NZ740585B2 - Portable nucleic acid extraction apparatus and method of using the same - Google Patents
Portable nucleic acid extraction apparatus and method of using the same Download PDFInfo
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- NZ740585B2 NZ740585B2 NZ740585A NZ74058516A NZ740585B2 NZ 740585 B2 NZ740585 B2 NZ 740585B2 NZ 740585 A NZ740585 A NZ 740585A NZ 74058516 A NZ74058516 A NZ 74058516A NZ 740585 B2 NZ740585 B2 NZ 740585B2
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- New Zealand
- Prior art keywords
- nucleic acid
- tip
- acid extraction
- portable
- aqueous
- Prior art date
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 222
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 220
- 238000000605 extraction Methods 0.000 title claims abstract description 98
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 69
- 239000000463 material Substances 0.000 claims abstract description 50
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- 239000004033 plastic Substances 0.000 claims abstract description 20
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- 239000000203 mixture Substances 0.000 claims description 41
- 239000002253 acid Substances 0.000 claims description 22
- 239000011324 bead Substances 0.000 claims description 21
- 239000000835 fiber Substances 0.000 claims description 19
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- 108090000623 proteins and genes Proteins 0.000 claims description 11
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- 239000002105 nanoparticle Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 54
- 239000012472 biological sample Substances 0.000 description 36
- 239000000523 sample Substances 0.000 description 36
- 239000012139 lysis buffer Substances 0.000 description 32
- 239000012062 aqueous buffer Substances 0.000 description 27
- 239000012148 binding buffer Substances 0.000 description 27
- 239000011534 wash buffer Substances 0.000 description 25
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- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 7
- 229910052708 sodium Inorganic materials 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- KRHYYFGTRYWZRS-UHFFFAOYSA-N HF Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 6
- FPKOPBFLPLFWAD-UHFFFAOYSA-N Trinitrotoluene Chemical compound CC1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1[N+]([O-])=O FPKOPBFLPLFWAD-UHFFFAOYSA-N 0.000 description 6
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- 239000005708 Sodium hypochlorite Substances 0.000 description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 4
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- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminum Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L1/00—Enclosures; Chambers
- B01L1/52—Transportable laboratories; Field kits
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
portable nucleic acid extraction apparatus for nucleic acid extraction is disclosed. The apparatus includes a handle, a rod attached to the handle, a tip attached to the rod. The handle, the rod, and the tip are formed as a single piece. The material that forms the handle, the rod, and the tip is selected from at least one of the group consisting of wood, plastic, polystyrene, functionalized polystyrene, glass, and a silica-derived material. The apparatus does not comprise specialized equipment selected from the group consisting of pipettes, centrifuges, and automated equipment. The apparatus also does not comprise a stationary nucleic acid binding medium. selected from at least one of the group consisting of wood, plastic, polystyrene, functionalized polystyrene, glass, and a silica-derived material. The apparatus does not comprise specialized equipment selected from the group consisting of pipettes, centrifuges, and automated equipment. The apparatus also does not comprise a stationary nucleic acid binding medium.
Description
Description
LE NUCLEIC ACID EXTRACTION APPARATUS AND METHOD OF USING
THE SAME
BACKGROUND
[0001] Methods for nucleic acid (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA)) extraction generally include four common elements: cell lysis for e of
nucleic acids, binding of the nucleic acid to a nucleic acid binding medium, washing of
the bound nucleic acid to remove cellular components other than the nucleic acids
from the bound nucleic acid, and n (release) of the bound nucleic acid from the
nucleic acid binding medium. Conventional nucleic acid extraction from a biological
sample is carried out by a variety of different tion techniques, including, organic
extraction, magnetic separation, silica-based extraction, and anion exchange. These
s typically utilize diverse equipment including centrifuges, pipettes, columns
and/or other equipment that requires technical expertise to use effectively. Further,
such conventional methods typically require anywhere from 12 to 24 steps to
complete, which are labor intensive, reducing efficiency.
Typically, the nucleic acid binding medium remains stationary and takes the
form of a solid medium, such as a silica-based and exchange column. The
biological sample is d to the nucleic acid binding medium via pipettes or other
automated s. Next, buffers are applied to the medium either manually or
through an automated system to first bind and then elute nucleic acids. These methods
generally employ centrifuges to separate the nucleic acid components from the
remaining cellular components. The use of pipettes, fuges, and other automated
equipment requires cal ise to operate, and may increases the time
required to extract nucleic acids. Methods of nucleic acid extraction that utilize such
equipment are not conducive to utilization in a field scenario (e.g. on a farm, ranch, or
at the point of care in an underserved area) and/or by an individual with little to no
technical ng.
17707096_1 (GHMatters) P108029.NZ
Another c acid extraction method utilizes magnetic separation to eliminate
the need of centrifuges to separate bound nucleic acid from the other cellular
components. However, the magnetic beads used in such methods are typically added
in free form to the sample, thus requiring the use of a magnet or other automated
machine to maneuver and contain the beads by a skilled operator. Further, this method
is time intensive, again inhibiting its widespread use in a field scenario by an individual
without technical expertise.
As molecular diagnostics are g rity, there have been world-wide
s to eliminate or reduce the limitations of existing nucleic acid extraction
techniques, particularly in field scenarios, as on-site extraction may reduce barriers to
uent analysis. It is therefore desirable to eliminate the need for technical
expertise, the use of specialized equipment, and the nary nucleic acid binding
medium used in nucleic acid extraction to make nucleic acid extraction available in a
field scenario or laboratory setting. r it is desirable to reduce the number of steps
necessary to extract nucleic acids to increase efficiency of methods to nucleic extract
nucleic acids.
SUMMARY
In a first , the present invention relates to a portable nucleic acid
extraction apparatus for nucleic acid extraction, comprising a handle, a rod attached
to the handle, a tip attached to the rod, n the tip is configured to bind nucleic
acids.
In another aspect, the present invention relates to a method for extracting
c acid from a biological sample sing nucleic acid containing cells, the
method sing the following steps (a) through (f): (a) adding lysis buffer to the
biological sample; (b) adding binding buffer to the e obtained in step (a); (c)
dipping the tip of a portable nucleic acid extraction apparatus according to the present
invention into the mixture obtained in step (b); (d) incubating the tip of the portable
nucleic acid extraction tus in the mixture obtained in step (b); (e) washing the
tip of the portable nucleic acid extraction apparatus with a washing buffer; and (f)
eluting nucleic acid bound to the tip of the portable nucleic acid tion apparatus
with an elution buffer.
17707096_1 (GHMatters) P108029.NZ
In a third aspect, the present invention relates to a portable nucleic acid
extraction kit comprising a portable nucleic acid extraction apparatus according to the
present invention; at least one lysis ; at least one binding buffer, at least one
washing , at least one elution buffer, and at least three kit sample tubes.
BRIEF DESCRIPTION OF THE DRAWINGS
The numerous advantages of the present invention may be better understood
by reference to the accompanying s in which:
Fig. 1.a. depicts a portable nucleic acid extraction apparatus.
Fig. 1.b. depicts a portable c acid extraction apparatus comprising nucleic
acid binding fibers.
Fig. 1.c. depicts a portable nucleic acid extraction apparatus sing c
acid binding beads.
Fig. 1.d. depicts a portable nucleic acid extraction apparatus comprising nucleic
acid binding proteins.
[0013] Fig. 1.e. depicts a portable nucleic acid extraction apparatus made of glass and
having an increased e area.
Fig. 2 illustrates a method for extracting a nucleic acid using the portable nucleic
acid extraction apparatus.
Fig. 3.a. izes the concentration [ng/μΐ] and purity [A260/280] of six
genomic DNA samples obtained with the portable nucleic acid extraction.
Fig. 3.b. illustrates the results of PCR amplifications using genomic DNA
samples obtained with the le nucleic acid extraction apparatus as DNA
template.
Fig. 3.c. illustrates the results of RCA using c DNA samples ted
with the portable nucleic acid extraction apparatus as a template.
17707096_1 (GHMatters) P108029.NZ
Fig. 4 depicts a portable nucleic acid extraction kit.
DETAILED DESCRIPTION
In a first aspect, the present invention relates to a le nucleic acid
extraction apparatus. The portable nucleic acid extraction apparatus comprises a
handle, a rod, and a tip. Typically, the rod is attached to the handle and the tip is
attached to the rod. The tip is configured to bind nucleic acids.
Within the context of the present invention, the term "binding nucleic acids"
means that free nucleic acids which are present in solution can be directly bound to
the tip of the portable nucleic acid extraction apparatus. "Free" nucleic acids means
that the nucleic acids are not t within a cell but have been released from a cell.
Within the context of the present invention, the term "binding nucleic acids" refers to
molecular binding and includes covalent and non-covalent binding of nucleic acids to
the tip of the portable nucleic acid tion apparatus. valent interactions that
may contribute to the binding of nucleic acids to materials such as silica, silica-derived
materials, polystyrene and glass but also to biomolecules such as certain proteins
include electrostatic interactions such as ionic interactions and hydrogen bonding and
Van der Waals forces. Within the t of the present invention, the term "mixture"
means a solution, a sion, or a mixture.
Within the context of the present invention, the term "configured to bind nucleic
acids" means that the tip is either made of material which is able to bind nucleic acids
that are present in solution or ses structures that are able to bind nucleic acids
that are present in solution. These structures may be made of nucleic acid binding
material (nucleic acid binding medium) such as glass, polystyrene, silica or silicaderived
materials, and may additionally be shaped such that the nucleic acid binding
surface of the tip is increased. For instance, the tip of the portable nucleic acid
extraction apparatus may comprise fibers, such as polystyrene fibers and silica ,
or beads such as polystyrene beads, silica beads and glass beads. The tip of the
le nucleic acid extraction apparatus may also se at least one nucleic acid
binding n, such as streptavidin.
17707096_1 (GHMatters) P108029.NZ
In particular embodiments, the portable nucleic acid extraction apparatus
es a nucleic acid binding medium attached to the tip, where nucleic acid g
medium is ured to bind nucleic acids.
In particular embodiments, the tip is made of a material which is able to bind
nucleic acids and/or the tip comprises at least one structure which is configured to bind
nucleic acids.
In ular embodiments, wherein the tip is made of a material selected from
at least one of the group consisting of wood, plastic, yrene, functionalized
yrene, glass, and a silica-derived material.
[0025] In particular embodiments, the handle, the rod, and the tip are formed as a
single piece. In particular such embodiments, the handle, the rod, and the tip are made
of a al selected from at least one of the group consisting of wood, plastic,
polystyrene, functionalized polystyrene, glass, and a silica-derived material.
Particularly, the handle, the rod and the tip are made of glass.
[0026] In particular ments, the tip and optionally the handle and/or the rod are
made of glass, and the tip and optionally the handle and/or the rod are sandblasted.
Sandblasting increases the surface area of the tip and optionally the handle and/or the
rod, and thereby increases the surface that may be bound by nucleic acids.
In particular embodiments, the material which is able to bind nucleic acids is
selected from at least one of the group consisting of polystyrene, functionalized
polystyrene, glass, and silica-derived material.
In particular embodiments, the at least one structure which is configured to bind
nucleic acids is selected from the group consisting of at least one fiber, at least one
bead, at least one protein and any combination thereof.
[0029] In particular embodiments, the at least one fiber is selected from a silica fiber,
a polystyrene fiber and a combination thereof.
17707096_1 (GHMatters) P108029.NZ
In particular embodiments, the at least one bead is selected from the group
consisting of a silica micro bead, a silica nano particle, a polystyrene micro bead, a
polystyrene nano particle, a sand-blasted glass bead, and any combination thereof.
In use, the tip of the portable nucleic acid extraction apparatus is inserted into
a first sample tube ning a biological sample comprising free nucleic acids, i.e.
nucleic acids that are not present within a cell. Typically, a biological sample
sing nucleic acid containing cells is first treated with lysis buffer to allow for cell
lysis and nucleic acid release into the sample solution to form a mixture. After lysis
buffer on, the tip of the portable nucleic acid extraction apparatus is inserted into
the mixture. The tip is then incubated in the mixture to allow for binding of the nucleic
acids to the tip. After incubation, the tip with nucleic acids bound to its e is
removed from the first sample tube and inserted into a second sample tube ning
washing buffer to wash the nucleic acids bound to the tip. The tip with washed nucleic
acids bound to its surface is then inserted into a third sample tube containing n
buffer for elution, i.e. to allow for release of the bound nucleic acids from the tip into
the elution buffer. The eluted nucleic acids may be further analyzed.
Thus, in a second aspect, the present invention relates to a method for
extracting nucleic acid from a ical sample comprising nucleic acid ning
cells, the method comprising the following steps: (a) adding lysis buffer to the
biological sample; (b) adding binding buffer to the mixture obtained in step (a); (c)
dipping the tip of a portable nucleic acid tion apparatus according to the present
invention into the mixture ed in step (b); (d) incubating the tip of the portable
nucleic acid extraction apparatus in the mixture obtained in step (b); (e) washing the
tip of the portable nucleic acid extraction apparatus with washing buffer; and (f) g
nucleic acid bound to the tip of the portable nucleic acid extraction apparatus with an
elution .
In particular embodiments, the method further comprises the step of adding
ethanol to the biological sample before or during step (a) and/or to the mixture obtained
in step (a) before or during step (b) and/or to the mixture obtained in step (b) before
or during step (c) or before or during step (d).
17707096_1 (GHMatters) P108029.NZ
In ular embodiments, the lysis buffer is an aqueous buffer comprising trishydrochloride
; the binding buffer is an aqueous buffer comprising sodium hypochlorite
and glycine-hydrochloride and having a pH of from about 2.0 to 4.0, preferably of about
3.0; the washing buffer is an aqueous buffer comprising glycine-hydrochloride and
ethanol; and the elution buffer is an aqueous buffer comprising tris-hydrochloride and
having a pH of from about 7.0 to about 10.0, preferably of about 8.8.
In particular such embodiments, the lysis buffer is an aqueous on of trishydrochloride
, optionally further comprising at least one substance selected from the
group consisting of lysozyme, proteinase K, sodium hydroxide and any combination
thereof; the binding buffer is a mixture of aqueous sodium hypochlorite and aqueous
glycine-hydrochloride having a pH of from about 2.0 to 4.0, preferably of about 3.0; the
washing buffer is a mixture of aqueous e-hydrochloride and aqueous ethanol;
and the elution buffer is aqueous tris- hydrochloride having a pH of from about 7.0 to
about 10.0, preferably of about 8.8.
[0036] In particular embodiments, the lysis buffer is selected from the group consisting
of an aqueous buffer sing from 0.2 to 5 t, preferably about 1 percent (v/v)
triton x-100, an aqueous buffer comprising from 2 to 50 millimolar, ably about 10
olar tris- hloride, an aqueous buffer comprising ethylenediaminetetraacetic
acid and having a pH of from about 5.5 to about 7.5, preferably of about 6.5,
and any combination thereof; the binding buffer is selected from the group consisting
of an aqueous buffer comprising sodium lorite and glycine-hydrochloride and
having a pH of from about 2.0 to about 4.0, preferably of about 3.0, an aqueous buffer
comprising tris-hydrochloride and sodium acetate/acetic acid and having a pH of from
3.6 to 5.6, an aqueous buffer comprising sodium citrate/citric acid and having a pH of
from 3.0 to 6.2, an aqueous buffer comprising sodium perchl orate, an s buffer
comprising sodium nitrate, an aqueous buffer comprising from 4 to 5 molar sodium
chloride, an aqueous buffer comprising from 4 to 5 molar potassium chloride, an
s buffer comprising from 4 to 6 molar guanidine-hydrochloride, an aqueous
buffer comprising from 4 to 6 molar guanidine thiocyanate, an aqueous buffer
comprising from 4 to 6 molar iodide, an aqueous buffer comprising from 4 to 6 molar
urea, and any ation thereof; the washing buffer is selected from the group
consisting of an aqueous buffer comprising from 5 to 7, preferably 6 molar sodium
17707096_1 (GHMatters) P108029.NZ
perchlorate, an aqueous buffer comprising from 5 to 7, preferably 6 molar sodium
nitrate, an aqueous buffer comprising from 4 to 5 molar sodium chloride, an aqueous
buffer comprising 4 to 5 molar potassium chloride, an aqueous buffer comprising 4 to
6 molar guanidine-hydrochloride, an aqueous buffer sing 4 to 6 molar guanidine
thiocyanate, an aqueous buffer comprising from 4 to 6 molar iodide, an aqueous buffer
comprising from 4 to 6 molar urea, an aqueous buffer comprising from 100 to 300
millomolar, preferably about 200 millimolar glycine-hydrochloride, and any
ation thereof; and/or the n buffer is selected from the group consisting of
ultrapure water, an aqueous buffer comprising tris-hydrochloride and having a pH from
7.4 to 9, an aqueous buffer comprising from 0.05 to 0.2 millimolar, preferably about
0.1 millimolar ethylenediaminetetra- acetic acid, and any ation thereof.
In particular such embodiments, the lysis buffer is selected from the group
consisting of 0.2 to 5 percent, preferably about 1 percent (v/v) triton x-100 in water,
aqueous 2 to 50 millimolar, preferably about 10 millimolar tris-hydrochloride, aqueous
ethyl enediaminetetra- acetic acid having a pH of from about 5.5 to about 7.5,
preferably of about 6.5, in each case ally further comprising at least one
substance ed from the group ting of lysozyme, proteinase K, sodium
hydroxide and any ation thereof; the binding buffer is selected from the group
consisting of a mixture of aqueous sodium hypochlorite and aqueous glycine-
hydrochloride having a pH of from about 2.0 to about 4.0, preferably of about 3.0, a
e of aqueous tris-hydrochloride and aqueous sodium acetate/acetic acid having
a pH of from 3.6 to 5.6, aqueous sodium citrate/citric acid and having a pH of from 3.0
to 6.2, aqueous sodium perchlorate, aqueous sodium nitrate, aqueous 4 to 5 molar
sodium chloride, aqueous 4 to 5 molar potassium chloride, aqueous 4 to 6 molar
guanidine-hydrochloride, aqueous 4 to 6 molar guanidine thiocyanate, aqueous 4 to 6
molar iodide, s 4 to 6 molar urea, and any ation thereof; the washing
buffer is selected from the group consisting of aqueous 5 to 7, preferably 6 molar
sodium orate, aqueous 5 to 7, preferably 6 molar sodium nitrate, aqueous 4 to
molar sodium chloride, aqueous 4 to 5 molar potassium chloride, aqueous 4 to 6
molar guanidine-hydrochloride, aqueous 4 to 6 molar guanidine thiocyanate, aqueous
4 to 6 molar iodide, aqueous 4 to 6 molar urea, aqueous 100 to 300 millomolar,
preferably about 200 millimolar glycine-hydrochloride, and any combination thereof;
and/or the elution buffer is selected from the group consisting of ultrapure water,
17707096_1 (GHMatters) P108029.NZ
aqueous tris -hydrochloride having a pH from 7.4 to 9, aqueous 0.05 to 0.2 millimolar,
preferably about 0.1 olar ethylenediaminetetra-acetic acid, and any combination
thereof.
In particular embodiments, the lysis buffer comprises at least one substance
selected from the group consisting of lysozyme, proteinase K, sodium hydroxide and
any combination thereof.
In a third aspect, the present invention relates to a portable nucleic acid
extraction kit sing: a portable nucleic acid extraction apparatus according to the
present invention; at least one lysis buffer; at least one binding buffer; at least one
washing buffer; at least one elution buffer; and at least three sample tubes.
In particular embodiments, the kit further comprises ethanol.
Preferably, the kit includes at least 50 le c acid extraction
apparatuses, at least 150 kit sample tubes, and a first bottle containing a volume of
lysis buffer to lyse at least 50 s, a second bottle containing a volume of binding
buffer to bind nucleic acids of at least 50 samples, a third bottle containing a volume
of g buffer to wash at least 50 samples, and a fourth bottle containing a volume
of elution buffer to elute nucleic acids of at least 50 samples.
Fig. 1 represents a le c acid extraction apparatus 100. The portable
nucleic acid apparatus 100 es a handle 101, a rod 102, and a tip 103 as shown
in Fig. l .a. The tip of the portable nucleic acid extraction apparatus 100 may further
comprise at least one structure 104 which is configured to bind nucleic acids, as
represented in Fig. lb. The handle 101 is proximally located on the portable nucleic
acid extraction apparatus 100. A distal end 110 of the handle 101 may be in
mechanical communication with a proximal end 1 11 of the rod 102. The handle 101
may be formed from the proximal end 111 of the rod 102, where the handle 101 and
the rod 102 are formed as a single piece. The handle 101 may be a geometric shape,
such as a sphere, ellipsoid, or cuboid. The handle 101 may be made of a non-reactive
material, such as wood or plastic (e.g. polypropylene). The handle 101 may be made
of a material that binds nucleic acids, such as polystyrene, functionalized yrene
(e.g. functionalized with hydrofluoric acid), glass, or a silica derived material.
17707096_1 (GHMatters) P108029.NZ
Preferably, the handle 101 is a material that binds nucleic acids when the handle 101
and rod 102 are formed as a single piece.
The rod 102 of the portable nucleic acid extraction apparatus 100 is distal to
the handle 101. The rod 102 may be a geometric shape, such as a , er,
or triangular prism. Preferably the rod 102 is cylindrical in shape. The rod 102 is a
length that allows portability of the portable c acid extraction apparatus 100, such
as from 1 to 20, particularly from 2 to 15, more particularly from 3 to 10, most
particularly about 5 cm in length. The rod 102 is a diameter that allows portability of
the portable nucleic acid extraction apparatus 100, such as 2 to 4 millimeters. The rod
102 may be made of a non-reactive material, such as wood or plastic (e.g. or
polypropylene). The rod 102 may be made of a material that binds nucleic acids, such
as polystyrene, functionalized polystyrene (e.g. functionalized with hydrofluoric acid),
glass, or a silica derived material.
The tip 103 is distal to the rod 102 of the portable nucleic acid extraction
apparatus 100 and is configured to bind nucleic acids. The tip 103 may be in
mechanical communication with the rod 102. The tip 103 may be formed from the distal
end of the rod 102, thus being contiguous with the rod 102, where the tip 103 and the
rod 102 are a single piece. The tip 103 may be a geometric shape, such as a sphere,
cylinder, or cuboid. The tip 103 may have a similar or the same geometric shape as
the rod 102. The tip 103 may be made of a active material, such as wood or
c (e.g. polypropylene).
atively, the tip 103 may be made of a material that binds nucleic acids,
such as polystyrene, onalized polystyrene (e.g. functionalized with hydrofluoric
acid), glass, or a silica derived material, in particular, when the portable c acid
extraction apparatus 100 does not include at least one ure 104 which is
configured to bind nucleic acids, such as shown in Fig. 1.e. When the portable nucleic
acid extraction apparatus 100 does not comprise at least one structure 104 which is
configured to bind nucleic acids, the tip 103 is made of a material which is able to bind
nucleic acids. The tip 103 may comprise an increased surface area as ed to
the respective perfect geometric shape, such as provided by sandblasting, for
facilitation of nucleic acid binding, such as shown in Fig. 1.e.
17707096_1 (GHMatters) P108029.NZ
The at least one structure 104 which is ured to bind nucleic acids may be
on the tip 103 of the portable c acid extraction apparatus 100. The at least one
structure 104 which is configured to bind nucleic acids may be made of a nucleic acid
binding medium such as polystyrene, silica, a silica tive, silicon oxide, or an
anion exchange material, which binds the nucleic acids of interest. The structure 104
may be a nucleic acid binding medium in the physical form of a fiber, a bead, or a
protein. The at least one structure 104 which is a nucleic acid binding medium may
further be a complementary nucleic acid sequence that is attached through
derivatization to the tip 103, which is complementary to a ular nucleic acid
sequence in the biological sample.
When the at least one ure 104 which is configured to bind c acids
is a structure in contact with the tip 103, the structure may be fibers, beads, or proteins.
The structure may be contacted to the tip with an adhesive that is non-inhibitory to
proteins. The structure may extend outward from the tip 103. Fig. 1.b. illustrates
structures 104 configured to bind nucleic acids in the physical form of fibers, where
the fibers may be silica or yrene. Fig. 1.e. illustrates structures 104 configured
to bind nucleic acids in the physical form of beads. The beads may be silica micro
beads, silica nano particles, polystyrene micro beads, yrene nano les, or
sand-blasted glass beads. Fig. 1.d. illustrates structures 104 configured to bind c
acids that are proteins. When the structure is a protein the nucleic acid may be
biotinylated to increase binding to the protein. For example, when the protein is
streptavidin, the nucleic acid is biotinylated for binding to the n.
Fig. 2 illustrates a method 200 for extracting nucleic acids from a biological
sample. The biological sample may be blood, muscle tissue, plasma, semen, cells,
cheek swabs, nasal swabs, hair follicles, buffer wash from a biological specimen, and
preserved biological samples including frozen samples. In 201, the biological sample
having nucleic acids is treated to extract c acids. The treating includes drawing
the biological sample into a first sample tube containing a lysis buffer configured for
cell lysis. The first sample tube is configured to hold liquids and is made of a non-
reactive material, such as glass, plastic, metal, polypropylene or ceramic. The lysis
buffer is configured for lysing cells in the biological sample. For example, the lysis
buffer may be 1% triton x-100 (volume triton x-100/volume water), aqueous 10
17707096_1 ters) P108029.NZ
millimolar (mM) TnsHydrochloride (Tris HCl), or aqueous 1 mM ethylenediaminetetraacetic
acid (EDTA) having a pH of about 6.5, and combinations thereof. The lysis
buffer may further include a lysozyme, Proteinase K, or another basic reagent (e.g.
sodium hydroxide) that digests cell wall material and helps in the release of nucleic
acids. The lysis buffer preferably is added in a volume approximately equal to the
volume of the ical sample. The ng may further include inverting the first
sample tube to mix the lysis buffer with the biological sample. When the biological
sample es durable tissue, such as muscle , ear notches, tail clippings, or
hair follicles, the treating may still further include incubating the first sample tube at a
temperature from 37 to 95 degrees Celsius for a period of time, such as 2 minutes to
3 hours. When a basic t, such as sodium hydroxide, is used in the lysis buffer,
the treating may further include neutralizing the buffer with an equal volume of acid,
such as hydrochloric acid.
In 202, the tip of the portable nucleic acid extraction apparatus as previously
described in regard to Fig. 1 binds a nucleic acid in the treated biological . The
binding includes adding a binding buffer to the treated biological sample in the first
sample tube. The binding buffer is configured to facilitate binding of nucleic acids to
the tip and/or the nucleic acid binding structures of the portable nucleic acid extraction
apparatus, and may be an e buffer, may be a e buffer, may comprise at
least one salt, may comprise at least one chaotropic salt, may comprise biotin and
reagents ured for biotinylated on nucleic acids, and any ation f.
For example, when the tip and/or the nucleic acid binding structure of the portable
nucleic acid extraction apparatus is made of glass or silica d material, and when
the biological sample is whole blood, the binding buffers may be as follows: a mixture
of s 6 molar (M) sodium perchlorate and aqueous 200 millimolar (mM) ehydrochloride
having a pH of about 3. For further example, an acetate binding buffer
may be a mixture of aqueous 50 mM Tris-hydro chloride (Tris-HCl) and aqueous
sodium acetate/acetic acid having a pH of about 3.6 to 5.6; a citrate buffer may be
aqueous sodium citrate/citric acid having a pH of about 3.0 to 6.2; a salt binding buffer
may be aqueous 6 M sodium perchlorate, aqueous 6 M sodium nitrate, aqueous 4-5
M sodium chloride, or aqeous 4-5 M potassium chloride, aqueous sodium iodide,
aqueous potassium chloride, aqueous Guanidine hydrochloride, and aqueous
ine thiocyanate; a chaotropic salt binding buffer may be aqueous 4-6 M
17707096_1 (GHMatters) P108029.NZ
ine-hydrochloride, aqueous 4-6 M guanidine thiocyanate, aqueous 4-6 M
Iodide, or aqueous 4-6 M urea, and combinations thereof. The binding buffer is added
in a volume that preferably is approximately 2.5 times the volume of the biological
sample.
[0050] The nucleic acid binding further includes contacting the tip of the portable
nucleic acid extraction apparatus with the first sample tube, where the contacting may
include moving the tip in any pattern, such as a circular motion (stirring), in the first
sample tube. When the biological sample is whole blood or muscle tissue the binding
may be done at room temperature (from 18 to 30 s Celsius).
[0051] In 203, the tip of the portable nucleic acid extraction tus is incubated in
the biological sample to which lysis buffer and binding buffer has been added to allow
for g of nucleic acids to the tip. The incubation preferably includes adding a
volume of 70 percent ethanol (volume ethanol/volume water) that is approximately 2.5
times the volume of the biological sample to the first sample tube and ting the
tip of the portable nucleic acid extraction apparatus with the first sample tube. The
incubation may r include incubating the tip of the portable nucleic extraction
apparatus bound with nucleic acid in the first sample tube at room temperature for at
least 10 minutes.
In 204, the tip of the portable nucleic extraction apparatus with c acids
bound to it is washed. The washing includes contacting the tip with nucleic acids bound
to it with a washing buffer in a second sample tube. The second sample tube is
configured for holding liquids and is made of a non-reactive material such as glass,
plastic, metal, polypropylene or ceramic. The washing buffer is configured to wash the
nucleic acid to remove cellular components, except nucleic acid. The washing buffer
may comprise a salt, a chaotropic salt, and combinations thereof. The washing buffer
may further comprise a chelating agent, such as styrene divinylbenzene copolymer
containing paired iminodiacetate ions. For example, the g buffer may be as
follows: a salt washing buffer may be s 6 M sodium orate, aqueous 6 M
sodium nitrate, aqueous 4-5 M sodium chloride, or aqueous 4-5 M potassium chloride;
a chaotropic salt g buffer may be aqueous 4-6 M guanidine-hydrochloride,
aqueous 4-6 M guanidine thiocyanate, aqueous 4-6 M , or aqueous 4-6 M urea.
When the biological sample is bovine blood, and the tip and/or nucleic acid binding
17707096_1 (GHMatters) P108029.NZ
structure are glass or a silica d mater, the washing buffer may include a mixture
of aqueous 200 mM e- hydrochloride in a volume approximately equal to 2 times
the volume of the biological sample, approximately 70 percent aqueous ethanol
(volume ethanol/volume of water) in a volume approximately equal to 2.5 times the
volume of the biological sample, and combinations f. The washing may further
include incubating the tip of the portable nucleic acid extraction apparatus in the
second sample tube for approximately 5 to 10 minutes. The washing may further
include repeating the washing.
In 205 the nucleic acid bound to the tip and/or nucleic acid g medium of
the portable nucleic acid extraction apparatus is eluted. The eluting includes
contacting the tip with nucleic acids bound to it with an elution buffer in a third sample
tube. The third sample tube is ured for holding liquids and is made of a nonreactive
material such as glass, c, metal, polypropylene or ceramic. The elution
buffer is configured for eluting the nucleic acid bound to the tip into the elution buffer,
i.e. for facilitating the release of the bound nucleic acids from the tip into the elution
buffer. The elution buffer may be a neutral solution of a neutral pH (from pH 7 to 8)
(e.g. ultrapure water), aqueous 1 mM Tris-HCl of having a of pH 7.4 to 9.0, or aqueous
0.1 mM EDTA, and combinations thereof. The eluting may further include incubating
the tip in the third sample tube from 37 to 95 degrees s for approximately 10
minutes.
In 206, the eluted nucleic acid is analyzed. is may include quantitative
and qualitative analysis, such as absorption analysis, rase chain on
(PCR) amplification optionally followed by gel electrophoresis, and rolling circle
amplification optionally followed by gel electrophoresis.
[0055] Fig. 4 depicts a nucleic acid extraction kit 400. The portable nucleic acid
extraction apparatus may be part of a nucleic acid extraction kit 400, which may be
used in the field close to the source of a biological sample (e.g. cattle, swine, poultry)
or in a laboratory. The c acid tion kit 400 includes at least one nucleic acid
extraction apparatus 100, at least one lysis buffer 402, at least one binding buffer 404,
at least one washing buffer 406, at least one elution buffer 408, and, optionally ethanol
410, and at least three kit sample tubes 411. The buffers are typically provided in
separate bottles 401, 403, 405, 407 and, optionally, ethanol is ed in a fifth bottle
17707096_1 (GHMatters) P108029.NZ
409. The portable nucleic acid extraction kit optionally further comprises a container
412. Preferably the nucleic acid extraction kit 400 includes at least 50 portable nucleic
acid tion tuses, at least 150 kit sample tubes, the first bottle having a
volume of lysis buffer to treat at least 50 biological samples, the second bottle having
a volume of binding buffer to bind nucleic acids of at least 50 biological s, a
third bottle having a volume of washing buffer to wash at least 50 biological samples,
and a fourth bottle having a volume of elution buffer to elute c acids of at least
50 ical samples.
The portable nucleic acid extraction apparatus 100 in the nucleic acid extraction
kit 400 may be wrapped in a material (not pictured) to prevent atmospheric
contaminants from contacting the portable nucleic acid extraction apparatus, such as
a polymeric wrap, paper, or aluminum foil.
The first bottle 401 is made of a non-reactive material, such as glass, plastic,
metal, polypropylene or ceramic. The first bottle 401 is configured to hold liquids when
inverted, such as by a cap, rubber stopper, screw cap, or dropper tube. The first bottle
401 may be flexible and configured to deliver the lysis buffer 401, such as, through a
Yorker tip by squeezing the first bottle.
The second bottle 403 is made of a non-reactive material, such as glass, c,
metal, polypropylene or ceramic. The second bottle 403 is configured to hold liquids
when inverted, such as by a cap, rubber stopper, screw cap, or r tube. The
second bottle 403 may be flexible and configured to deliver the binding buffer 403,
such as, through the Yorker tip by squeezing the second bottle.
The third bottle 405 is made of a non-reactive material, such as glass, plastic,
metal, polypropylene or ceramic. The third bottle 405 is configured to hold s when
inverted, such as by a cap, rubber stopper, screw cap, or dropper tube. The third bottle
405 may be flexible and ured to deliver the washing buffer through the Yorker
tip, such as by squeezing the third bottle.
The fourth bottle 407 is made of a non-reactive material, such as glass, plastic,
metal, polypropylene or ceramic. The fourth bottle 407 is configured to hold liquids
when ed, such as by a cap, rubber stopper, screw cap, or dropper tube. The
96_1 (GHMatters) P108029.NZ
fourth bottle 407 may be flexible and ured to deliver the lysis buffer through the
Yorker tip, such as by squeezing the fourth bottle.
The fifth bottle 409 is made of a non-reactive material, such as glass, plastic,
metal, polypropylene or ceramic. The fifth bottle 409 is configured to hold liquids when
inverted, such as by a cap, rubber stopper, screw cap, or dropper tube. The fifth bottle
409 may be flexible and configured to deliver the ethanol, such as, h the Yorker
tip by squeezing the fifth bottle.
The sample kit tubes 41 1 are configured to hold s and are made of a non
-reactive material, such as glass, plastic, metal, polypropylene or c. The sample
kit tubes may further be configured to hold s when inverted, such as through a
cap, lid, or rubber stopper.
The container 412 is configured to contain at least one nucleic acid tion
apparatus 100, a first bottle 401 containing a lysis buffer 402, a second bottle 403
containing a binding buffer 404, a third bottle 405 containing a washing buffer 406, a
fourth bottle 407 containing an elution buffer 408, optionally a fifth bottle 409 containing
ethanol 410, and at least three kit sample tubes 411 (contents of the kit). The container
may be of any non-reactive material, such as cardboard, plastic, or polystyrene. The
container may be of any geometric shape configured for the holding the contents of
the kit, such as a , cylinder, or triangular prism.
[0064] The container may further include a first compartment 413 configured to hold
the first bottle 401, a second compartment 414 configured to hold the second bottle
403, a third compartment 415 configured to hold the third bottle 405, a fourth
compartment 416 configured to hold the fourth bottle 407, optionally a fifth
compartment 417 configured to hold the fifth bottle 409, a sixth compartment 418
configured to hold the at least one nucleic acid extraction apparatus 100, and a
seventh container 419 configured to hold the at least three kit sample tubes 411. The
first compartment 413, second compartment 414, third compartment 415, fourth
compartment 416, fifth compartment 417, sixth compartment 418, and seventh
compartment 419 may be formed by any active material such as cardboard,
plastic, or polystyrene. The container may further include a closure (not pictured) to
17707096_1 ters) P108029.NZ
keep the contents of the kit in the container when inverted. The closure may be of any
non-reactive material, such as cardboard, plastic, or polystyrene.
The present invention further relates to the following embodiments.
In particular embodiments, the present invention relates to a portable nucleic
acid tion kit comprising: a container, the container ing, a portable nucleic
acid extraction apparatus comprising, a handle, a rod ed to the handle, and a
tip attached to the rod, where the tip is a material selected from at least one of the
group consisting of wood, plastic, polystyrene, functionalized yrene, glass, and
a silica-derived material; a lysis buffer in a first bottle; a binding buffer in a second
bottle; a washing buffer in a third ; an elution buffer in a fourth bottle; and at least
three kit sample tubes.
In particular embodiments, the container r includes a volume ethanol in a
fifth bottle.
In particular embodiments, the portable nucleic acid tion tus
further comprises a nucleic acid binding , where the nucleic acid binding
medium is configured to bind c acids.
In ular embodiments, the present invention relates to a portable nucleic
acid extraction apparatus for nucleic acid extraction, comprising: a handle; a rod
attached to the handle; and a tip attached to the rod, where the tip is a material
selected from at least one of the group consisting of wood, plastic, polystyrene,
functionalized polystyrene, glass, and a silica- derived material.
In particular embodiments, the portable nucleic acid extraction apparatus
further comprises a nucleic acid binding medium attached to the tip, where the nucleic
acid binding medium is configured to bind nucleic acids.
[0071] In particular embodiments, the handle, the rod, and the tip are formed as a
single piece, where the material that forms the handle, the rod, and the tip is selected
from at least one of the group consisting of wood, plastic, polystyrene, functionalized
polystyrene, glass, and a silica- derived material.
96_1 ters) P108029.NZ
In particular embodiments, the handle, the rod, and the tip are glass.
In ular embodiments, the handle, the rod, and the tip are glass
sandblasted to increased surface area.
In particular embodiments, the tip is ured to bind nucleic acids and is
selected from at least one of the group consisting of polystyrene, functionalized
polystyrene, glass, and silica- derived material.
In particular embodiments, the tip is glass sandblasted to increased surface
area.
In particular embodiments, the nucleic acid binding medium is in the form of a
fiber.
In particular embodiments, the fiber is a silica fiber.
In particular ments, the fiber is a polystyrene fiber.
In particular embodiments, the nucleic acid g medium is in the form of a
bead.
[0080] In particular embodiments, the bead is selected from the group consisting of
silica micro beads, silica nano particles, polystyrene micro beads, polystyrene nano
particles, sand-blasted glass beads, and combinations thereof. [0081] In particular
embodiments, the nucleic acid binding medium is a protein.
In particular embodiments, the present invention relates to a nucleic acid
analysis method for analyzing nucleic acids using a portable nucleic acid extraction
apparatus, comprising: treating a ical sample having a c acid with a lysis
buffer to lyse cells in the biological ; binding the nucleic acid in the treated
biological sample to a tip of a portable nucleic acid extraction apparatus with a binding
buffer; incubating the tip of the portable nucleic acid extraction apparatus bound with
the nucleic acid; g the tip of the portable c acid tion apparatus
bound with the nucleic acid with a washing buffer; eluting the nucleic acid bound to
17707096_1 (GHMatters) P108029.NZ
the tip of the portable nucleic acid extraction apparatus with an elution buffer; and
analyzing the eluted nucleic acid.
In particular embodiments, the incubating further includes contacting the tip of
the portable nucleic acid extraction apparatus with a volume of ethanol.
[0084] In particular embodiments, the lysis buffer is an aqueous on of 10
millimolar tris- hydrochloride; the binding buffer is a combination of aqueous 6 molar
sodium lorite and aqueous 200 millimolar glycine-hydrochloride having a pH
from 2.0 to 4.0; the washing buffer is a combination of s glycine-hydrochloride
and aqueous ethanol (volume ethanol/volume of water); and the elution buffer is
aqueous tris-hydrochloride having a pH from 7.0 to 10.0.
In particular embodiments, the lysis buffer further comprises a substance
chosen from the group consisting of lysozyme, proteinase k, and sodium hydroxide.
In particular embodiments, the lysis buffer is selected from the group consisting
from 0.2 to 5 percent triton x-100 (volume triton x-100/volume water), from 2 to 50
millimolar aqueous tris-hydrochloride, and aqueous 1 millimolar ethylenediaminetetraacetic
acid having a pH from 5.5 to 7.5, and combinations f; the g buffer
is selected from the group consisting of a mixture of aqueous sodium lorite and
glycine-hydrochloride having a pH from 2.0 to 4.0, a mixture of aqueous trishydrochloride
and aqueous sodium acetate/acetic acid having a pH from 3.6 to 5.6;
aqueous sodium citrate/citric acid having a pH from 3.0 to 6.2, aqueous 5 to 7 molar
sodium perchlorate, aqueous 6 molar sodium nitrate, aqueous 4 to 5 molar sodium
chloride, aqueous 4 to 5 molar potassium chloride, aqueous 4 to 6 molar guanidinehydrochloride
, aqueous 4 to 6 molar guanidine thiocyanate, aqueous 4 to 6 molar
iodide, and aqueous 4 to 6 molar urea, and combinations thereof; the washing buffer
is selected from the group consisting of aqueous 5 to 7molar sodium orate,
aqueous 5 to 7 molar sodium nitrate, aqueous 4 to 5 molar sodium chloride, aqueous
4 to 5 molar potassium chloride, s 4 to 6 molar ine-hydrochloride,
aqueous 4 to 6 molar ine thiocyanate, aqueous 4 to 6 M iodide, s 4 to 6
molar urea, and aqueous 100 to 300 millimolar glycine- hloride, and
combinations thereof; the elution buffer is selected from the group consisting of
17707096_1 (GHMatters) P108029.NZ
ultrapure water, aqueous tris-hydrochloride having a pH from 7.4 to 9, and from 0.05
to .2 millimolar aqueous ethylenediaminetetra-acetic acid, and combinations thereof.
The following examples are ed to illustrate one or more red
embodiments of the invention. Numerous variations can be made to the ing
examples that lie within the scope of the invention.
e 1. Determining concentration of extracted genomic DNA. Fig. 3. a.
illustrates a concentration and purity determination of extracted genomic DNA.
Genomic DNA from a biological sample was extracted using the method 200. The
biological sample was six 100 microliter aliquots of bovine blood. The lysis 201 was
performed with 0.01 mM Tris-HCl at pH 6.8 as lysis buffer. While a specific lysis buffer
was used in this instance other lysis buffers may be used. The g 202 ed
the addition of 6M Sodium hypochlorite in 200 mM e- hydrochloride at pH 3.0 as
binding buffer. While a specific binding buffer was used in this instance other binding
buffers may be used. The incubation 203 included adding ethanol and stirring the
treated biological sample for 10 s. The washing 204 was performed with 200
microliters of 200 mM glycine-hydrochloride and 250 uL of 70% (volume/volume)
ethanol as washing buffer and included ng the treated biological sample for
between 2 to 5 minutes. While a specific washing buffer was used in this instance
other washing buffers may be used. The n 205 was performed with 10 mM Tris-
HCl buffer at pH 8.8 as elution buffer and included incubation for 10 minutes at 90
degrees Celsius. While a specific elution buffer was used in this instance other n
buffers may be used. The analysis 206 included the measurement of DNA
concentration and purity. DNA concentration was measured by DNA absorbance of
ultra violet visible light by a spectrophotometer and purity was measured by calculating
the ration of absorbance reading at 260 nanometers (nm) and 280 nm. Fig. 3. a.
demonstrates that the portable nucleic acid extraction apparatus and method 200 of
tion yields DNA of a quality that is accepted as good (e.g. having an absorbance
ration from 1.3 to 1.8) and suitable for further application and quantitative analysis.
Example 2. PCR amplification and gel electrophoresis. Fig. 3.b. illustrates PCR
amplification and gel electrophoresis of the bovine glyceraldehyde 3 -phosphate
dehydrogenase (GAPDH) gene, using genomic DNA extracted with method 200 as a
template. Six 100 microliter aliquots of bovine blood were used as starting material.
96_1 (GHMatters) P108029.NZ
The method 200 of Example 1 was used to t the genomic DNA of these
biological samples. The analysis 206 included PCR amplification of the GAPDH gene
using 1 microliter eluate obtained from each of the 6 samples in 15 microliter PCR
reactions. The 6 PCR reaction products were run on a 1% agarose gel and stained
with SYBR green to ize the extracted DNA. Lanes 1 through 6 correspond to
each of the 6 PCR products obtained. Lane N is a negative control. Lane P is a positive
l, which used 40 nanograms bovine genomic DNA as template for PCR
amplification of the GAPDH gene. Fig. 3.b. illustrates a distinct PCR product of about
650 base pairs in each of lanes 1 through 6 indicating that the portable nucleic acid
extraction apparatus and method 200 yield extracted genomic DNA which is suitable
for subsequent PCR amplification, e.g., to qualitatively determine the presence or
absence of a given DNA sequence.
Example 3. Rolling Circle ication (RCA) and gel electrophoresis. Fig. 3.c.
illustrates RCA and gel electrophoresis for a region of the bovine beta casein gene,
using genomic DNA extracted with the method 200 as template. Six 100 microliter
ts of bovine blood were used as starting material. . The method 200 of Example
1 was used to extract genomic DNA from these ical samples. The analysis 206
included ligation of specific padlock probes to the bone beta casein gene in the
extracted c DNA from the biological sample and amplification of the ligated
padlock probe specific primers using Bst DNA polymerase. The six amplification
products were run on a 1% agarose gel and d with SYBR green to visualize the
ied DNA. The lane marked MWS is a size standard l lane ed a 100
base pair molecular weight ladder. Fig. 3.c. illustrates a visualization of banding
patterns in lanes 1 through 6 that is tive of the amplification of the padlock probe
specific to the bovine beta casein gene, which indicates that the portable nucleic acid
extraction apparatus and method 200 yield extracted genomic DNA that is suitable for
subsequent Rolling Circle Amplification, e.g., to qualitatively determine the presence
or absence of a given DNA sequence.
17707096_1 (GHMatters) P108029.NZ
Claims (11)
1. A le c acid tion apparatus for nucleic acid extraction, comprising: 5 a handle; a rod attached to the handle; a tip attached to the rod; wherein the handle, the rod, and the tip are integrally formed as a single piece, where the material that forms the handle, the rod and the tip is selected from at least one of the group consisting of wood, plastic, 10 polystyrene, functionalized polystyrene, glass, and a silica-derived material; n the apparatus does not se specialized equipment selected from the group consisting of pipettes, centrifuges, and automated equipment; and wherein the portable nucleic acid extraction apparatus does not comprise a stationary nucleic acid binding medium.
2. The portable c acid extraction tus of claim 1, wherein the handle, the rod, and the tip are glass.
3. The portable nucleic acid tion apparatus of claim 1 or claim 2, wherein 20 the handle, the rod, and the tip are glass sandblasted to increased surface area.
4. The portable nucleic acid extraction apparatus of any one of claims 1 to 3, wherein the tip is configured to bind nucleic acids in that the tip is either made of material which is able to bind nucleic acids that are present in solution or the 25 tip comprises structures that are able to bind nucleic acids that are present in on and wherein the tip is selected from at least one of the group consisting of polystyrene, functionalized polystyrene, glass, and silica-derived material.
5. The portable nucleic acid extraction apparatus of claim 4, wherein the tip is 30 glass sandblasted to increased surface area. 17707096_1 (GHMatters) P108029.NZ
6. The le nucleic acid extraction apparatus of any one of claims 1 to 5, wherein the nucleic acid binding medium is in the form of a fiber.
7. The portable nucleic acid extraction apparatus of claim 6, wherein 5 the fiber is a silica fiber.
8. The portable nucleic acid extraction apparatus of claim 6, wherein the fiber is a polystyrene fiber. 10
9. The portable nucleic acid extraction tus of any one of claims 1 to 5, wherein the nucleic acid g medium is in the form of a bead.
10. The portable c acid extraction apparatus of claim 9, wherein the bead is selected from the group consisting of silica micro beads, silica nano particles, 15 polystyrene micro beads, polystyrene nano particles, sand-blasted glass beads, and combinations thereof.
11. The portable nucleic acid extraction apparatus of any one of claims 1 to 10, wherein the nucleic acid g medium is a protein. 17707096_1 (GHMatters) P108029.NZ WO 27538 SUBSTITUTE SHEET (RULE 26) vs. «a» \ ram» , «msmxa . 333 EB, Sm «3:? «35 n 934% EERQ‘Q. §,Q§§§ 29..“K3“. SUBSTITUTE SHEET (RULE 26)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562203707P | 2015-08-11 | 2015-08-11 | |
US62/203,707 | 2015-08-11 | ||
PCT/US2016/046243 WO2017027538A1 (en) | 2015-08-11 | 2016-08-10 | Portable nucleic acid extraction apparatus and method of using the same |
Publications (2)
Publication Number | Publication Date |
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NZ740585A NZ740585A (en) | 2021-06-25 |
NZ740585B2 true NZ740585B2 (en) | 2021-09-28 |
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