U.S. patent application number 11/841069 was filed with the patent office on 2008-03-20 for method and device for obtaining or amplifying nucleic acid from a cell using a nonplanar solid substrate.
This patent application is currently assigned to SAMSUNG ELECTRONICS CO., LTD.. Invention is credited to Jung-im HAN, Kyu-youn HWANG, Sung-young JEONG, Joon-ho KIM.
Application Number | 20080070282 11/841069 |
Document ID | / |
Family ID | 38980858 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080070282 |
Kind Code |
A1 |
HWANG; Kyu-youn ; et
al. |
March 20, 2008 |
METHOD AND DEVICE FOR OBTAINING OR AMPLIFYING NUCLEIC ACID FROM A
CELL USING A NONPLANAR SOLID SUBSTRATE
Abstract
The present invention relates to a method of isolating a nucleic
acid from a microorganism cell. The method comprises contacting a
nonplanar solid substrate with a cell-containing sample in a liquid
medium having a pH of 3.0 to 6.0 so that the microorganism cell is
bound to the nonplanar solid substrate, and lysing the
microorganism cell bound to the nonplanar solid substrate. The
invention also relates a device including the nonplanar solid
substrate for isolating and amplifying a nucleic acid.
Inventors: |
HWANG; Kyu-youn; (Yongin-si,
KR) ; JEONG; Sung-young; (Yongin-si, KR) ;
KIM; Joon-ho; (Yongin-si, KR) ; HAN; Jung-im;
(Yongin-si, KR) |
Correspondence
Address: |
CANTOR COLBURN, LLP
20 Church Street
22nd Floor
Hartford
CT
06103
US
|
Assignee: |
SAMSUNG ELECTRONICS CO.,
LTD.
416, Maetan-dong, Yeongtong, gu
Suwon-si
KR
|
Family ID: |
38980858 |
Appl. No.: |
11/841069 |
Filed: |
August 20, 2007 |
Current U.S.
Class: |
435/91.2 ;
435/306.1; 435/91.1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
B01L 2300/0636 20130101; B01L 7/52 20130101; C12N 2533/30 20130101;
C12N 15/1006 20130101 |
Class at
Publication: |
435/091.2 ;
435/306.1; 435/091.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12M 1/00 20060101 C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 21, 2006 |
KR |
10-2006-0079053 |
Aug 21, 2006 |
KR |
10-2006-0079054 |
Aug 21, 2006 |
KR |
10-2006-0079055 |
Aug 21, 2006 |
KR |
10-2006-0079056 |
Claims
1. A method of obtaining a nucleic acid from a cell, the method
comprising: contacting a nonplanar solid substrate with a
cell-containing sample in a liquid medium having a pH of 3.0 to 6.0
so that the cell is bound to the nonplanar solid substrate, wherein
the nonplanar solid substrate is hydrophobic and has a water
contact angle of 70.degree. to 95.degree. or the nonplanar solid
substrate has at least one amine-based functional group at its
surface; and lysing the cell bound to the nonplanar solid substrate
to obtain a nucleic acid from the lysed cell.
2. The method of claim 1, wherein the cell is a bacteria, a fungus,
or a virus.
3. The method of claim 1, wherein the cell-containing sample is a
biological sample.
4. The method of claim 3, wherein the cell-containing sample is
blood, urine, or saliva.
5. The method of claim 1, wherein the cell-containing sample is a
solution diluted with a phosphate buffer or an acetate buffer.
6. The method of claim 5, wherein the cell-containing sample is
diluted in a ratio of 1:1 to 1:10.
7. The method of claim 5, wherein the cell-containing sample has a
salt concentration of 10 mM to 500 mM.
8. The method of claim 7, wherein the cell-containing sample has a
salt concentration of 50 mM to 300 mM.
9. The method of claim 1, wherein the nonplanar solid substrate is
selected from the group consisting of a solid substrate having a
surface comprising a pillar structure formed of a plurality of
pillars, a bead-shaped solid substrate, and a sieve-shaped solid
substrate having a surface comprising pores.
10. The method of claim 9, wherein each of the pillars has an
aspect ratio of 1:1 to 20:1.
11. The method of claim 9, wherein, in the pillar structure, a
ratio of a height of the pillars to a distance between adjacent
pillars is in the range of 1:1 to 25:1.
12. The method of claim 9, wherein, in the pillar structure, a
distance between adjacent pillars is in the range of 5 .mu.m to 100
.mu.m.
13. The method of claim 1, wherein the hydrophobic nonplanar solid
substrate is obtained by coating the nonplanar solid substrate with
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS).
14. The method of claim 1, wherein the nonplanar solid substrate
having at least one amine-based functional group is prepared by
coating the nonplanar solid substrate with
polyethyleneiminetrimethoxysilane (PEIM).
15. The method of claim 1, further comprising washing the nonplanar
solid substrate with a wash buffer to remove materials in the
cell-containing sample which are not bound to the nonplanar solid
substrate.
16. The method of claim 1, wherein lysing the cell is performed by
boiling lysis, laser lysis, lysis using a chemical material, or
electrochemical lysis.
17. The method of claim 1, wherein lysing is performed in a liquid
medium having a pH of 3.0 to 8.0 so that the nucleic acid obtained
from the cell binds to the nonplanar solid substrate.
18. The method of claim 17, further comprising washing the
nonplanar solid substrate to remove materials which are not bound
to the nonplanar solid substrate.
19. The method of claim 17, further comprising eluting the nucleic
acid bound to the nonplanar solid substrate.
20. The method of claim 1, wherein lysing the cell is performed in
a liquid medium having a pH of 11 to 14.
21. A method of amplifying a target nucleic acid comprising
amplifying a target nucleic acid using the nucleic acid obtained
according to the method of claim 1 as a template.
22. The method of claim 21, wherein the nucleic acid is DNA or
RNA.
23. The method of claim 21, wherein amplifying the target nucleic
acid is performed in a first chamber comprising the nonplanar solid
substrate or in a second chamber in fluid communication with the
first chamber.
24. The method of claim 21, wherein amplifying is performed through
PCR.
25. A device for isolating and amplifying a nucleic acid, the
device comprising: a reaction chamber comprising a nonplanar solid
substrate, wherein the nonplanar solid substrate is hydrophobic and
has a water contact angle of 70.degree. to 95.degree. or the
nonplanar solid substrate has at least one amine-based functional
group at its surface; a heating unit which heats the reaction
chamber; and a temperature controlling unit which controls the
heating unit.
26. The device of claim 25, further comprising a nucleic acid
amplification chamber in fluid communication with the reaction
chamber comprising the nonplanar solid substrate.
27. The device of claim 25, wherein the nonplanar solid substrate
is selected from the group consisting of a solid substrate having a
surface comprising a pillar structure formed of a plurality of
pillars, a bead-shaped solid substrate, and a sieve-shaped solid
substrate having a surface comprising pores.
28. The device of claim 27, wherein each of the pillars has an
aspect ratio of 1:1 to 20:1.
29. The device of claim 27, wherein, in the pillar structure, a
ratio of a height of the pillars to a distance between adjacent
pillars is in the range of 1:1 to 25:1.
30. The device of claim 27, wherein, in the pillar structure, a
distance between adjacent pillars is in the range of 5 .mu.m to 100
.mu.m.
31. The device of claim 25, wherein the nonplanar solid substrate
is hydrophobic and has a water contact angle of 70.degree. to
95.degree..
32. The device of claim 31, wherein the hydrophobic nonplanar solid
substrate is obtained by coating the nonplanar solid substrate with
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS).
33. The device of claim 25, wherein the nonplanar solid substrate
has at least one amine-based functional group at its surface.
34. The device of claim 33, wherein the nonplanar solid substrate
having at least one amine-based functional group is prepared by
coating the nonplanar solid substrate with
polyethyleneiminetrimethoxysilane (PEIM).
Description
[0001] This application claims priority to Korean Patent
Application Nos. 10-2006-0079056, 10-2006-0079053, 10-2006-0079054,
and 10-2006-0079055, each filed on Aug. 21, 2006, and all the
benefits accruing therefrom under 35 U.S.C. .sctn. 119, the
disclosure of each is incorporated herein in its entirety by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method of isolating a
nucleic acid from a cell using a nonplanar solid substrate, a
method of amplifying the isolated nucleic acid as a template, and a
device comprising the nonplanar solid substrate for isolating and
amplifying a nucleic acid.
[0004] 2. Description of the Related Art
[0005] Several conventional methods of purifying a nucleic acid
using a solid phase are known. For example, U.S. Pat. No. 5,234,809
discloses a method of purifying a nucleic acid using a solid phase
to which the nucleic acid is bound. This method, however, is
time-consuming and complex, and thus, is unsuitable for a
lab-on-a-chip (LOC). In addition, this method requires use of a
chaotropic substance. That is, when a chaotropic substance is not
used, a nucleic acid does not bind to the solid phase.
[0006] U.S. Pat. No. 6,291,166 discloses a method of archiving a
nucleic acid using a solid phase matrix. According to this method,
the nucleic acid is irreversibly bound to the solid phase matrix.
Such irreversible binding enables delayed analysis or repeated
analysis after a nucleic acid-solid phase matrix composite is
generated and stored. However, in this method, a substance having a
positively charged surface, such as alumina, is activated by a base
substance, such as NaOH, and then the nucleic acid is irreversibly
bound to the activated alumina. As a result, the bound nucleic acid
cannot be isolated from the alumina.
[0007] U.S. Pat. No. 5,705,628 discloses a method of reversibly and
non-specifically binding DNA in a DNA-containing solution
containing a salt and polyethylene glycol to a magnetic
microparticle having a carboxyl group-coated surface. This method
uses a magnetic microparticle having a carboxyl group-coated
surface, a salt, and polyethylene glycol, in order to isolate
DNA.
[0008] In the conventional methods of isolating and purifying a
nucleic acid described above, addition of a high-concentration
reagent is required for DNA binding. However, such addition can
affect a subsequent process, such as a polymerase chain reaction
(PCR) and cannot be used on a lab-on-a-chip (LOC). In addition,
these conventional methods of isolating and purifying a nucleic
acid are performed independently from a method of purifying or
concentrating a cell. Furthermore, a method of purifying or
concentrating a cell and then, in the same reaction vessel,
isolating a nucleic acid from the resulting purified or
concentrated cell is not known.
[0009] Accordingly, there is a need to develop a method in which a
cell is isolated or concentrated by binding the cell to a solid
surface, such as a substrate, and then, in the same reaction
vessel, a nucleic acid derived from the cell can be purified,
isolated and concentrated due to high affinity with respect to the
nucleic acid.
BRIEF SUMMARY OF THE INVENTION
[0010] In one embodiment, the invention is directed to a method of
obtaining a nucleic acid from a cell, the method comprising
contacting a nonplanar solid substrate with a cell-containing
sample in a liquid medium having a pH of 3.0 to 6.0 so that the
cell is bound to the nonplanar solid substrate, wherein the
nonplanar solid substrate is hydrophobic and has a water contact
angle of 70.degree. to 95.degree. or the nonplanar solid substrate
has at least one amine-based functional group at its surface; and
lysing the cell bound to the nonplanar solid substrate to obtain a
nucleic acid from the lysed cell.
[0011] In another embodiment, the invention is directed to a device
for isolating and amplifying a nucleic acid, the device comprising
a reaction chamber comprising a nonplanar solid substrate, wherein
the nonplanar solid substrate is hydrophobic and has a water
contact angle of 70.degree. to 95.degree. or the nonplanar solid
substrate has at least one amine-based functional group at its
surface; a heating unit which heats the reaction chamber; and a
temperature controlling unit which controls the heating unit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The above and other features and advantages of the present
invention will become more apparent by describing in detail
exemplary embodiments thereof with reference to the attached
drawings in which:
[0013] FIG. 1 is a graph showing concentration of DNAs, indicated
by fluorescence intensity, in a NaOH solution of pH 12.0 and in a
phosphate buffer of pH 7.0.
[0014] FIG. 2 is a graph showing the degree of DNA collection with
respect to pH of a DNA sample after the DNA sample is pumped
through a fluidic device with a pillar array.
[0015] FIG. 3 is a graph showing the concentration of a cell with
respect to the concentration of DNA, indicated by fluorescence
intensity, present in a cell lysate obtained by cell lysis.
[0016] FIG. 4 is a graph illustrating DNA elution efficiency
measured according to a method of the present invention in which an
E. coli-containing sample is pumped through a fluidic device
including a chamber having an octadecyldimethyl (3-trimethoxysilyl
propyl)ammonium (OTC)-coated surface and a pillar array, the bound
cell is lysed, and then the resultant lysate is collected to
measure the DNA elution efficiency.
[0017] FIG. 5 is a graph showing the concentration of amplified DNA
measured according to a method in which the E. coli-containing
sample is pumped through a chamber having a surface with a pillar
array of a fluidic device, undergoes cell lysis, cell washing, and
real time PCR amplification, and electrophoresis.
[0018] FIG. 6 is a graph illustrating results of real time PCR
amplification after an E. coli-containing urine sample is pumped
through a chamber having a pillar array of a fluidic device and
undergoes cell lysis and cell washing.
[0019] FIG. 7 is a graph illustrating results of real time PCR
amplification after an E. coli-containing whole blood sample is
pumped through a chamber having a pillar array of a fluidic device
and undergoes cell lysis and DNA.
[0020] FIG. 8 is a graph illustrating results of electrophoresis
after an E. coli-containing whole blood sample is pumped through a
chamber having a pillar array of a fluidic device and undergoes
cell lysis, DNA extraction, and real time PCR amplification.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention will now be describe more fully hereinafter
with reference to the accompanying drawings, in which embodiments
of the invention are shown. The invention may, however, be embodied
n may different forms and should not be construed as limited to
embodiments set forth herein. Rather these embodiments are provided
so that this disclosure will be through and complete, and will
fully convey the scope of the invention to those skilled in the
art.
[0022] In one embodiment, the invention provides a method of
obtaining a nucleic acid from a cell. The method comprises
contacting a nonplanar solid substrate with a cell-containing
sample in a liquid medium having a pH of 3.0 to 6.0 so that the
cell is bound to the nonplanar solid substrate; and lysing the cell
bound to the nonplanar solid substrate to obtain a nucleic acid
from the lysed cell.
[0023] The method comprises contacting a nonplanar solid substrate
with a cell-containing sample in a liquid medium having a pH of 3.0
to 6.0 so that the cell is bound to the nonplanar solid substrate.
The cell binds to the nonplanar solid substrate by the
contacting.
[0024] As used herein, the term "cell" means a prokaryotic or
eukaryotic cell, a plant cell, a bacteria cell, a pathogenic cell,
a yeast cell, an aggregate of cells, a virus, a fungus, or other
nucleic acid containing biological material, such as, for example,
an organelle.
[0025] As used herein, the term "nucleic acid" means DNA or RNA, or
a combination of both. The DNA or RNA can be in any possible
configuration, i.e., in the form of double-stranded (ds) nucleic
acid, or in the form of single-stranded (ss) nucleic acid, or as a
combination thereof (in part ds or ss).
[0026] As used herein, the term "cell-binding" means the ability to
bind a cell or other biomaterial, such as, for example a nucleic
acid.
[0027] In a liquid medium containing a solid substrate, a cell,
such as, for example, a bacteria cell, can exist in the liquid
medium or can be bound to the solid substrate. Whether the cell
exists in the liquid medium or is bound to the solid substrate is
determined by a difference in surface tensions of the liquid medium
and the cell. For example, when the liquid medium has greater
surface tension than the cell, the cell may be easily bound to a
solid substrate having low surface tension, that is, to a
hydrophobic solid substrate. When the surface tension of the liquid
medium is less than the surface tension of the cell, the cell may
be easily bound to a solid substrate having greater surface
tension, that is, to a hydrophilic solid substrate. When the liquid
medium and cell have the same surface tension, the surface tension
does not affect the binding of cell to the solid substrate and
other interaction factors, such as electrostatic interaction, may
affect such binding (see Applied and Environmental Microbiology,
July 1983, p. 90-97). In addition, it is known that cell can be
bound to the solid substrate by electrostatic attraction as well as
by a thermodynamic approach based on the surface tension. However,
such bindings occur very slowly and its bound quantity was
minimal.
[0028] In an attempt to address the problems described above, the
inventors of the present invention found that a large amount of
cells could be bound to a nonplanar solid substrate by contacting
the nonplanar solid substrate with a cell-containing sample in a
liquid medium having a pH of 3.0 to 6.0. The use of a nonplanar
solid substrate provides increased surface area upon which the
cells bind, relative to a planar surface. Therefore, without being
held to theory, it is believed that a large amount of cells can be
bound because the surface area of a nonplanar solid substrate is
increased, relative to a planar surface. Furthermore, by using a
liquid medium having a pH 3.0 to 6.0, the cell membrane of the cell
is denatured and thus the cell is less soluble with respect to a
solution and therefore relatively more cells can be bound to the
solid surface. However, the present invention is not limited to
such a technique.
[0029] During the contacting step, the sample can be any sample
containing a cell. For example, the sample can be a biological
sample containing a cell, a clinical sample containing a cell, or a
lab sample containing a cell.
[0030] As used herein, the term "biological sample" a sample that
comprises or is formed of a cell or tissue, such as a cell or
biological liquid isolated from an animal or plant. In one
advantageous embodiment, the animal can be a human. The biological
sample can be saliva, sputum, blood, blood cells (for example, red
blood cells or white blood cells), amniotic fluid, serum, semen,
bone marrow, tissue or a micro needle biopsy sample, urine,
peritoneum fluid, pleura fluid, or cell cultures. In addition, the
biological sample can be a tissue section, such as a frozen section
taken for a histological object. Preferably, the biological sample
is a clinical sample obtained from a human patient. More
preferably, the biological sample is blood, urine, saliva, or
sputum. Furthermore, the term "biological sample" means a sample
that is formed comprising an organism, group of organisms from the
same or different species, cells or tissues, obtained from the
environment, such as from a body of water, from the soil, or from a
food source or an industrial source.
[0031] In one embodiment, the method comprises, during the
contacting step, the biological sample can be diluted with a
solution or buffer that may buffer the cell with a low pH. The
buffer can be, for example, a phosphate buffer, such as sodium
phosphate of pH 3.0 to 6.0, or an acetate buffer, such as sodium
acetate of pH 3.0 to 6.0. The degree of dilution is not limited,
and, for example, the biological sample can be diluted in a range
of 1:1 to 1:1,000, and preferably, 1:1 to 1:10.
[0032] In another embodiment, the method comprises, during the
contacting step, the sample may have a salt concentration of 10 mM
to 500 mM, and preferably, 50 mM to 300 mM. That is, the sample may
have an acetate or phosphate ion concentration of 10 mM to 500 mM,
preferably 50 mM to 300 mM.
[0033] In one embodiment, during the contacting process, the solid
substrate contacted with the cell-containing sample has a nonplanar
shape such that the surface area of the nonplanar solid substrate
can be increased compared to a planar surface. For example, the
nonplanar solid substrate may have a corrugated surface. As used
herein, the term "corrugated surface" refers to a non-level surface
having grooves and ridges. The corrugated surface can be a surface
having a plurality of pillars or a sieve-shaped surface having a
plurality of pores. However, the corrugated surface is not limited
thereto, and may comprise other shapes.
[0034] For example, the nonplanar solid substrate can be a solid
substrate having a surface comprising a plurality of pillars, a
bead-shaped solid substrate, and a sieve-shaped solid substrate
having a plurality of pores in its surface. The solid substrate can
be a single solid substrate or a combination of one or more solid
substrates, such as a solid substrate assembly that fills a tube or
container.
[0035] In one embodiment, the nonplanar solid substrate may form an
inner wall of a microchannel or microchamber of a microfluidic
device. Accordingly, the method of obtaining a nucleic acid from a
cell according the present invention can be used in a fluidic
device or microfluidic device having at least one inlet and outlet
connected through a channel or microchannel.
[0036] As used herein, the term "microfluidic device" incorporates
the concept of a microfluidic device that comprises microfluidic
elements such as, e.g., microfluidic channels (also called
microchannels or microscale channels). As used herein, the term
"microfluidic" refers to a device component, e.g., chamber,
channel, reservoir, or the like, that includes at least one
cross-sectional dimension, such as depth, width, length, diameter,
etc. of from about 0.1 micrometer to about 1000 micrometer. Thus,
the term "microchamber" and "microchannel" refer to a channel and a
chamber that includes at least one cross-sectional dimension, such
as depth, width, and diameter of from about 0.1 micrometer to about
1000 micrometer, respectively.
[0037] According to the current embodiment, during the contacting
process, the nonplanar solid substrate used in the contacting step
may have a surface having a plurality of pillars. Methods of
forming pillars on a solid substrate is well known in the art. For
example, micro pillars can be formed in a high density structure
using a photolithography process used in a semiconductor
manufacturing process. The micro pillars can have an aspect ratio
of 1:1 to 20:1. However, the aspect ratio of the micro pillars is
not limited thereto. As used herein, the term "aspect ratio" refers
to a ratio of a cross-sectional diameter to height of a pillar. In
the pillar structure, a ratio of the height of the pillars to a
distance between adjacent pillars may be in the range of 1:1 to
25:1. The distance between adjacent pillars may be in the range of
5 .mu.m to 100 .mu.m.
[0038] In another embodiment, cell during the contacting process,
the nonplanar solid substrate used during the contacting step can
be hydrophobic and can have a water contact angle of 70.degree. to
95.degree.. In one embodiment, the hydrophobic property of the
nonplanar solid substrate having a water contact angle of
70.degree. to 95.degree. can be obtained by coating
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS) on a solid
substrate. More specifically, the surface of a solid substrate
having a water contact angle of 70.degree. to 95.degree. can be
obtained by self-assembled molecule (SAM) coating
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS) on a SiO.sub.2
layer of the solid substrate.
[0039] In this application, the term "water contact angle" refers
to water contact angle measured by a Kruss prop Shape Analysis
System type DSA 10 Mk2. A droplet of 1.5 .mu.l deionized water is
automatically placed on the sample. The droplet was monitored every
0.2 seconds for a period of 10 seconds by a CCD-camera and analyzed
by prop Shape Analysis software (DSA version 1.7, Kruss). The
complete profile of the droplet was fitted by the tangent method to
a general conic section equation. The angles were determined both
at the right and left side. An average value is calculated for each
drop and a total of five drops per sample are measured. The average
of the five drops is taken the contact angle.
[0040] According to one embodiment, the nonplanar solid substrate
used during the contacting step can have at least one amine-based
functional group at its surface. The surface with the amine-based
functional group may be obtained by coating
polyethyleneiminetrimethoxysilane (PEIM) on a solid substrate. For
example, the coated surface can be obtained by self-assembled
molecule (SAM) coating polyethyleneiminetrimethoxysilane (PEIM) on
a SiO.sub.2 layer of the solid substrate.
[0041] In one embodiment of the method of obtaining a nucleic acid
from a cell, the nonplanar solid substrate, during the contacting
step, the nonplanar solid substrate can be a substrate formed of
any kind of material that has the water contact angle described
above, or has at least one amine-based functional group at its
surface. For example, the nonplanar solid substrate can be formed
of glass, silicon water, plastic, or the like, but is not limited
thereto. When a nonplanar solid substrate with a surface having a
water contact angle of 70.degree. to 95.degree. or a surface having
at least one amine-based functional group is contacted with a
sample containing a cell, it is assumed that the cell is bound to
the nonplanar solid substrate. However, the present invention is
not limited to such a specific mechanism.
[0042] According to the current embodiment, the method of obtaining
a nucleic acid from a cell may further include, after the
contacting step, washing the cells bound to the nonplanar solid
substrate by introducing a washing solution to the nonplaner solid
substrate to wash other materials which are not bound to the
nonplanar solid substrate, whereby the washing step does not remove
the target cell or target nucleic acid bound to the nonplaner solid
substrate. During the washing step, any solution that does not
liberate the target cell bound to the nonplanar solid substrate
from the nonplanar solid substrate and removes impurities that may
adversely affect subsequent processes can be used. For example, the
washing solution can be an acetate buffer or phosphate buffer,
which can also used as a binding buffer, can be used as the washing
solution. In one embodiment, the washing solution can have a pH of
3.0 to 6.0.
[0043] As used herein, "isolation of a microorganism cell" means
concentrating the cell in the sample as well as purely separating
the cell.
[0044] According to the current embodiment the method comprises
lysing the cell bound to the nonplanar solid substrate.
[0045] The step of lysing of the microorganism cell can be
performed using any lysing method known in the art. For example,
the lysis method can be boiling lysis, laser lysis, lysis using a
chemical material, or electrochemical lysis, such as electrolysis,
but is not limited thereto.
[0046] In the current embodiment, the cell can be lysed in any
liquid medium having a pH of 3.0 to 6.0 to bind a nucleic acid
derived from the cell to the nonplanar solid substrate. In one
embodiment, the lysis can be performed in a phosphate buffer or an
acetate buffer.
[0047] In the current embodiment, the nucleic acid derived from the
lysed microorganism cell is bound to the nonplanar solid substrate.
The bound nucleic acid can be isolated by removing cell debris, and
the like, which are not bound to the nonplanar solid substrate.
[0048] Accordingly, the method according to the current embodiment
further comprises, after the lysing step, washing the nonplanar
solid substrate with a washing buffer to remove materials which are
not bound to the nonplanar solid substrate.
[0049] During the washing step, the washing buffer can be any
solution which does not liberate the bound nucleic acid from the
nonplanar solid substrate and removes materials which are not bound
to the nonplanar solid substrate. More specifically, the washing
solution can be a solution having these properties described above
and which removes impurities that may adversely affect subsequent
processes. Therefore, the washing solution used to wash bound
nucleic acid can be the same as the wash buffer used to wash the
cells bound to the nonplaner solid substrate, but is not limited
thereto. In one embodiment, the washing buffer can be an acetate
buffer or phosphate buffer which can also be used as the binding
buffer. The washing buffer can be a buffer having a pH of 3.0 to
6.0.
[0050] In the current embodiment, the nucleic acid bound to the
nonplanar solid substrate can be used in its bound form, or the
bound nucleic acid can be extracted from the nonplanar solid
substrate.
[0051] Accordingly, the according to the current embodiment, the
method may further comprise extracting the nucleic acid bound to
the nonplanar solid substrate.
[0052] During the extracting step, the extracting solution may be
any solution known in the art which can liberate the nucleic acid
bound to the nonplanar solid substrate from the nonplanar solid
substrate. For example, the extracting solution can be a solution
having a high pH. Without being bound to theory, it is believed
that by using a solution having a high pH, hydroxide ions (OH--)
make the surface of the nonplanar solid substrate anionic such that
anionic DNA can be liberated from the nonplanar solid substrate. In
one embodiment, the extracting solution can be a solution having a
pH 11 or more, such as, for example, a NaOH solution.
[0053] According to another embodiment of the invention, the step
of lysing the cell in a solution of pH 11 to 14. Lysis of the cell
in a solution having a high pH can minimize the binding of DNA to a
nonplanar solid substrate so that DNA can be easily extracted. By
using a solution having a high pH, hydroxide ions (OH--) make the
surface of the nonplanar solid substrate anionic such that anionic
DNA can be liberated after the lysis is not bound to the nonplanar
solid substrate. That is, if the object is to bind a cell to a
nonplanar solid substrate and to extract DNA derived from the cell,
it is desired that the cell is lysed in a solution having a high
pH, followed by an extraction step performed using the same
solution having a high pH. Therefore, according to the current
embodiment, the method of isolating a nucleic acid from a cell can
further comprise purifying the nucleic acid from a lysate obtained
during the lysing step. The purifying method can be any method
known in the art.
[0054] In another embodiment, the present invention also provides a
method of amplifying a nucleic acid using as a template the nucleic
acid that is isolated using the method of isolating a nucleic acid
from a cell according to an embodiment of the present
invention.
[0055] The amplifying of a nucleic acid step can be performed using
any amplifying method known in the art, for example, using PCR. The
method of isolating a nucleic acid from a microorganism cell is
described above.
[0056] In the method of amplifying a nucleic acid, the nucleic acid
can be DNA or RNA, or a combination of both, and preferably
DNA.
[0057] According to the current embodiment, the nucleic acid can be
isolated and amplified in the same container that includes the
nonplanar solid substrate. The container can be a microchannel, a
microchamber, or a tube, but is not limited thereto. For example,
the container may be a microchamber of a microfluidic device which
is equipped with a PCR device. In one embodiment, the PCR device
includes a heater and a cooler. Therefore, in the method of
amplifying a nucleic acid according to the current embodiment, a
nucleic acid is extracted in a microchamber and the extracted
nucleic acid is amplified in the same microchamber.
[0058] In another embodiment of the method of amplifying a nucleic
acid, the isolation and amplification of the nucleic acid can be
performed in different containers. For example, the isolation of
the nucleic acid can be performed in a container that includes the
nonplanar solid substrate, and the amplification of the nucleic
acid can be performed in a different container that may or may not
in fluid communication with the container that includes the
nonplanar solid substrate. The different container can be a
microchannel, a microchamber, or a tube, but is not limited
thereto. For example, the isolation of the nucleic acid can be
performed in a microchannel or microchamber of a microfluidic
device, and the isolated nucleic acid is then transported to a
different microchannel or microchamber and extracted for
amplification.
[0059] In one embodiment, the invention comprises a device for
isolating and amplifying a nucleic acid The device for isolating
and amplifying nucleic acid comprises: a reaction chamber including
a nonplanar solid substrate; a heating unit which heats the
reaction chamber; and a temperature controlling unit which controls
the heating unit.
[0060] According to one embodiment, the device for isolating and
amplifying a nucleic acid comprises a solid substrate having a
nonplanar surface that has a greater surface area than a planar
solid substrate. The nonplanar solid substrate may have a
corrugated surface. In the present specification, the corrugated
surface refers to a non-level surface having grooves and ridges.
The corrugated surface can be a surface with a plurality of pillars
or a sieve-shaped surface with a plurality of pores. However, the
corrugated surface is not limited thereto, and may have other
shapes.
[0061] In one embodiment, the device for isolating and amplifying a
nucleic acid comprises a nonplanar solid substrate, which can have
various shapes. For example, the nonplanar solid substrate can be
selected from a solid substrate having a surface with a plurality
of pillars, a bead-shaped solid substrate, and a sieve-shaped solid
substrate having a plurality of pores in its surface. The solid
substrate can be a single solid substrate or a combination of solid
substrates, such as a solid substrate assembly which fills a tube
or container.
[0062] In one embodiment, the device for isolating and amplifying a
nucleic acid, the nonplanar solid substrate of the device for
isolating and amplifying a nucleic acid can form an inner wall of a
microchannel or microchamber of a microfluidic device. Accordingly,
the device for isolating and amplifying a nucleic can be a fluidic
device or microfluidic device having at least one inlet and outlet
connected through a channel or microchannel. That is, the device
for isolating and amplifying a nucleic acid according to the
current embodiment can be a fluidic device or microfluidic device
in which the isolation and PCR of a nucleic acid can be performed
in the same chamber.
[0063] According to the current embodiment of the present
invention, the nonplanar solid substrate can have a surface having
a plurality of pillars. Methods of forming pillars on a solid
substrate is well known in the art. For example, micro pillars can
be formed in a high density structure using a photolithography
process used in a semiconductor manufacturing process. The micro
pillars can have an aspect ratio of 1:1 to 20:1. However, the
aspect ratio of the micro pillars is not limited thereto. As used
herein, the term "aspect ratio" refers to a ratio of a
cross-sectional diameter to height of a pillar. In the pillar
structure, a ratio of the height of the pillars to a distance
between adjacent pillars may be in the range of 1:1 to 25:1. The
distance between adjacent pillars may be in the range of 5 .mu.m to
100 .mu.m.
[0064] According to the current embodiment, the nonplanar solid
substrate may be hydrophobic, having a water contact angle of
70.degree. to 95.degree.. In one embodiment, the hydrophobic
property of the nonplanar solid substrate having a water contact
angle of 70.degree. to 95.degree. can be obtained by coating
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS) on the nonplanar
solid substrate. More specifically, the nonplanar solid substrate
having a water contact angle of 70.degree. to 95.degree. can be
obtained by self-assembled molecule (SAM) coating
octadecyldimethyl(3-trimethoxysilyl propyl)ammonium (OTC) or
tridecafluorotetrahydrooctyltrimethoxysilane (DFS) on a SiO.sub.2
layer of the non planar solid substrate.
[0065] According to the current embodiment of the present
invention, the nonplanar solid substrate can have at least one
amine-based functional group at its surface. The surface with the
amine-based functional group may be obtained by coating
polyethyleneiminetrimethoxysilane (PEIM) on the nonplanar solid
substrate. For example, the coated surface can be obtained by
self-assembled molecule (SAM) coating
polyethyleneiminetrimethoxysilane (PEIM) on a SiO.sub.2 layer of
the nonplanar solid substrate.
[0066] In the device for isolating and amplifying a nucleic acid
according to the current embodiment, the heating unit can be any
device known in the art which can be used to heat the chamber. The
heating unit can be a heater or a micro heater.
[0067] In the device for isolating and amplifying a nucleic acid
according to the current embodiment, the temperature controlling
unit can be any controller known in the art which can control the
heating unit to generate a temperature cycle used in PCR. The
temperature controlling unit can include a temperature sensor that
senses the temperature of the chamber and a device that controls
on/off operation of the heating unit.
[0068] The present invention will now be described in further
detail with reference to the following examples. These examples are
for illustrative purposes only and are not intended to limit the
scope of the present invention.
EXAMPLES
Example 1
Binding Properties of DNA to Solid Substrate Having Pillar
Structure
[0069] In the current example, binding properties of DNA were
determined by loading a DNA sample into a fluidic device including
an inlet, an outlet, and a chamber having a pillar array on a 10
mm.times.23 mm substrate. In the pillar array, the distance between
adjacent pillars was 12 .mu.m, the height of each pillar was 100
.mu.m, and a sectional surface of each pillar was a regular square
with sides of 25 .mu.m.
[0070] In the pillar array, each pillar had a surface coated with
PEIM having at least one amine-based functional group, or a surface
coated with OTC having a water contact angle of 80.degree..
Coatings of OTC having a water contact angle of 70.degree. to
95.degree. give similar results.
[0071] DNA in 0.01N NaOH (pH 12) or DNA in 100 mM NaH.sub.2PO.sub.4
(pH 7.0) was used as a DNA sample. A volume of 200 .mu.l of the DNA
sample was pumped through a fluidic device at a flow rate of 200
.mu.l/minute.
[0072] The DNA bound to the pillar array in the fluidic device was
measured using a PICOGREEN.RTM. kit (obtained from Molecular Probes
Inc) and a spectroscope according to the instructions provided by
the manufacturer.
[0073] FIG. 1 is a graph showing fluorescence intensity as a
function of the concentration of DNA in NaOH solution of pH 12.0 or
in a phosphate buffer of pH 7.0. Referring to FIG. 1, the graph
demonstrates that the concentration of DNA is proportional to the
fluorescence intensity, and thus it was found that the
concentration of DNA could be determined using an equation based on
the proportional relation between a fluorescence intensity and DNA
concentration.
[0074] FIG. 2 is a graph showing the degree of DNA collection as a
function of pH of the DNA sample after the DNA sample was pumped
through a fluidic device with a pillar array coated with OTC having
a water contact angle of 80.degree.. Referring to FIG. 2, when DNA
was in NaOH solution of pH 12.0, 95% or more of the DNA was
re-collected, that is, only 5% or less of the DNA in the sample
bound to the substrate surface. On the other hand, when DNA was in
a phosphate buffer of pH 7.0, about 40% or less of the DNA was
re-collected, with 60% or more of the DNA bound to the substrate
surface. These results show that when the pH of the sample solution
is high, such as in the range of 11 to 14, DNA does not bind
efficiently to the solid substrate, but when pH of the sample
solution is relatively low, such as in the range of 3 to 8,
preferably 3 to 6, DNA is efficiently bound to the solid substrate.
Accordingly, it was determined that by using solutions having
different pH values, DNA can be easily bound to or eluted from a
solid substrate having a pillar structure coated with PEIM having
at least one amine-based functional group or coated with OTC having
a water contact angle of 70.degree. to 95.degree.. That is, binding
of DNA to a solid substrate should be performed in low pH, and
eluting of DNA from the solid substrate should be performed in high
pH.
Example 2
Binding of a Cell to a Solid Substrate with a Pillar Array in the
Fluidic Device, Cell Lysis, and DNA Elution
[0075] In this example, a cell sample was loaded into a fluidic
device including an inlet, an outlet, and a chamber having a pillar
array formed on a 10 mm.times.23 mm substrate, and then the cell
was lysed to elute DNA. In the pillar array, a distance between
adjacent pillars was 12 .mu.m, the height of each pillar was 100
.mu.m, and a sectional surface of each pillar was a regular square
having sides of 25 .mu.m.
[0076] The pillar array had a surface coated with OTC having a
water contact angle of 80.degree..
[0077] The cell sample was E. coli in LB medium (pH 7.2), with an
OD.sub.600 value of 0.01. 250 .mu.l of the cell sample was pumped
through the fluidic device at a flow rate of 200 .mu.l/minute.
[0078] Subsequently, E. coli that was bound to the surface of the
pillar array was lysed. The cell lysis was performed in one of
three ways: (1) by pumping through 50 .mu.l of 0.01N NaOH aqueous
solution (pH 12.0) to the chamber (chamber volume 5 ul) at a flow
rate of 25 .mu.l/minute for 2 minutes (hereinafter referred to as
0.01N NaOH 2), (2) by pumping 50 .mu.l of 0.01N NaOH solution to
the chamber at a flow rate of 5 .mu.l/minute for 10 minutes
(hereinafter referred to as 0.01N NaOH.sub.--10), or (3) by loading
3.5 .mu.l of 0.01N NaOH to the chamber at a flow rate of 200
.mu.l/minute, and then raising the temperature to 100.degree. C.
and maintained for 2 minutes, and then pumping through 46.5 .mu.l
of 0.01N NaOH solution at a flow rate of 200 .mu.l/minute
(hereinafter referred to as 0.01N NaOH/boiling 3).
[0079] 40 .mu.l of the resulting lysate obtained by the cell lysis
was collected and the concentration of DNA therein was measured
using a PICOGREEN.RTM. kit (obtained from Molecular Probes Inc) and
a spectroscope. The DNA elution efficiency was measured using the
following formula: (fluorescence intensity of the
sample/fluorescence intensity when 100% of the cells are
lysed).times.100.
[0080] A reference plot for determining DNA concentration in the
cell lysates was obtained by lysing samples containing E. coli
having a known concentration using NaOH in a tube, measuring the
fluorescence intensity of the lysed sample, and generating a
standard curve of the fluorescence intensity as a function of the
cell concentration.
[0081] FIG. 3 is a graph showing fluorescence intensity of DNA
present in a cell lysate as a function of the concentration of the
cells lysed. Referring to FIG. 3, the fluorescence intensity of the
cell lysate is proportional to the concentration of the cells.
[0082] The expected DNA concentration when 100% of the cells were
lysed was obtained by following conversion process. The amount of
input cells=10.sup.7 cells/ml (assuming 1.0 D.sub.600 as 10.sup.9
cell/ml).times.0.25 ml=2.5.times.10.sup.6 cells, (assuming that the
binding efficiency was 100%). Therefore, the expected maximum
amount of eluted DNA=2.5.times.10.sup.6 cells.times.5 fg DNA/E.
coli cell=12.5 ng. Then, the amount of eluted DNA was divided by
about 50 .mu.l, which was the entire volume of NaOH used as the
eluting solution. As a result, the concentration of DNA obtained
from the total cells was 0.25 ng/.mu.l.
[0083] FIG. 4 is a graph illustrating DNA elution efficiency
measured according to a method in which an E. coli-containing
sample was pumped through a fluidic device including a chamber
having a octadecyldimethyl (3-trimethoxysilyl propyl)ammonium
(OTC)-coated surface and a pillar array, the bound cells from the
sample were lysed by one of the three methods described above, and
then the resultant cell lysate was collected. As illustrated in
FIG. 4, DNA elution efficiency was highest when the DNA was eluted
after using the method designated NaOH/boiling 3. When 0.01N
NaOH/boiling.sub.--3 was performed, 32% of DNA was collected,
assuming 1.0 D.sub.600 represents 10.sup.9 cell/ml. As such, it was
found that the DNA collection rate after the cell lysis was in the
range of 30-60% based on the total DNA present in the cells, even
if considering a cell concentration correspond to 1.0 OD600 value
varies, generally 5.times.10.sup.8 to 10.sup.9 cell/ml.
Example 3
Binding of Cells to a Solid Substrate with a Pillar Array in a
Fluidic Device, Cell Lysis, and DNA Purification and
Amplification
[0084] In the current example, a cell sample was pumped through a
fluidic device including an inlet and an outlet and having a pillar
array on a 7.5 mm.times.15 mm substrate, the cells were lysed to
bind DNA derived from the cells to the substrate, materials that
were not bound to the substrate were removed by washing, and DNA
amplification was performed using the DNA bound to the substrate as
a template. In the pillar array, a distance between adjacent
pillars was 15 .mu.m, the height of each pillar was 100 .mu.m, and
a sectional surface of each pillar was a regular square having
sides of 25 .mu.m.
[0085] The pillar array had a surface coated with OTC having a
water contact angle of 80.degree..
[0086] The cell sample was an E. coli-containing sample of 0.01
OD.sub.600 in a LB medium. The E. coli-containing sample was
adjusted to have a pH of 4.0 by using 100 mM sodium acetate buffer,
and was pumped into a fluidic device from the inlet to the outlet
through a chamber at a flow rate of 100 .mu.l/minute for five
minutes.
[0087] Then, E. coli bound to the surface of the pillar array was
lysed. More specifically, the chamber was filled with 100 mM
phosphate buffer (pH 4.0), treated at 95.degree. for 2 minutes, and
then the temperature was decreased to room temperature. This lysis
step was repeated five times. After cell lysis, impurities which
were not bound to the surface of the pillar array were washed using
200 .mu.l of 100 mM phosphate buffer (pH 4.0) at a flow rate of 200
.mu.l/minute.
[0088] Subsequently, PCR was performed using DNA bound to the
surface of the pillar array of the solid substrate as a template
for a TAQMAN.RTM. probe.
[0089] Table 1 shows a Ct value of real time PCR amplification of
DNA obtained from an E. coli-containing sample which was pumped
through the chamber having a pillar array of a fluidic device.
TABLE-US-00001 TABLE 1 Sample No. Treatment Ct 1 dPCR1 14.14 2
dPCR2 14.13 3 Purified PCR1 13.29 4 Purified PCR2 14.63 5 0.01 OD
cell 25.72 6 Negative Control group 25.23-
[0090] In Table 1, purified PCR refers to a PCR using an E.
coli-containing sample that was lysed and washed (samples 3 and 4
of Table 1 or Lanes 3 and 4 of the electrophoresis gel depicted in
FIG. 5), while dPCR refers to a PCR using an E. coli-containing
sample that was not lysed and washed (samples 1 and 2 of Table 1 or
Lanes of 1 and 2 of the electrophoresis gel shown in FIG. 5). 0.01
OD cell refers to a PCR using 0.01 OD E. coli cells in a LB medium
that was not flew the microfluidic device The threshold cycle (Ct)
is the cycle number in the PCR at which the reporter dye emission
intensity rises above background noise. The Ct is inversely
proportional to the copy number of the target template; the higher
the template concentration, the lower the threshold cycle measured.
In the present invention, the Ct is practically defined as a cycle
number at the reflection point of real time PCR product curve.
[0091] As shown in Table 1, in the case when the E. coli-containing
sample (at OD600=0.01) was pumped through the chamber, underwent
cell lysis, washing, and subsequent real time PCR amplification
(samples 3 and 4), an increase in fluorescence intensity above the
baseline was detected around 15 PCR cycles, while when a 0.01 OD600
reference E. coli-containing sample was used in a PCR as template,
Ct was observed at around 25 (sample 5), thus DNA was concentrated
about 1,000 times (that is, .times.2.sup.10) by the isolation
process. As shown in Table 1, the E. coli cells which underwent
binding, cell lysis, washing, and PCR (samples 3 and 4) showed
similar Ct value to the E. coli which was bound and underwent PCR
without a separate cell lysis process (samples 1 and 2). Such
results may result from high concentration and purification effect
at a cell level. That is, it is assumed that the purification
effect at a DNA level may be negligible. The negative control
(sample 6) was sample without containing E. coli cell.
[0092] FIG. 5 is an electrophoresis gel showing amplified DNA
obtained according to a method in which the E. coli-containing
sample was pumped through the chamber, and subsequently underwent
cell lysis, cell washing, real time PCR amplification, and
electrophoresis. Referring to FIG. 5, Lanes 1 and 2 show results
obtained when the E. coli was bound and then underwent PCR without
a separate cell lysis process (samples 1 and 2 of Table 1), Lanes 3
and 4 shows results obtained when E. coli was bound and underwent
cell lysis and PCR (samples 3-4 of Table 1), and Lane 6 shows
results obtained from PCR using a 0.01 OD.sub.600 E.
coli-containing sample as a template (sample 5 of Table 1). Lane 5
presents sample without containing E. coli cells. The concentration
of the target PCR product is shown in Table 2. The Lapchip
instrument (Agilent) was used to measure the DNA concentration
automatically. The concentration of the target PCR product
increased by 70% or more when cell lysis was performed on the bound
E. coli sample even though the two types of samples showed similar
Ct values. TABLE-US-00002 TABLE 2 PCR using 0.01 OD Cell binding/
E. coli- Cell Lysis/ Cell containing Washing/PCR Binding/PCR sample
DNA concentration (ng/.mu.l) 24.45 17.35 7.5 Increase rate compared
to 226% 131% 1 when PCR using 0.01 OD E. coli-containing sample was
performed
Example 4
Binding of Cells from a Clinical Mimic Sample to a Solid Substrate
with a Pillar Array in a Fluidic Device, Followed by Cell Lysis,
DNA Purification, and Amplification
[0093] In the current example, a urine sample containing E-coli was
loaded into a fluidic device including an inlet and an outlet and
having an pillar array on a 7.5 mm.times.15 mm substrate, the cell
was lysed and DNA derived from the cell was bound to the substrate,
materials that were not bound to the substrate were removed by
washing, and DNA was amplified using the DNA bound to the substrate
as a template. In the pillar array, a distance between adjacent
pillars was 15 .mu.m, the height of each pillar was 100 .mu.m, and
a sectional surface of each pillar was a regular square having
sides of 25 .mu.m.
[0094] The pillar array had a surface coated with OTC having a
water contact angle of 80.degree..
[0095] The cell sample was a urine sample which was diluted with a
sodium acetate buffer (pH 4.0) in a 4:1 ratio and contained E. coli
at a concentration yielding a measured value of 0.01 OD.sub.600.
The E. coli-containing urine sample was pumped into the fluidic
device from the inlet to the outlet through a chamber at a flow
rate of 100 .mu.l/minute for five minutes. The reference sample
used was a 0.01 OD.sub.600 E. coli-containing sample in sodium
acetate buffer (pH 4).
[0096] Then, E. coli bound to the surface of the pillar array was
lysed. More specifically, the chamber was filled with 100 mM
phosphate buffer (pH 4.0), treated at 95.degree. C. for 2 minutes,
and then the temperature was decreased to room temperature. The
lysis process was repeated five times. After the cell lysis,
impurities which were not bound to the surface of the pillar array
were washed using 200 .mu.l of 100 mM phosphate buffer (pH 4.0) at
a flow rate of 200 .mu.l/minute. Subsequently, PCR was performed
using DNA bound to the surface of the pillar array of the solid
substrate as a template and SYBR.RTM. Green.
[0097] FIG. 6 is a graph illustrating results of the real time PCR
amplification of various E. coli-containing urine samples pumped
through the chamber. Referring to FIG. 7, samples according to
Table 3 were used. TABLE-US-00003 TABLE 3 Sample No. Treatment Ct 1
Purified PCR1 11.43 2 Purified PCR2 11.21 3 dPCR1 11.94 4 dPCR2
13.16 5 Negative Control group 1 23.76 6 Negative Control group 2
23.38
[0098] In Table 3, purified PCR refers to a PCR using a
lysed/washed sample and dPCR refers to a PCR using a sample that
was not lysed or washed. In the Negative Control groups 1 and 2,
PCR was conducted using the 0.01 OD E. coli-containing sample as
template, without loading into the fluidic device and undergoing
the process of concentrating and purifying the cells in the urine
sample).
[0099] Referring to FIG. 6, the Ct value was about one cycle lower
(1.23) when an E. coli-containing urine sample was loaded through
the chamber, and subsequently underwent cell lysis, cell washing,
and real time PCR than when an E. coli-containing urine sample was
loaded through the chamber, and subsequently underwent real time
PCR without cell lysis or washing.
Comparative Example 4
Binding of Cells in a Clinical Mimic Sample to a Solid Substrate
with a Pillar Array in a Fluidic Device, Cell Lysis, and DNA
Elution and Amplification
Comparative Example with Respect to Commercially Available DNA
Purification Kit
[0100] In the current example, an E. coli-containing whole blood
sample was loaded into a fluidic device including a 10 mm.times.23
mm substrate with a pillar array, and then subsequently underwent
cell lysis to elute DNA from the substrate. Then, the eluted DNA
was used as a template for amplification. DNA purified by the
disclosed method was compared to DNA purified using a commercially
available DNA purification kit produced by Qiagen Inc.
[0101] In the pillar array, the distance between adjacent pillars
was 12 .mu.m, the height of each pillar was 100 .mu.m, and the
sectional surface of each pillar was a regular square having sides
of 25 .mu.m. The pillar array had a surface coated with OTC having
a water contact angle of 80.degree..
[0102] A clinical mimic cell sample containing E. coli was prepared
by adding 10 .mu.l of E. coli (1.0 OD.sub.600) to 1 ml of whole
blood. 200 .mu.l of the clinical mimic cell sample containing E.
coli was diluted with sodium acetate buffer (pH 3.0, 100 mM) in a
1:1 ratio and then pumped into the chamber of the fluidic device at
a flow rate of 200 .mu.l/minute. E. coli cells bound to the surface
of the pillar array was lysed. In order to perform the cell lysis,
5 .mu.l of 0.01N NaOH was added to the chamber and then boiled for
2 minutes. Subsequently, 45 .mu.l of 0.01N NaOH was added to the
chamber at a flow rate of 200 .mu.l/min to elute the DNA from the
lysate.
[0103] As a control sample, 200 .mu.l of the clinical mimic sample
was prepared using a Blood & Cell Culture DNA Mini Kit (Qiagen
Inc., Cat 13323) according to the protocol provided by the
manufacturer.
[0104] Then, DNA purified from the clinical mimic cell sample
containing E. coli using the method of the invention ("SAIT") or
purified using the commercial control ("Qiagen") were subjected to
PCR using SYBRO.RTM. Green to detect amplification. The
amplification results for the samples are summarized in Table 4.
The concentration of the PCR product was determined from the
electrophoresis analysis illustrated in FIG. 8. The target bands
are located between ladder 150 and 200. As shown in Table 4, the
two samples (SAIT and Qiagen samples) showed similar Ct values and
PCR product concentration. That is, it was found that the quality
of DNA prepared according to the method of the present invention
was high enough to undergo PCR, not requiring additional DNA
purification. These methods, however required different times to
prepare DNA. The method of the present invention required about 10
minutes, while the Qiagen method required about 45 minutes. In
addition, the method of the present invention can be easily
automated because cell capture and DNA elution can be performed in
a single chip. Therefore, the method of the present invention can
be implemented on a lap-on-a-chip (LOC). In Table 4, the sample
labeled "Negative Control Group" represents a sample for which PCR
was conducted using the E. coli-containing whole blood sample as
the template, without undergoing any purification process.
TABLE-US-00004 TABLE 4 Chip Ct Concentration (ng/.mu.l) SAIT1 18.7
11.8 SAIT2 18.9 13.1 Qiagen1 18.4 13.0 Qiagen2 17.8 11.5 Negative
Control Group 28.8 --
[0105] FIG. 7 is a graph illustrating the results of real time PCR
amplification for the samples listed in Table 4.
[0106] FIG. 8 shows the g results of electrophoresis of PCR
products generated with the samples listed in Table 4.
[0107] In summary, in the method of isolating a nucleic acid from a
cell according to the present invention, isolation of a cell and
isolation of a nucleic acid can be efficiently performed at the
same time. In addition, a nucleic acid separator, such as a
chaotropic substance, is not required for isolation of the nucleic
acid. Furthermore, the method can be usefully realized using a
small device, such as lap-on-a-chip (LOC).
[0108] Further, in the method of amplifying DNA according to the
present invention, isolation of a cell and isolation of a nucleic
acid derived from the cell can be performed in rapid succession so
that the nucleic acid can be effectively amplified using a small
device, such as a LOC.
[0109] In addition, by using a device for isolating and amplifying
a nucleic acid according to the present invention, isolation of a
cell, isolation of a nucleic acid derived from the cell, and
amplification of the nucleic acid derived from the cell can be
performed in the same container or in different containers.
[0110] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. The terms "a" and "an" do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item. The term "or" means "and/or". The terms
"comprising", "having", "including", and "containing" are to be
construed as open-ended terms (i.e., meaning "including, but not
limited to").
[0111] Recitation of ranges of values are merely intended to serve
as a shorthand method of referring individually to each separate
value falling within the range, unless otherwise indicated herein,
and each separate value is incorporated into the specification as
if it were individually recited herein. The endpoints of all ranges
are included within the range and independently combinable.
[0112] All methods described herein can be performed in a suitable
order unless otherwise indicated herein or otherwise clearly
contradicted by context. The use of any and all examples, or
exemplary language (e.g., "such as"), is intended merely to better
illustrate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention as used
herein. Unless defined otherwise, technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs.
[0113] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
While the present invention has been particularly shown and
described with reference to exemplary embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the present invention as defined by
the following claims.
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