cells were introduced into respective sterile glass containers at
standard temperature and pressure. 125 .mu.l of guanidinium
thiocyanate solutions were added to each respective sample where
the concentrations of guanidinium thiocyanate were 2M at reference
702, 3M at reference 703, 4M at reference 704, 5M at reference 705,
and 6M at reference 706. The guanidinium thiocyanate solution also
contained 1% Triton X-100, 10 mM EDTA, and 20 mM Tris, and the pH
was adjusted to 6. Graphene coated magnetic beads as shown in FIG.
5 are then added to the respective samples. A solution of 70%
ethanol is added to each sample to a constant 56% of the solution
by volume, making the concentration of the guanidinium thiocyanate
0.4M at reference 702, 0.6M at reference 703, 0.8M at reference
704, 1M at reference 705, and 1.2M at reference 706 respectively.
The graphene coated magnetic beads are 20 nm to 100,000 nm in size
and have a carbon layer 1 angstrom to 50 nm thick. The carbon
layers can be visualized in FIG. 5 as rings around the magnetic
metal core. The magnetic metal core may be any material where Fe,
Co, or Ni individually or combined compose more than 30% of
material by atomic percent. The supernatant was separated from the
carbon-coated magnetic beads complexed with nucleic acids and
washed three times with a solution of 70% ethanol. 100 .mu.l of
purified water was then added and the single stranded nucleic acids
were released into this water.
[0062] Portions of 10 .mu.l of each respective sample was removed
and subjected to electrophoresis. An agarose gel was cast and the
samples were mixed with an ethenium bromide loading dye. The gel
was run at 90V for 1 h.
[0063] This non-limiting example exemplifies how the process is
industrially applicable by demonstrating RNA isolation from cells
without DNA contamination. This non-limiting example also
exemplifies the unexpected result that single stranded nucleic
acids are extracted without contamination from double stranded
nucleic acids when the concentration of chaotropic salts is between
0.1M and 1M.
Example II
[0064] To further illustrate the nucleic acid separation method
described herein, the process was used to separate RNA from samples
of 1 million MCF7 breast cancer cells with various process
parameters and the RNA separated was quantified with gel
electrophoresis in FIG. 8. Samples of 1 million MCF7 breast cancer
cells were introduced into respective sterile glass containers at
standard temperature and pressure. 125 .mu.l of 2M guanidinium
thiocyanate solutions were added to each respective sample. A
solution of 70% ethanol is added such that the concentration of
ethanol is 0% at reference numeral 801, 31% by volume at reference
numeral 802, 43% by volume at reference numeral 803, 49% by volume
at reference numeral 804, and 56% by volume at reference numeral
805. It is observed that when 6M guanidinium thiocyanate solution
is provided with carbon coated magnetic beads but without ethanol
as previously described, single stranded nucleic acids are
separated from the mixture but with poorer yield. The addition of
ethanol has the unexpected result of enhancing recovery yield.
[0065] Portions of 10 .mu.l of each respective sample was removed
and subjected to electrophoresis. An agarose gel was cast and the
samples were mixed with an ethidium bromide loading dye. The gel
was run at 90V for 1 h.
[0066] This non-limiting example exemplifies how the process is
industrially applicable by demonstrating RNA isolation from cells
without DNA contamination. This non-limiting example also
exemplifies the unexpected result that single stranded nucleic
acids are extracted without contamination from double stranded
nucleic acids when the concentration of alcohols is between 20% and
80% of the solution by volume.
Example III
[0067] FIG. 9 compares RNA extraction from 1 million MCF7 breast
cancer cells using process outlined in Example I in the first lane
901 to the Thermo Fisher MirVana Total RNA Extraction Kit using the
procedure described in that kit in the second lane at reference
numeral 902. The Thermo Fisher MirVana Total Extraction Kit is a
state-of-the-art kit from one of the largest life science suppliers
in the world. The most noticeable difference is the absence of DNA
contamination. DNA contamination is a problem for RNA analysis
because PCR amplification cannot be performed with DNA
contamination. Heretofore, the DNA contamination had to be removed
with DNase, an enzyme which destroys DNA. This is both expensive
and slow. The second noticeable difference is improved yield of
small RNA, which is important for applications which analyze short
RNA such as Micro RNA for diagnostic and research applications.
[0068] While the invention has been described in connection with
what is presently considered to be the most practical and preferred
embodiment, it is to be understood that the invention is not to be
limited to the disclosed embodiments but, on the contrary, is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims, which
scope is to be accorded the broadest interpretation so as to
encompass all such modifications and equivalent structures as is
permitted under the law.
* * * * *