U.S. patent application number 13/562959 was filed with the patent office on 2012-11-22 for rotational pcr equipment and pcr method using the same.
This patent application is currently assigned to KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY (KAIST). Invention is credited to Seok Jin Chol, Jae Hwan Jung, Tae Seok Seo.
Application Number | 20120295312 13/562959 |
Document ID | / |
Family ID | 44710118 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120295312 |
Kind Code |
A1 |
Seo; Tae Seok ; et
al. |
November 22, 2012 |
ROTATIONAL PCR EQUIPMENT AND PCR METHOD USING THE SAME
Abstract
A rotational PCR apparatus, a PCR chip for the same and a
rotational PCR method using the same. The rotational PCR apparatus
includes: a PCR chip where PCR is performed; a rotating means
connected to the PCR chip and rotating the PCR chip; and a
temperature zone forming means spaced apart from the PCR chip,
capable of applying thermal energy to the PCR chip and allowing the
rotating PCR chip to pass through different temperature zones. The
rotational PCR apparatus and method allow performance of PCR with
wanted temperature condition and cycles by rotating the chip
containing the target substance. Accordingly, a high-efficiency PCR
process may be accomplished at low cost. Further, since the target
substance can be effectively separated and purified utilizing the
centrifugal force resulting from the rotating platform, separation
and purification may be achieved economically without requiring
additional equipments.
Inventors: |
Seo; Tae Seok; (Daejeon,
KR) ; Jung; Jae Hwan; (Daejeon, KR) ; Chol;
Seok Jin; (Daejeon, KR) |
Assignee: |
KOREA ADVANCED INSTITUTE OF SCIENCE
AND TECHNOLOGY (KAIST)
Daejeon
KR
|
Family ID: |
44710118 |
Appl. No.: |
13/562959 |
Filed: |
July 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12965585 |
Dec 10, 2010 |
|
|
|
13562959 |
|
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|
Current U.S.
Class: |
435/91.2 |
Current CPC
Class: |
B01L 2400/0677 20130101;
B01L 3/502761 20130101; B01L 2300/0819 20130101; B01L 2300/161
20130101; B01L 2300/0867 20130101; B01L 2300/0864 20130101; B01L
7/5255 20130101; B01L 2400/088 20130101; B01L 2300/0803 20130101;
B01L 2400/0409 20130101 |
Class at
Publication: |
435/91.2 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2010 |
KR |
10-2010-0028294 |
Aug 17, 2010 |
KR |
10-2010-0079474 |
Claims
1. A PCR method using a PCR chip, comprising: performing PCR by
allowing a PCR chip containing a target substance to pass through a
plurality of temperature zones at different temperatures.
2. The PCR method according to claim 1, wherein the plurality of
temperature zones are arranged such that temperatures required for
PCR are repeated.
3. The PCR method according to claim 1, which comprises: performing
pretreatment of separating the target substance from a sample
solution by sequentially flowing the sample solution, a washing
buffer and an elution buffer from a pretreatment unit of the PCR
chip to silica beads; introducing the separated target substance
into a PCR unit connected at the rear end of the pretreatment unit;
and performing PCR by rotating the target substance introduced into
the PCR unit through a plurality of temperature zones.
4. The PCR method according to claim 3, wherein said performing
pretreatment comprises: rotating the chip at or above a first
speed, so that the sample solution flows from a sample chamber to
the silica beads; rotating the chip at or above a second speed, so
that the washing buffer flows to the silica beads; and rotating the
chip at or above a third speed, so that the elution buffer flows to
the silica beads.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of co-pending
U.S. application Ser. No. 12/965,585, filed Dec. 10, 2010, the
disclosure of which is incorporated herein by reference. This
application claims priority benefits under 35 U.S.C. .sctn.119 to
Korean Patent Application No. 10-2010-0079474 filed on Aug. 17,
2010 and Korean Patent Application No. 10-2010-0028294 filed on
Mar. 30, 2010, the disclosures of which are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to a rotational PCR
apparatus, a PCR chip for the same and a rotational PCR method
using the same. More particularly, the disclosure relates to a
rotational PCR apparatus capable of performing PCR processes under
desired temperature conditions by rotating a PCR chip and capable
of effectively performing separation and purification of sample by
rotating the chip, the PCR chip for the same and a rotational PCR
method using the same.
BACKGROUND
[0003] DNA amplification techniques are widely utilized in the
fields of bioscience, genetic engineering and medicine for the
purposes of research, development and diagnosis. Especially, the
DNA amplification technique based on polymerase chain reaction
(PCR) is widely employed. PCR is used to amplify a particular DNA
sequence as desired. The first step of PCR is to denature DNA. A
double-stranded DNA is split by heating. Each separated DNA strand
serves as a template. The second step of PCR is annealing. In this
step, primers are annealed to the template DNA. The annealing
temperature is an important factor determining the accuracy of the
reaction. If the temperature is too high, the quantity of amplified
DNA products decreases drastically because the primers are too
weakly bound to the template DNA. And, if the temperature is too
low, unwanted DNA may be amplified due to nonspecific binding of
the primers. The third PCR step of PCR is elongation. At this step,
a thermostable DNA polymerase synthesizes new DNA from the template
DNA. The PCR may be classified into DNA PCR and RNA PCR. Usually,
the purpose of amplifying genes by PCR is to observe particular
sequences in the genes, not the entire genes. In such PCR
techniques, it is very important to form accurate temperature
gradients for the respective PCR steps and to maintain them.
SUMMARY
[0004] The present disclosure is directed to providing a rotational
PCR apparatus which performs PCR by rotating a chip including a
target sample to be analyzed and the PCR chip for the same.
[0005] The present disclosure is also directed to providing a
rotational PCR method allowing to perform pretreatment of the
target sample and PCR on the same platform.
[0006] In one general aspect, the present disclosure provides a PCR
apparatus including: a PCR chip where PCR is performed; a rotating
means connected to the PCR chip and rotating the PCR chip; and a
temperature zone forming means spaced apart from the PCR chip,
capable of applying thermal energy to the PCR chip and allowing the
rotating PCR chip to pass through different temperature zones.
[0007] In another general aspect, the present disclosure provides a
PCR method using a PCR chip, comprising: performing PCR by allowing
a PCR chip containing a target substance to pass through a
plurality of temperature zones at different temperatures.
[0008] The present disclosure also provides a PCR method using a
PCR chip having a pretreatment unit and a PCR unit, which includes:
performing pretreatment of separating the target substance from a
sample solution by sequentially flowing the sample solution, a
washing buffer and an elution buffer from the pretreatment unit of
the PCR chip to silica beads; introducing the separated target
substance into the PCR unit connected at the rear end of the
pretreatment unit; and performing PCR by rotating the target
substance introduced into the PCR unit through a plurality of
temperature zones.
[0009] Other features and aspects will be apparent from the
following detailed description, the drawings, and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The above and other objects, features and advantages of the
present disclosure will become apparent from the following
description of certain exemplary embodiments given in conjunction
with the accompanying drawings, in which:
[0011] FIG. 1 is a front view of a temperature zone forming means
100 according to an embodiment of the present disclosure;
[0012] FIGS. 2 to 4 are perspective views of a PCR apparatus
comprising the temperature zone forming means 100 of FIG. 1 and a
PCR chip 220;
[0013] FIG. 5 is a front view of a PCR apparatus according to
another embodiment of the present disclosure;
[0014] FIG. 6 is a partial schematic view of a pretreatment unit of
a PCR chip according to an embodiment of the present
disclosure;
[0015] FIG. 7 shows configurations of solution chambers and
hydrophobic channels according to an embodiment of the present
disclosure;
[0016] FIG. 8 is a perspective view of an integrated PCR chip
according to an embodiment of the present disclosure;
[0017] FIGS. 9 and 10 are cross-sectional views of a pretreatment
unit of a PCR chip according to an embodiment of the present
disclosure; and
[0018] FIG. 11 is a flow diagram of a PCR method using a PCR chip
according to another embodiment of the present disclosure.
[0019] It should be understood that the appended drawings are not
necessarily to scale, presenting a somewhat simplified
representation of various preferred features illustrative of the
basic principles of the disclosure. The specific design features of
the disclosure as disclosed herein, including, for example,
specific dimensions, orientations, locations and shapes, will be
determined in part by the particular intended application and use
environment.
[0020] In the figures, reference numerals refer to the same or
equivalent parts of the disclosure throughout the several figures
of the drawings.
DETAILED DESCRIPTION OF EMBODIMENTS
[0021] The advantages, features and aspects of the present
disclosure will become apparent from the following description of
the embodiments with reference to the accompanying drawings, which
is set forth hereinafter. The present disclosure may, however, be
embodied in different forms and should not be construed as limited
to the embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the present disclosure to those
skilled in the art. The terminology used herein is for the purpose
of describing particular embodiments only and is not intended to be
limiting of the example embodiments. As used herein, the singular
forms "a", "an" and "the" are intended to include the plural forms
as well, unless the context clearly indicates otherwise. It will be
further understood that the terms "comprises" and/or "comprising",
when used in this specification, specify the presence of stated
features, integers, steps, operations, elements, and/or components,
but do not preclude the presence or addition of one or more other
features, integers, steps, operations, elements, components, and/or
groups thereof.
[0022] The present disclosure provides a PCR apparatus and a PCR
method allowing a chip (hereinafter, "PCR chip") containing a
target substance (i.e., DNA or RNA) of PCR to pass through a
plurality of temperature zones at different temperatures. For this,
the PCR chip is rotated to pass thorough the temperature zones
arranged in circular shape.
[0023] As used herein, the term "temperature gradient zone" or
"temperature zone" refers to a spatial region where a specific
temperature is maintained. In an embodiment of the present
disclosure, the temperature zones may be formed by a heating metal
block, as a temperature zone forming means, spaced apart above
and/or below from the PCR chip and capable of applying thermal
energy to the PCR chip. In another embodiment of the present
disclosure, a light source capable of generating light energy such
as infrared rays may be used as the temperature zone forming means.
However, the present disclosure is not limited to those examples,
and any means capable of applying a certain level of thermal energy
to the rotating PCR chip may be used as the temperature zone
forming means, which is included within the scope of the present
disclosure.
[0024] Hereinafter, exemplary embodiments of the present disclosure
will be described in detail with reference to the accompanying
drawings.
[0025] FIG. 1 is a front view of a temperature zone forming means
100 according to an embodiment of the present disclosure. FIG. 2 is
a perspective view of a PCR apparatus comprising the temperature
zone forming means 100 of FIG. 1 and a PCR chip 220, FIG. 3 is a
perspective view of a chip module comprising three PCR chips, and
FIG. 4 is a perspective view of a PCR chip module coupled with a
rotating means.
[0026] Referring to FIG. 1, the temperature zone forming means of
the PCR apparatus according to this embodiment may be in the form
of a wheel or disc comprising a plurality of heating metal blocks
(hereinafter, "heating blocks") the temperature of which is
independently controllable. Between the heating blocks 100a, 100b,
100c, an insulator 100d and/or a cooling block for preventing
thermal conduction between the heating blocks may be provided. In
an embodiment of the present disclosure, heat may be applied to the
heating blocks by means of an electric heater using a resistor, but
the scope of the present disclosure is not limited thereto.
[0027] The temperature zone forming means 100 comprises a plurality
of independent heating means (i.e., the heating blocks). By varying
the temperature condition of the heating blocks, a rotating PCR
chip spaced apart from the heating blocks may be heated at
different temperatures.
[0028] Referring to FIGS. 2 and 4, the PCR apparatus comprises the
rotating means to rotate the PCR chip of FIG. 3. In an embodiment
of the present disclosure, the apparatus may comprise a motor (not
shown) and a shaft 230, which is connected to the motor and
rotates, as the rotating means.
[0029] In an embodiment of the present disclosure, one or more
rotating PCR chip (s) 220 rotating as the shaft rotates may be
coupled with the shaft 230. In FIG. 3, a chip module comprising
three PCR chips 220a, 220b, 220c is illustrated.
[0030] In the PCR chip 220, a PCR cocktail solution containing a
target sample (e.g., DNA or RNA) to be amplified, a primer, etc.
flows. For this, the PCR chip 220 may be equipped with an inlet and
an outlet through which the sample solution is introduced and
discharged. A chamber unit wherein PCR occurs may be provided
between the inlet and the outlet.
[0031] A heating block 210 in the form of a disc, which is the
temperature zone forming means, is provided spaced apart from the
PCR chip 220. One or more of the temperature zone forming means 210
may be provided for one PCR apparatus. In an embodiment, two
temperature zone forming means 210 may face each other with the
chip therebetween. However, the scope of the present disclosure is
not limited thereto.
[0032] In an embodiment of the present disclosure, the heating
block may comprise at least one heating block group(s) comprising
three unit heating blocks so as to form at least three temperature
zones. It is because one cycle of a PCR procedure passes through
three temperature steps, in general. In an embodiment of the
present disclosure, three temperature zones are formed by the
plurality of heating blocks, at temperatures 95.degree. C.,
72.degree. C. and 55.degree. C., respectively. As the rotating PCR
chip passes through the temperature zones, PCR occurs under the
corresponding temperature conditions. Although the number of the
temperature gradient zones formed by the heating block shown in the
embodiment with reference to FIGS. 2 to 4 is three, it may be
increased further. In this case, a plurality of PCR cycles may
occur while the PCR chip rotates 360.degree.. Thus, a plurality of
PCR cycles may be performed at once simply by rotating a plurality
of PCR chips once.
[0033] FIG. 5 is a front view of a PCR apparatus according to
another embodiment of the present disclosure.
[0034] Referring to FIG. 5, a temperature zone forming means 310 of
a PCR apparatus according to this embodiment comprises a plurality
of heating block groups comprising three unit heating blocks. A
first heating block group 310a, 310b, 310c is controlled under a
temperature condition corresponding to one PCR cycle and a second
heating block group 310d, 310e, 310f is also controlled under the
same temperature condition corresponding to one PCR cycle. The
remaining heating block groups 310g to 310l are also controlled
under the same temperature condition.
[0035] As a PCR chip rotates from 310a to 310l, the sample in the
PCR chip passes through four PCR cycles. If a plurality of PCR
chips are rotated from 310a, 310d, 310g and 310j (These correspond
to the temperature zones where PCR is initiated.), four samples
pass through four PCR cycles with just one rotation. As such, the
present disclosure provides a highly efficient chip-based PCR
apparatus.
[0036] The PCR apparatus according to the present disclosure also
provides a new concept of performing separation and purification of
the sample (sample pretreatment), which is required for PCR
analysis, on the same platform using the centrifugal force
occurring as the PCR chip rotates.
[0037] In general, a pretreatment process of separating and
purifying the target substance, e.g. DNA or RNA, is required for a
PCR procedure. Usually, a solid-phase capture approach using a
capturing means capable of selectively capturing the target sample
only, for example, silica beads, is employed. This pretreatment
method comprises: a first step of flowing a sample containing the
target substance to be captured to a capturing means (e.g., silica
beads) so as to adsorb the target substance onto the silica beads;
a second step of removing components other than the target
substance to be amplified from the capturing means by washing; and
a third step of separating the target substance captured by the
capturing means. In general, the second step is performed by
flowing a washing buffer to the silica beads, and the third step is
performed by flowing an elution buffer to the silica beads.
[0038] In a PCR process according to an embodiment of the present
disclosure, the pretreatment for separating the target substance
such as RNA and DNA is performed by flowing a mixture solution
(sample solution) containing the target substance to a capturing
means such as silica beads. In particular, noting that the mixture
solution as well as the washing buffer and the elution buffer is
flown toward one direction, i.e. toward the capturing means (silica
beads), the cross-sectional areas of channels of the solutions from
the respective solution chambers holding and storing the solutions
are set differently, so that only the wanted solution may flow
toward the capturing means by varying the rotation speed. For this,
in an embodiment of the present disclosure, the solution channels
are hydrophobically treated, so that the aqueous solutions may flow
through the hydrophobic channels only when a force exceeding a
predetermined value is applied thereto. However, the scope of the
present disclosure is not limited thereto, and any possible means
allowing selective control of the flow of the solutions based on
the difference in centrifugal force are included within the scope
of the present disclosure.
[0039] The PCR chip according to the present disclosure may further
comprise, in addition to the PCR unit where PCR occurs, and a
pretreatment unit connected to the fore end of the PCR unit, where
the target substance is separated from the sample solution. FIG. 6
is a partial schematic view of a pretreatment unit of a PCR chip
according to an embodiment of the present disclosure.
[0040] Referring to FIG. 6, a pretreatment unit of a PCR chip
according to an embodiment of the present disclosure has three
solution chambers 410a, 410b, 410c each containing different
solution. Fluid channels 420a, 420b, 420c connected to the chambers
are hydrophobically treated. As such, the three aqueous solutions
(sample solution, washing buffer and elution buffer) cannot
normally flow through the hydrophobically treated channels, unless
a force exceeding a predetermined value is applied thereto. The
enlarged portion of the hydrophobic channels 420a, 420b, 420c in
FIG. 6 shows that the channels from the solution chambers are
hydrophobically treated with a siloxane-based compound. However,
the scope of the present disclosure is not limited thereto.
[0041] It is to be noted that the force needed to move the
solutions changes depending on the size (cross-sectional area) of
the hydrophobically treated fluid channels. The force needed to
move the liquids is obtained from the centrifugal force resulting
from the rotation of the PCR chip comprising the pretreatment unit,
which will be described in detail hereinbelow.
[0042] The hydrophobic channels 420a, 420b, 420c are commonly
connected to silica beads 430 to which the target substance is
selectively bound. The solutions flowing through the hydrophobic
channels 420a, 420b, 420c are introduced to the silica beads 430.
Thereafter, the target substance captured by the silica beads 430
moves, as a valve 440 that can be selectively opened/closed by heat
is opened, through an outlet 450 to the PCR unit at the rear end
due to the centrifugal force resulting from the rotation of the PCR
chip. Then, PCR proceeds as described referring to FIGS. 1 to
5.
[0043] A discharge port 460 may be further provided to discharge
the solution remaining after washing the silica beads to outside.
That is, while the valve 440 is closed, the solution (e.g., washing
buffer) discharged from the silica beads 430 is discharged to
outside through the discharge port 460.
[0044] FIG. 7 shows configurations of solution chambers and
hydrophobic channels according to an embodiment of the present
disclosure.
[0045] Referring to FIG. 7, if the hydrophobic channel 420 has a
large cross-sectional area, the fluid in the solution chamber (the
upper chamber in FIG. 7) may be flown with a relatively smaller
force (1.5 kPa). However, if the cross-sectional area is smaller, a
larger force is required for the fluid to flow. In the present
disclosure, the force needed to move the solution is attained from
the centrifugal force resulting from the rotation of the PCR chip
including the pretreatment unit.
[0046] First, when the PCR chip including the pretreatment unit
rotates relatively slowly, a relatively smaller force is applied to
the solution chamber 410. Thus, the solution in the solution
chamber (the upper chamber in FIG. 7) connected to the hydrophobic
channel with the largest cross-sectional area flows first. For
example, whereas the solution in the lower solution chamber 410c of
FIG. 6 may flow through the hydrophobic channel with a rotation
speed of 2690 rpm, the intermediate solution chamber 410b and the
upper solution chamber 410a may respectively require rotation
speeds of 4100 rpm and 5800 rpm.
[0047] Accordingly, in an embodiment of the present disclosure, the
hydrophobic channel for the sample solution which needs to be flown
to the silica beads first has the largest cross-sectional area. As
such, when the rotation speed of the PCR chip becomes equal to or
greater than a first speed, the sample solution flows from the
sample solution chamber to the silica beads. The hydrophobic
channel for the washing buffer which needs to be flown secondly has
the second largest cross-sectional area. As such, when the rotation
speed of the PCR chip exceeds the first speed and becomes equal to
or greater than a second speed, the washing buffer flows through
the hydrophobic channel to the silica beads. As a result, all the
components other than the target substance adsorbed to the silica
beads are removed and discharged to outside through the discharge
port 460. Then, the elution buffer for separating the target
substance adsorbed to the silica beads is flown to the silica
beads. The hydrophobic channel for the elution buffer has a
cross-sectional area smaller than those of the channels for the
sample solution and the washing buffer. Thus, when the rotation
speed of the PCR chip exceeds the second speed and becomes equal to
or greater than a third speed, the elution buffer flows through the
hydrophobic channel connected to the corresponding chamber to the
silica beads. As a result, the target substance such as DNA or RNA
adsorbed to the silica beads is separated from the silica
beads.
[0048] Noting that both the PCR process and the pretreatment
process are carried out as the PCR chip rotates, the present
disclosure provides a new-concept integrated PCR chip wherein a PCR
unit and a pretreatment unit are integrated in a single chip.
[0049] FIG. 8 is a perspective view of an integrated PCR chip
according to an embodiment of the present disclosure.
[0050] Referring to FIG. 8, a pretreatment unit 610 described in
FIG. 6 and a PCR unit 620 corresponding to the PCR chip 220 in FIG.
2 are coupled with each other. Between the pretreatment unit 610
and the PCR unit 620, a thermoreactive polymer valve 630 is
provided. As the thermoreactive polymer valve 630 is opened, the
separated and purified target substance is introduced from an
outlet of the pretreatment unit 610 to the PCR unit 620. Then, PCR
is carried out in a PCR chamber 640. As the PCR chip rotates, a
plurality of PCR cycles occur, as described earlier.
[0051] As described, the PCR chip according to this embodiment has
the pretreatment unit for separating and purifying DNA or RNA and
the PCR unit where PCR occurs. The target substance separated by
the pretreatment unit flows through the thermoreactive polymer
valve 630, which is opened at low temperature, and is introduced
into the PCR unit. Thereafter, the target substance introduced to
the PCR unit is heated by heating blocks and PCR occurs. In a PCR
apparatus according to an embodiment of the present disclosure, the
valve 630 connecting the pretreatment unit and the PCR unit is
closed as temperature rises. In this case, backflow of the target
substance to the pretreatment unit may be prevented during the PCR
process occurring at high temperature.
[0052] A more detailed description will be given about the
thermoreactive valve and the pretreatment unit.
[0053] In an embodiment of the present disclosure, the
thermoreactive valve comprises a thermoreactive polymer. More
specifically, Fluorinert FC40 available from 3M may be used. The
thermoreactive polymer expands above a predetermined temperature,
e.g. about 40.degree. C., thereby pushing a flexible membrane
contacting with the thermoreactive polymer toward a fluid channel.
As a result, the fluid channel is blocked. Conversely, below a
predetermined temperature, e.g. about 40.degree. C., the
thermoreactive polymer is shrunken and the pretreatment unit is
communicated with the PCR unit again.
[0054] FIGS. 9 and 10 are cross-sectional views of a pretreatment
unit of a PCR chip according to an embodiment of the present
disclosure.
[0055] Referring to FIG. 9, the PCR chip comprises three layers--a
sample layer 710 with a channel allowing the flow of a target
substance formed, a polymer layer 730 comprising a thermoreactive
polymer expanding/shrinking depending on the temperature condition,
and a flexible membrane 720 provided between the sample layer 710
and the polymer layer 730 and comprising a flexible material that
can move elastically according to the expansion/shrinkage of the
polymer layer, such as polydimethylsiloxane (PDMS). As seen in FIG.
9, the thermoreactive polymer layer 730 does not expand at room
temperature. Thus, a sample solution may through the channel of the
sample layer 710 therebelow.
[0056] However, referring to FIG. 10, the thermoreactive polymer
layer 730 expands as temperature rises. As the polymer layer 730
expands, the flexible membrane 720 therebelow also expands
downward. As a result, the fluid channel of the sample layer 710 is
blocked and closed by the flexible membrane 720 expanding downward.
Since PCR occurs at temperatures above 40.degree. C.,
thermoreactive valve remains closed during the PCR process.
Accordingly, the PCR process occurs stably in the PCR unit without
leakage of fluid to the pretreatment unit.
[0057] According to the PCR method of the present disclosure using
the PCR chip, PCR occurs while the PCR chip containing the target
substance rotates and passes through a plurality of temperature
zones at different temperatures. For this, in an embodiment of the
present disclosure, the plurality of temperature zones is arranged
such that temperatures required for PCR are repeated. The
temperatures of the temperature zones may be maintained by a
heating means (e.g. heating blocks) spaced apart from the PCR chip,
as described earlier.
[0058] FIG. 11 is a flow diagram of a PCR method using a PCR chip
according to another embodiment of the present disclosure.
[0059] Referring to FIG. 11, first, a PCR chip comprising a
pretreatment unit where a target substance is separated and a PCR
unit where PCR occurs for the separated target substance is rotated
at or above a first speed, with the chip temperature maintained
above a certain temperature. Under this temperature condition, a
thermoreactive valve connecting the pretreatment unit and the PCR
unit is closed, and no solution flows from the pretreatment unit to
the PCR unit.
[0060] As the chip rotates at or above the first speed, only a
sample solution (an aqueous mixture solution containing the target
substance) flows from a sample solution chamber having a
hydrophobic channel with the largest cross-sectional area to silica
beads, which are a capturing means. Then, the chip is rotated at or
above a second speed. As the chip rotates at or above the second
speed, a washing buffer (a solution for removing components other
than the target substance from the silica beads) flows to the
silica beads. The components other than the target substance are
removed from the silica beads by the washing buffer. Then, the chip
is rotated at or above a third speed. As the chip rotates at or
above the third speed, an elution buffer flows through a
hydrophobic channel with the smallest cross-sectional area to
silica beads. As a result, the target substance is separated from
the silica beads. The separated target substance is introduced to
the PCR unit. In order to introduce the target substance into the
PCR unit, the temperature of the PCR chip needs to be lowered to
open the thermoreactive valve between the PCR unit and the
pretreatment unit. The valve may be opened before, after or during
the flow of the elution buffer, as long as the target substance
separated from the silica beads by the elution buffer may be
introduced into the PCR unit.
[0061] Then, PCR occurs as the PCR chip rotates. As described with
reference to FIGS. 1 to 5, the PCR process occurs while the PCR
chip passes through a plurality of temperature gradient zones. At
this time, in order to prevent backflow of the solution to the
pretreatment unit, the thermoreactive valve is closed as the PCR
chip is maintained above a predetermined temperature.
[0062] The present disclosure further provides a PCR system
comprising the above-described integrated PCR chip and a rotating
platform with a plurality of temperature zones formed thereon.
[0063] The rotational PCR apparatus and method according to the
present disclosure allow performance of PCR with wanted temperature
condition and cycles by rotating the chip containing the target
substance. Accordingly, a high-efficiency PCR process may be
accomplished at low cost. Further, since the target substance can
be effectively separated and purified utilizing the centrifugal
force resulting from the rotating platform, separation and
purification may be achieved economically without requiring
additional equipments.
[0064] While the present disclosure has been described with respect
to the specific embodiments, it will be apparent to those skilled
in the art that various changes and modifications may be made
without departing from the spirit and scope of the disclosure as
defined in the following claims.
* * * * *