Patent Grant
8697433
1. Field
The present disclosure relates to polymerase chain reaction (hereinafter, simply referred to as PCR) modules and multiple PCR systems using the same, and more particularly, to PCR modules with a combined PCR thermal cycler and PCR product detector, and multiple PCR system using the same.
2. Description of the Related Art
The science of genetic engineering originated with the discovery of restriction enzymes. Similarly, PCR technology led to an explosive development in the field of biotechnology, and thus, it may be said that the PCR technology is a contributor to the golden age of biotechnology. PCR is a technology to amplify DNA copies of specific DNA or RNA fragments in a reaction chamber. Due to a very simple principle and easy applications, the PCR technology has been extensively used in medicine, science, agriculture, veterinary medicine, food science, and environmental science, in addition to pure molecular biology, and its applications are now being extended to archeology and anthropology.
PCR is performed by repeated cycles of three steps: denaturation, annealing, and extension. In the denaturation step, a double-stranded DNA is separated into two single strands by heating at 90° C. or more. In the annealing step, two primers are each bound to the complementary opposite strands at an annealing temperature of 55 to 60° C. for 30 seconds to several minutes. In the extension step, primer extension occurs by DNA polymerase. The time required for the primer extension varies depending on the density of template DNA, the size of an amplification fragment, and an extension temperature. In the case of using Thermusaquaticus (Taq) polymerase, which is commonly used, the primer extension is performed at 72° C. for 30 seconds to several minutes.
Generally, PCR products are separated on a gel and the approximate amount of the PCR products is estimated. However, faster and more accurate quantification of PCR products is increasingly necessary. Actually, an accurate measurement of the amount of target samples in gene expression (RNA) analysis, gene copy assay (quantification of human HER2 gene in breast cancer or HIV virus burden), genotyping (knockout mouse analysis), immuno-PCR, etc. is very important.
However, conventional PCR is end-point PCR for qualitative assay of amplified DNA by gel electrophoresis, which causes many problems such as inaccurate detection of the amount of DNA. To overcome the problems of the conventional end-point PCR, a quantitative competitive (QC) PCR method was developed. The QC-PCR is based on co-amplification in the same conditions of a target and a defined amount of a competitor having similar characteristics to the target. The starting amount of the target is calculated based on the ratio of a target product to a competitor product after the co-amplification. However, the QC-PCR is very complicated in that the most suitable competitor for each PCR has to be designed, and multiple experiments at various concentrations for adjusting the optimal ratio range (at least a range of 1:10 to 10:1, 1:1 is an optimal ratio) of the target to the competitor has to be carried out. The success probability for accurate quantification is also low.
In view of these problems of the conventional PCR methods, there has been introduced a real-time PCR method in which each PCR cycle is monitored to measure PCR products during the exponential phase of PCR. At the same time, there has been developed a fluorescence detection method for quickly measuring PCR products accumulated in a tube at each PCR cycle, instead of separation on a gel. UV light analysis of ethidium bromide-containing target molecules at each cycle and detection of fluorescence with a CCD camera were first reported by Higuchi et al. in 1992. Therefore, an amplification plot showing fluorescent intensities versus cycle numbers may be obtained.
However, in a conventional real-time PCR system, all wells or chips have to be set to the same temperature conditions due to use of metal blocks such as peltier elements. Even though it may be advantageous to carry out repeated experiments using a large amount of samples at the same conditions, there are limitations on performing PCR using different samples at different temperature conditions. Also, since metal blocks such as peltier elements are used for temperature maintenance and variation, a temperature transition rate is as low as 1-3° C./sec, and thus, a considerable time for temperature transition is required, which increases the duration of PCR to more than 2 hours. In addition, the temperature accuracy of ±0.5° C. limits fast and accurate temperature adjustment, which reduces the sensitivity and specificity of PCR.
Provided are polymerase chain reaction (PCR) modules in which co-amplification of different samples at different temperature conditions may be carried out and monitored in real time.
Provided are multiple PCR systems using the PCR module.
Provided are real-time PCR monitoring methods using the PCR modules or the multiple PCR systems.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments.
According to an aspect of the present invention, a PCR module includes a heater including a heater wire and a temperature sensor; a first housing for fixing the heater; a PCR tube thermally contacting with the heater and comprising a PCR chamber containing a PCR solution; a second housing fixed to the first housing, for fixing the PCR tube; and a detection unit detecting a PCR product signal.
According to an aspect of the present invention, a multiple PCR system includes the PCR module; and a host computer controlling the PCR module, wherein the PCR module and the host computer are electrically connected through a wire or wireless mode.
According to an aspect of the present invention, a multiple PCR system includes the PCR module; and a host computer controlling the PCR module, wherein the PCR module includes a computing unit and the computing unit of the PCR module and the host computer are electrically connected through a wire or wireless network.
According to an aspect of the present invention, a real-time PCR monitoring method includes (a) loading a PCR solution in a PCR chamber of a PCR tube received in each of one or more PCR modules; (b) performing PCR independently in the PCR chamber of the PCR tube of each PCR module having an independently determined temperature condition; (c) detecting a PCR product signal based on PCR performed in each PCR module; and (d) displaying data about the PCR product signal of each PCR module.
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description.
Here, the “the PCR tube 10” indicates a disposable or reusable device that is detachable from the PCR module 40, generally a microchip-type PCR tube. For example, the PCR tube 10 is mainly made of silicon. Therefore, heat generated by a heater 20 may be rapidly transferred, and thus, a temperature transition rate may be remarkably enhanced, relative to a conventional technology. Furthermore, PCR may be performed for smaller amounts of unconcentrated samples, unlike a conventional technology. For example, the PCR chamber 11 has a capacity of several tens microliters or less. If the capacity of the PCR chamber 11 exceeds several tens microliters, the content of a sample increases, thereby remarkably retarding PCR and increasing the size of the PCR tube 10. In this respect, the PCR chamber 11 with the capacity of more than several tens micrometers is not appropriate with a view to the capacity of the entire system.
The PCR tube 10 includes the PCR chamber 11 as described above. The PCR chamber 11 contains a PCR solution. The PCR solution may be controlled to an appropriate temperature range by feedbacking the temperature of the PCR solution accurately measured by the temperature sensor. An impedance measurement sensor is used to measure impedance in a PCR solution as a PCR product signal using a chip (10 of
The heater 20 contained in the PCR module 40 is separately disposed from the PCR tube 10 and contacts with a lower surface of the PCR tube 10 to apply heat to the PCR tube 10.
The temperature sensor 21 is positioned on a central portion of the heater 20, and detects a temperature change. A resistance change of the temperature sensor 21 due to the temperature change may be converted into a voltage, and then may be transmitted to a computing unit through four terminals connected to the temperature sensor 21. In this case, a four-point measuring method, or a three-point measuring method may be used. In the four-point measuring method, central two terminals of the four terminals are used to measure a voltage while allowing a predetermined current to flow through external two terminals of the four terminals. In the three-point measuring method, a bridge is formed by using only any three terminals of the four terminals.
The heat wire 22 may have a winding shape. In addition, on/off of the heat wire 22 may be controlled according to a temperature that is measured by the temperature sensor 21. For example, the heater 20 is a microplate heater.
The temperature sensor 21 and the heat wire 22 may be formed on the lower surface of the heater 20, and may be formed of the same material.
As such, since the heater 20 and the PCR tube 10 are separately formed, only the PCR tube 10 may be replaced, and thus temperature calibration of the heater 20 does not have to be repeatedly performed, thereby improving the durability and lifetime of the heater 20.
Since the heater 20 and the PCR tube 10 are separately formed, a separate housing is required in order to fix the heater 20 and the PCR tube 10.
Likewise, the heater 20 is also fixed to a first housing. As shown in
The PCR tube 10 and the heater 20 may directly contact each other. Alternatively, a heat-transfer facilitating layer may be further provided between the PCR tube 10 and the heater 20, in order to uniformly transfer heat. A graphite sheet may be used as the heat-transfer facilitating layer.
After the second housing including the PCR tube 10 is coupled to the first housing including the heater 20, a sealing member may be positioned on the second housing. The sealing member may be fixed to the second housing by a fixer so as to seal an entrance of the PCR tube 10.
The PCR module 40 may further include a power supply unit 51 so that a fixed voltage is applied to the heater 20. The heater 20 may apply a uniform temperature to the PCR tube 10 for stable thermal transfer by electric power supplied from the power supply unit 51. However, in some cases, the power supply unit 51 may apply an electric power to the heater 20, together with another power supply unit connected to another device.
For example, the PCR module 40 may further include a cooler 43, in addition to the heater 20, so that the PCR solution in the PCR tube 10 is set to a desired temperature. That is, the cooler 43 is used to perform thermal circulation cycles by rapid temperature transition. As the cooler 43, there may be used a cooling fan for cooling an ambient air of the PCR module 40 to adjust the temperature of the PCR solution or a peltier device attached to the PCR tube 10 or the module 40 to adjust the temperature of the PCR solution. A water cooler may also be used. If necessary, an airguide or a heatsink may be installed to enhance thermal conductivity.
The detection unit 30 of the PCR module 40 includes an optical source 31 or an alternating power element 33 and is used to detect a PCR product signal based on the amount of a PCR product. The principle and construction of the detection unit 30 will be described later.
The computing unit 41 is operated according to a software program stored in the auxiliary memory 44. The auxiliary memory 44 is not particularly limited provided that it is that commonly used in the computation related field. For example, there may be used one or more selected from a hard disk, a floppy disk, an optical disk (CD, DVD, MD, etc.), a magnetic disk, and a flash memory card. CD used as the auxiliary memory 44 is used through a CD-ROM drive and a flash memory card used as the auxiliary memory 44 is used through a memory reader. The flash memory card is most appropriate because of its small size, easy use, and low power consumption. The flash memory card may be optionally selected from those known in the pertinent art. All types of flash memory cards such as Compact Flash (CF), Secure Digital (SD), Micro Drive (MD), memory stick, and eXtreme Digital (XD) may be used.
For example, a PCR software program for operating the computing unit 41 is stored in the auxiliary memory 44 as described above and used if necessary. The auxiliary memory 44 also stores various data about user-defined parameters for PCR, i.e., PCR temperature and cycle number. A separate power supply unit may be connected to the computing unit 41.
The multiple PCR systems 1 according to the present embodiment include one or more PCR modules 40 and are used for PCR for different samples at different PCR conditions. That is, the multiple PCR systems 1 are used to independently and simultaneously perform the real-time control of several PCR procedures, thereby enhancing PCR efficiency.
With respect to a multiple PCR system 1 shown in
The host computer 50 includes a CPU 52, an auxiliary memory 54, a RAM 55, and an input/output unit 60 and controls a PCR procedure according to a software program stored in the auxiliary memory 54. As described above, the auxiliary memory 54 may be one or more selected from a hard disk, an optical disk, a floppy disk, and a flash memory card. The software program stored in the auxiliary memory 54 has an additional management function for independently controlling the PCR modules 40, unlike the above-described computing unit 41 that has only a necessary function for controlling constitutional elements of the module 40. That is, the software program stored in the auxiliary memory 54 may independently control the detection unit 30, the heater 20, and the PCR tube 10 contained in each of the PCR modules 40 so that PCR for different samples may be controlled at the different conditions. Furthermore, parameter values optionally defined by a user are stored in the auxiliary memory 54.
The host computer 50 includes the input/output unit 60, unlike the computing unit 41. The input/output unit 60 serves to input user-defined parameter values or display in real time various data received from the PCR modules 40. According to the input or displayed data, a PCR procedure may be appropriately controlled by changing or modifying in real time the user-defined parameter values. For example, a liquid crystal display is used as a display portion of the input/output unit 60 with a view to power consumption or dimension. In this case, it is more appropriate to install a touch screen type input element on the display portion. Of course, a common keyboard, CRT, etc. may also be used.
The host computer 50 communicates with the PCR modules 40 via a data communication unit (not shown) through a wire or wireless mode. Common wire or wireless modes known in the pertinent art may be unlimitedly used. For example, a serial port such as RS-232C, a parallel port, a USB port, a 1394 port, etc. may be used for communication through the wire mode. It is appropriate to use a USB port considering extendability. A radio frequency (RF) mode may be used for wireless communication.
In particular, the detection unit 30 in each of the PCR modules 40 detects a PCR product signal in the PCR tube 10 and transmits the detected signal to the host computer 50 through a wire or wireless mode. For example, the PCR product signal may be a fluorescence signal emitted from the PCR chamber 11 disposed in the PCR tube 10. The detection unit 30 acts as a fluorescence detector that detects a fluorescence signal and transmits the detected signal to the host computer 50. For this, the detection unit 30 includes an optical source 31 for applying light to the PCR solution. When light from the optical source 31 is applied to the PCR solution, the fluorescence emitted from the PCR solution is concentrated on a lens (not shown) and recorded after passing through a filter.
The PCR product signal may also be an electrical signal. In this case, the detection unit 30 includes a sensor (not shown) for sensing an electrical signal. The sensor is disposed in the PCR tube 10. The sensor detects a PCR product signal emitted when an alternating current is applied to the PCR solution in the PCR chamber 11 disposed in the PCR tube 10 and transmits the detected signal to the host computer 50. The received data is displayed on the display portion of the input/output unit 60 to be viewed by a user. For this, the detection unit 30 includes an alternating power element 33.
The host computer 50 may include a separate power supply unit (not shown) for stable power supply. The power supply unit may simultaneously perform power supply to the constitutional elements of the PCR modules 40. That is, the host computer 50 and the PCR modules 40 may receive an electric power from individual power supply units or a single common power supply unit. This is also applied to the detection unit 30 and the heater 20 contained in each of the PCR modules 40.
As described above, the host computer 50 and the computing units 41 have respective auxiliary memories 54 and 44. The auxiliary memories 54 and 44 store software programs for PCR control and the software programs execute their functions. In particular, the software programs may be connected through wire or wireless network such as a pier-pier network or a server-client network. For example, a LAN transmission technology using a common network interface card or hub may be used through a wire or wireless mode. Through such a connection system, the PCR modules 40 are controlled remotely by the host computer 50 through real-time data communication, thereby independently controlling the PCR modules 40. As described above, the computing units 41 may independently control constitutional elements in the respective PCR modules 40.
In particular, in the multiple PCR system 1 shown in
A real-time PCR monitoring method using the multiple PCR system 1 according to an embodiment of the present invention in which a PCR product signal is a fluorescence signal emitted from the PCR chamber 11 will now be described in detail with reference to
A real-time PCR monitoring method using a multiple PCR system in which a PCR product signal according to another embodiment of the present invention is a signal corresponding to impedance measured from a PCR product will now be described with reference to
Hereinafter, one or more embodiments of the present invention will be described in detail with reference to the following examples. However, these examples are not intended to limit the purpose and scope of the one or more embodiments of the present invention.
To minimize difference between PCR experiments, other reagents except DNA samples were mixed to prepare a two-fold concentrated master mixture. Then, the master mixture was mixed with the DNA samples (1:1, by volume) to obtain a PCR solution.
The composition of the master mixture is as follows:
To investigate the effect of a thermal transfer rate and a temperature ramping rate on PCR, PCR was carried out on micro PCR chips with the dimension of 7.5 mm×15.0 mm×1.0 mm. The micro PCR chips were made of silicon and had fast thermal transfer in reactants, and so on due to several hundreds times faster thermal conductivity than conventional PCR tubes, a fast temperature ramping rate, and maximal thermal transfer due to use of a trace of DNA samples. The micro PCR chips were fixed to the second housing illustrated in
1 μl of the PCR solution of Example 1 was loaded in each of the micro PCR chips, and a PCR cycle of 92° C. for 1 second and 63° C. for 15 seconds was then repeated for 40 times. The experimental resultants were quantified using Labchip (Agilent) and amplification was identified on a 2% TAE agarose gel.
In this Example, a signal emitted from a PCR solution (Promega) was measured in real time using the following multiple PCR system 1 as shown in
Specifications of a host computer 50 and a computing unit 41 were as follows:
I. Host Computer
Industrial embedded board (manufactured by Transmeta Co., Ltd., model: AAEON Gene 6330) was used.
The GENE-6330 is thinnest board in the AAEON SubCompact Board series. It has a Mini-PCI slot, an onboard SMI 712 LynxEM+ graphic chip provides TFT and DSTN panel support and comes with one 10/100 Mbps Ethernet connector, four USB ports and a CompactFlash slot, offering great connectivity. Functional flexibility is enhanced through the choice of either a Type II PCMCIA and Type III Mini PCI slot.
Auxiliary memory: 2.5 inch 30 GB HDD (manufactured by Hitachi Co., Ltd.)
Network interface: RTL 8139DL, 10/100Base-T RJ-45
Input unit: 15.1 inch touch screen (manufactured by 3M Co., Ltd.)
Output unit: 15.1 inch LCD monitor (manufactured by BOE Hydis Co., Ltd.)
Operating System: MS Windows 2000 professional
II. Computing Unit
The computing unit used C8051 F061 (manufactured by Silicon Laboratories Co., Ltd.)
The Silicon Laboratories, Inc. C8051 F061 is a 25 MIPS Mixed-Signal 8051 with 24 I/O Lines, 5 Timers, Watchdog Timer, PCA, SPI, SMBus, I2C, 2 UARTS, CAN 2.0B, 2 Channel (16-bit) ND, 8 Channel (10-bit) A/D, 2 Channel (12-bit) D/A, 3 Analog Comparators, On-Chip Temperature Sensor, 64K Byte In-System Programmable FLASH, 256 Bytes RAM, 4K Bytes XRAM.
The host computer 50 and the computing unit 41 were connected through a hub over the Ethernet wire. A power supply unit installed at the host computer 50 supplied an electric power to the PCR modules 40 each including the computing unit 41. Further, the ambient temperature of the PCR modules 40 each including the PCR tube 10 was cooled by the cooler 43.
A microplate heater provided with the temperature sensor 21 and the heat wire 22 was used as the heater 20. The heater 20 was fixed to the first housing, as shown in
To minimize difference between PCR experiments, the PCR solution was prepared as follows: other reagents except DNA samples were mixed to prepare a two-fold concentrated master mixture and then the master mixture was mixed with the DNA samples (1:1, by volume) to obtain the PCR solution.
The composition of the master mixture is presented in Table 1 below.
The temperature and duration conditions for PCR were the same as those used in conventional PCR tubes as follows: 1 cycle of 50° C. for 120 seconds and 91° C. for 180 seconds; 1 cycle of 92° C. for 1 second and 63° C. for 180 seconds; 44 cycles of 92° C. for 1 second and 63° C. for 15 seconds; and 1 cycle of 63° C. for 180 seconds.
To measure impedance values, first, 1 μl of the PCR solution as prepared previously was loaded in each of micro PCR chips via a sample inlet as shown in
Two-stage thermal cycling for the PCR solution of Example 1 was performed according to the PCR conditions presented in Table 2 below. The same apparatus as in Example 1 was used as the multiple PCR system 1 except that the detection unit 30 including the optical source 31 was used for signal detection.
First, 1 μl of the PCR solution of Example 1 was loaded in each of micro PCR chips via a sample inlet as shown in
As described above, a multiple PCR system according to one or more embodiments of the present invention includes a plurality of PCR modules, each of which includes a microchip-type PCR tube having a PCR solution-containing PCR chamber, a heater, a detection unit that detects a PCR product signal based on the amount of a PCR product in the PCR solution, and a computing unit that adjusts the temperature of the PCR chamber of the PCR tube; and a host computer electrically connected to the modules. The computing unit of each PCR module independently controls the detection unit and the temperature of the PCR chamber of the PCR tube received in each PCR module. Therefore, PCR for different samples may be carried out at different temperature conditions at the same time and may be monitored in real time.
As described above, according to the one or more of the above embodiments of the present invention, in a PCR module, a multiple PCR system using the same, and a PCR monitoring method, co-amplification of different samples at different temperature conditions may be carried out and monitored in real time
Furthermore, PCR may be performed for smaller amounts of unconcentrated samples at an enhanced temperature transition rate using a microchip-type PCR tube made of silicon with excellent conductivity.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2003-0089352 | Dec 2003 | KR | national |
| 10-2004-0102738 | Dec 2004 | KR | national |
This is a continuation-in-part application of U.S. patent application Ser. No. 11/080,705, filed on Mar. 15, 2005 and issued as U.S. Pat. No. 7,799,557 on Sep. 21, 2010, which in turn is a continuation-in-part application of U.S. patent application Ser. No. 10/890,350, filed on Jul. 13, 2004 and issued as U.S. Pat. No. 7,767,439 on Aug. 3, 2010, and which claims priority to Korean Patent Application Nos. 10-2003-0089352 filed on Dec. 10, 2003 and 10-2004-0102738 filed on Dec. 8, 2004 under 35 U.S.C. §119, the disclosures of which are incorporated herein in their entirety.
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| Number | Date | Country | |
|---|---|---|---|
| 20100311070 A1 | Dec 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10890350 | Jul 2004 | US |
| Child | 12843552 | US | |
| Parent | 11080705 | Mar 2005 | US |
| Child | 10890350 | US |
