Patent Grant
9551665
1. Field of the Invention
The present invention relates to a method for detecting mitochondria gene alterations and, more particularly, to a method for detecting mitochondria gene alterations by means of a single primer pair.
2. Description of Related Art
Mitochondria are the main sites in cells where oxidative phosphorylation occurs and adenosine triphosphate is synthesized, and energy to human and animal cells comes from. Furthermore, mitochondria not only can provide energy, they also work to take part in apoptosis, cellular differentiation and signaling, as well as cell growth and cell cycle. Hence, whether the mitochondria genes are defective or not is instrumental to the functioning of human and animal cells.
Recent studies have found that mitochondria genes can be made susceptible to gene variations, including mutations and deletions, when exposed to attack of free radicals that are generated during oxidation. Such alterations in mitochondria genes can heavily influence biological functions related to mitochondria or can even lead to cell death. An example of known diseases or symptoms incurred by mitochondria gene alterations includes degenerative diseases. Some studies have also suggested that link between mitochondria gene alterations and cancers. Hence, detection for alterations in mitochondria genes would prove to be helpful for diagnosing diseases in clinical practices.
The majority of currently understood methods for detecting mitochondria gene alterations are focused on using direct DNA sequencing; however, the technicality of direct DNA sequencing is complicated and the material and the device required in its operation are expensive, so this technique has not been widely received by testing services and institutions.
In response, it is desirable to provide a rapid and simple device and method for detecting mitochondria gene alterations, which can be applied to clinical uses to examine mitochondria gene related diseases, and from which to use as a reference for treating or diagnosing diseases.
An object of the present invention is to provide a method for detecting mitochondria gene (mtDNA) alterations, which can not only determine occurrence of mitochondria gene alterations, but also quantify the same.
Another object of the present invention is to provide a device and a system for detecting mtDNA alterations, wherein the device has a three dimensional structure. Hence, extraction and detection processes can be performed on the mitochondria genes in a single device, and making it possible to determine in a rapid and precise fashion whether there are gene alterations in the mitochondria genes. On a particular note, the device and the system of the present invention are the resulting device and system realized from using the present invention's method for detecting mtDNA alterations.
To achieve the above-identified object, the device of the present invention comprises: a purification unit containing a first reaction chamber and a separation-element disposed chamber, wherein the separation-element storage chamber connects to the first reaction chamber through a first pump; and a detection unit disposed under the purification unit, wherein the detection unit comprises: a second reaction chamber and a detecting-sample chamber, the second reaction chamber connects to the first reaction chamber, and the detecting-sample chamber connects to the second reaction chamber through a second pump. Herein, the first reaction chamber and the second reaction chamber connect to each other. More specifically, the first reaction chamber and the second reaction chamber connect to each other to form a reaction chamber.
In the aforementioned device of the present invention, when a sample contained with mitochondria genes is placed into the first reaction chamber, a separation element contained in the separation-element storage chamber can be introduced into the first reaction chamber by the first pump. Then, the separation element is mixed with the sample to perform an extraction process on the mitochondria genes. The first reaction chamber and the second reaction chamber connect to each other, so the sample contained in the reaction chamber can be later introduced into the detecting-sample chamber by the second pump after the extraction process and then a process for detecting mitochondria gene alterations is performed on the sample introduced into the detecting-sample chamber. Then, due to the interconnection between the first reaction chamber and the second reaction chamber, the detecting-sample obtained from the extraction process of the separation unit can be introduced into the detecting-sample chamber through the help of the second pump for carrying out detection for mitochondria genes alterations and the like.
According to the aforementioned device of the present invention, the extraction and detection processes can be performed on the mitochondria genes in a single device. Hence, in contrast to the case with a conventional device that the extraction process has to be performed in conjunction with another extraction device, the whole series of detection steps can be further simplified by using the device of the present invention.
In the device for detecting mtDNA alterations of the present invention, the purification unit may further comprises a washing-solution disposed camber, which connects to the first reaction chamber through a third pump. When the separation element contained in the separation-element storage chamber is mixed with the sample, a washing solution contained in the washing-solution storage chamber can be introduced into the first reaction chamber by the third pump to remove other matrix in cells except for the mitochondria genes. Herein, the washing solution is not particularly limited, and can be any cell-washing solution generally used in the art, such as PBS.
In addition, the device for detecting mtDNA alterations of the present invention may further comprise a micro-mixture unit connecting to the first reaction chamber in order to extract and wash the sample more uniformly.
In one aspect of the device for detecting mtDNA alterations of the present invention, the detection unit may further comprise: a comparison chamber connecting to the second reaction chamber through the second pump. Herein, DNA amplified reactions can be selectively performed on the samples contained in the comparison chamber and the detecting-sample chamber. Then, DNA signals emitted from the comparison chamber are compared with those emitted from the detecting-sample chamber to determine whether the mitochondria genes are alternated or not.
In another aspect of the device for detecting mtDNA alterations of the present invention, the detection unit may further comprise: a mitochondria-gene checking chamber and a temporary chamber, wherein the mitochondria-gene checking chamber and the temporary chamber connect to the second reaction chamber through a fourth pump, and the temporary chamber also connects to the detecting-sample chamber through the second pump. Herein, the DNA amplified reaction can be firstly performed on the sample contained in the mitochondria-gene checking chamber to determine whether the mitochondria genes are extracted by the separation element or not. After the checking process confirmed that the extraction process is successful, the sample contained in the temporary chamber is then introduced into the detecting-sample chamber through the second pump to perform the sequential detection process.
In the aforementioned aspect of the device for detecting mtDNA alterations, the detection unit may further comprise: a comparison sample chamber, which connects to the temporary chamber through the second pump. Hence, the sample contained in the temporary chamber can be introduced into the detecting-sample chamber and the comparison sample chamber by the second pump, and then DNA amplified reactions and other reactions (such as restriction enzyme digestion) can be selectively performed on the samples contained in the detecting-sample chamber and the comparison sample chamber. Finally, the signals emitted from the comparison sample chamber are compared with those emitted from the detecting-sample chamber to determine whether the mitochondria genes are alternated or not.
The devices for detecting mtDNA alterations according to all the aforementioned aspects of the present invention may further comprise plural gas inlets, wherein each gas inlets connects to each elements of the purification unit or the detection unit such as the first pump, the second pump, the third pump, the forth pump, the micro-mixture unit, and the temporary chamber by corresponding connection paths.
In addition, in the devices for detecting mtDNA alterations according to all the aforementioned aspects of the present invention, the first pump, the second pump, the third pump and the forth pump can be any micro-pump generally used in the art such as peristaltic micropumps and suction-type micropumps. Preferably, the first pump and the third pump are peristaltic micropumps respectively, and the second pump and the forth pump are suction-type micropumps respectively.
Furthermore, in the devices for detecting mtDNA alterations according to all the aforementioned aspects of the present invention, all the elements are in micro-sized, so the length, the weight and the height of the whole devices can be confined to several tens of millimeters (mm). Hence, the devices of the present invention can be designed as portable and disposable devices.
Except for the aforementioned devices for detecting mtDNA alterations of the present invention, the present invention further provides a system using the aforementioned devices. The system for detecting mtDNA alterations of the present invention comprises: the aforementioned device for detecting mtDNA alterations; a temperature controller disposed surrounding the periphery of the device to control a temperature of the device; and an analysis device disposed over the device to detect signals emitting from the device.
In the system of the present invention, the temperature controller is used to control the temperature of the device, in order to perform the detection process such as the DNA amplified reaction. Herein, the temperature controller may comprise: a sensing module and a control module. The sensing module can detect the temperature of the device, and then the temperature of the device is increased or decreased by the control module.
In addition, the system for detecting mtDNA alterations of the present invention may further comprise: a heater/cooler device, which is disposed under the device for detecting mtDNA alterations and connects to the temperature controller. The heater/cooler device can change the temperature of the device rapidly. In the present invention, the heater/cooler device is not particularly limited, and can be any heater/cooler device generally used in the art, such as a thermoelectric cooler (TE cooler), a hot plate and a MEMS heater. Preferably, the heater/cooler device used in the present invention is a TE cooler. It is because that the TE cooler can increase and decrease temperature rapidly. In addition, the TE cooler further can increase and decrease temperature in large area, and the temperature range thereof is broad (−10° C. to 200° C.).
Furthermore, in the system for detecting mtDNA alterations of the present invention, the analysis device can be selected according to the DNA signal to be detected. The analysis device used in the present invention can be any analysis device generally used in the art, such as an electrophoresis device, a fluorescence device and a UV-light detecting device. Preferably, the analysis device used in the system of the present invention is a fluorescence device. More preferably, the analysis device used in the system of the present invention is a fluorescence device equipped with a photomultiplier tube (PMT).
When magnetic beads are used to extract the mitochondria genes in the device for detecting mtDNA alterations of the present invention, the system for detecting mtDNA alterations of the present invention may further comprise an electromagnetic controller, which provides a magnetic field to the device for detecting mtDNA alterations. Hence, when the electromagnetic controller provides a magnetic field to the device, the magnetic beads can be absorbed by the magnetic field. In this case, it is unnecessary to perform an additional separation step to obtain the purpose of extracting the mitochondria genes.
Except for the aforementioned device and system for detecting mtDNA alterations, the present invention further provides a method for detecting mtDNA alterations, and this method is particularly suitable for the device and the system of the present invention. However, the method of the present invention is not limited to be applied to the device and the system of the present invention, and it can also be applied to other devices and systems.
The method for detection mtDNA alterations of the present invention comprises the following steps: (A) providing a separation element and a sample contained with mitochondria genes, wherein the separation element is modified with a DNA fragment for recognizing mitochondria genes; (B) mixing the sample and the separation element to separate the mitochondria genes from the sample through a binding between the DNA fragment of the separation element and the mitochondria genes in the sample, and sequentially obtaining a detecting sample contained with the mitochondria genes; (C) dividing the detecting sample into a comparison group and a detection group; (D) adding an amplification solution into both the comparison part and the detection part respectively and further adding a restriction enzyme into the detection group to perform a DNA amplified reaction, wherein the amplification solution comprises a labeling reagent and a primer pair; and (E) detecting amounts of the labeling reagent in the comparison group and the detection group respectively after the DNA amplified reaction, wherein when the amount of the labeling reagent in the comparison part is different from that in the detection group, it indicates that a mtDNA alteration is present in the sample.
It should be noted that the primer pairs added into the comparison group and the detection group are the same primer pairs. Hence, when the method of the present invention is used, the purpose of detecting mtDNA alterations can also be accomplished at the expense of only a single primer pair. However, when the conventional method is used, two primer pairs has to be used, wherein one primer pair is paired with normal sequence of a target gene region, and the other primer pair is paired with mutated sequence of the target gene region. Hence, the method of the present invention is more convenient than the conventional method.
More specifically, in the method of the present invention, when the amount of the labeling reagent in the detection group is less than that in the comparison group, it indicates that a mtDNA alteration is present in the sample.
In addition, the restriction enzyme used in the step (D) can be a restriction enzyme capable of digesting DNAs in a target gene region of the mitochondria genes, and the target gene region can be a mutation region to be detected in the mitochondria genes. Herein, the restriction enzyme is selected according to the sequence of the target gene region in the mitochondria genes, and can be any restriction enzyme generally used in the art.
In the method for detection mtDNA alterations of all aspects of the present invention, the primer pair used in the step (D) is a primer pair to amplify a target gene region of the mitochondria genes. Herein, any DNA amplified reaction such as a polymerase chain reaction (PCR) and a real-time polymerase chain reaction (real-time PCR) can be used in the method of the present invention to amplify the mitochondria genes.
In addition, the labeling reagent used in the step (D) can be a dye detected with fluorescence or UV-light. Preferably, the labeling reagent is a fluorescent dye. More preferably, the labeling reagent is a fluorescent dye such as SybrGreen that can emit fluorescence when it chelates into major grooves of DNAs.
The methods for detection mtDNA alterations of all aspects of the present invention may further comprise a step (B′) after the step (B): dividing the detecting sample into a mitochondria gene-checking group and a detecting sample group, and detecting whether the mitochondria genes are present in the mitochondria gene-checking group or not. In this case, the step (C) is: dividing the detecting sample group into a comparison group and a detection group. Only when it is confirmed that the mitochondria genes are present in the mitochondria gene-checking group, the sequential steps (C) and (D) are performed. In the step (B′), any conventional method generally used in the art such as a PCR and a real-time PCR can be used to check whether the mitochondria genes are present in the mitochondria gene-checking group or not. When the PCR or the real-time PCR are used to check the mitochondria genes in the mitochondria gene-checking group, the used primer pair is not particularly limited, as long as it can amplify the mitochondria gene. Preferably, the used primer pair can pair with a D-loop region of the mitochondria genes.
In the method for detecting mtDNA alterations of all aspects of the present invention, the separation element used in the step (A) can be any separation element generally used in the art, such as a magnetic bead or a polymer bead. When the magnetic bead is used as the separation element, the mitochondria genes can be extracted with a magnetic field. When the polymer bead is used as the separation unit, the extraction of the mitochondria gene can be accomplished by gravitational or centrifugal force.
The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.
In the following embodiments of the present invention, the figures are simplified perspective views. However, only the elements relate to the present invention are shown in these figures. These shown embodiments are not actual performance aspects. The numbers, the shapes and the sizes of the shown elements are only one selective design, and they may be more complicated.
Embodiment 1
As shown in
In addition, in the device for detecting mtDNA alterations of the present embodiment, the purification unit 21 further comprises a washing-solution storage chamber 213, which connects to the first reaction chamber 211 through a third pump 215. In addition, a connection path 217 is disposed between the third pump 215 and the washing-solution storage chamber 213, and a washing solution (such as water) contained in the washing-solution storage chamber 213 is passed through the connection path 217 and introduced into the first reaction chamber 211 by the third pump 215. Herein, the third pump 215 is a peristaltic micropump.
In the device for detecting mtDNA alterations of the present embodiment, the detection unit 22 further comprises: a mitochondria-gene checking chamber 223 and a temporary chamber 222, wherein the mitochondria-gene checking chamber 223 and the temporary chamber 222 connect to the second reaction chamber 221 through a fourth pump 226, and the temporary chamber 222 also connects to the detecting-sample chamber 224 through the second pump 228. Herein, connection paths between the mitochondria-gene checking chamber 223/the temporary chamber 222 and the fourth pump 226 are back pressure paths 227, and the sample contained in the second reaction chamber 221 can be divided equally into the mitochondria-gene checking chamber 223 and the temporary chamber 222. In addition, connection paths between the temporary chamber 222 and the second pump 228 and between the second reaction chamber 221 and the fourth pump 226 are back pressure paths.
Furthermore, in the device for detecting mtDNA alterations of the present embodiment, the detection unit 22 further comprises: a comparison sample chamber 225, which connects to the temporary chamber 222 through the second pump 228. Herein, connection paths between the comparison sample chamber 225/the detecting-sample chamber 224 and the second pump 228 are back pressure paths, and the sample contained in the temporary chamber 222 can be divided equally into the comparison sample chamber 225 and the detecting-sample chamber 224 to perform the sequential detection process.
In addition, in the device for detecting mtDNA alterations of the present embodiment, the detection unit 22 further comprises: plural gas inlets 23, wherein each gas inlets 23 respectively connects to the first pump 214, the second pump 228, the third pump 215, the forth pump 226, the micro-mixture unit 216, the connection path between the second reaction chamber 221 and the fourth pump 226, the connection path between the mitochondria-gene checking chamber 223 and the temporary chamber 222, the connection path between the temporary chamber 222 and the second pump 228, and the connection path between the detecting-sample chamber 224 and the comparison sample chamber 225. Compressed gas can be introduced from the gas inlets 23 to facilitate the sample flowing between each connection path.
Then, as shown in
In addition, the temperature controller 4 of the present embodiment comprises: a temperature sensing module 41, a power supply 42, and a control module 43. The temperature sensing module 41 can detect the temperature of the device 2. The control module 43 can control the power supply 42 providing power to the heater/cooler device 3 based on the temperature detected by the temperature sensing module 41 to increase or decrease the temperature of the device 2. Herein, the detailed structure of the temperature controller 4 is only one embodiment of the present invention, and the present invention is not limited thereto.
Furthermore, the analysis device 5 of the present invention is a fluorescence device, which comprises: a processor 51 and a photomultiplier tube 52. The photomultiplier tube 52 provides an excitation light to the device 2 and receives signals emitted from the device 2, and the signals received by the photomultiplier tube 52 are outputted by the processor 51. The photomultiplier tube 52 can convert photo signals into electric signals, and amplify the electric signals to obtain the signals emitted from the device 2. Herein, the detailed structure of the analysis device 5 is only one embodiment of the present invention, and the present invention is not limited thereto.
The system for detecting mtDNA alterations of the present embodiment further comprises an electromagnetic controller 6, which provides a magnetic field to the device 2. Herein, the electromagnetic controller 6 comprises: a power supply 61, a vacuum pump 62, a digital controller 63, and a solenoid valve 64. The power supply 61 provides power to the vacuum pump 62 and the digital controller 63, and then signals are transmitted to the solenoid valve 64 to output a magnetic field to the device 2. Herein, the detailed structure of the electromagnetic controller 6 is only one embodiment of the present invention, and the present invention is not limited thereto.
Furthermore, the system for detecting mtDNA alterations of the present embodiment further comprises an object lens 7. The excitation light emitted from the analysis device 5 can be focused on a detection region by the object lens 7, and then the object lens 7 can receive signals emitted from the excited sample to improve the detection effect of the analysis device 5.
Hereafter, the device, the system and the method for detecting mtDNA alterations of the present embodiment are explained accompanied with
First, as shown in
Next, as shown in
As shown in
The device, the system and the method for detecting mtDNA alterations according to Embodiment 1 are used in the present example. The device, the system and the method used in the present example are the same as those in Embodiment 1, so the detailed description is omitted herein.
Mutated Mitochondria Gene (mtDNA) Group
Magnetic beads (1.08 μm, Dynabeads® MyOne™ Carboxylic Acid, Invetrogen, USA) surface-immobilized with specific DNA fragments were used, wherein the sequence of the DNA fragments was TGGTATTTTCGTCTGGGGGGTATG (SEQ ID NO: 1); the washing solution was de-ionized water (DI); the cell lines were Lu03 (cell lines with A3243G point-mutated mtDNA); the primer pair of the amplification solution added into the mitochondria-gene checking chamber had sequences shown in the following Table 1 (as shown in SEQ ID NOs: 4 and 5); the used restriction enzyme is ApaI, which can digest the sequence with A3243G point-mutated mtDNA but not digest normal mtDNA without mutations); the DNA amplified reaction was a PCR; the analysis device comprised a PMT system (C3830, R928; Hamamatsu Photonics, Japan); eight electromagnetic valves (EMVs; S070M-5BG-32, SMC, Japan) were used; the heater/cooler device was a TE cooler, wherein the working temperatures were set 60° C. for cell lysis, 37° C. for enzyme digestion and 95° C., 58° C. and 72° C. for PCR amplification; the labelling reagent was a fluorescent reagent contained in KAPA SYBR® FAST qPCR kits (KK4603, Kapa Biosystems, MA, USA); and the target region of the mitochondria genes was A3243G.
Normal mtDNA Group
The conditions of this group were the same as those in the mutated mtDNA group, except that the cell lines were Lu02 (cell lines with normal mtDNA).
The purpose of the present comparative example is to confirm that the method, the device and the system for detecting mtDNA alterations of Embodiment 1 can accomplish similar effect as those generally used in the art. In the present comparative example, a conventional real-time PCR machine was used, in which the reaction temperature, the sample, the sequences of the primer pair including the mutated mtDNA group and the normal mtDNA group, and the restriction enzyme were the same as those used in Example 1. The results are shown in
As show in
The device, the system and the method for detecting mtDNA alterations according to Embodiment 1 were used in the present example to detect mtDNA with 0, 30, 60, 90, 100% mutation degree. The mutation degree means the ratio of mutated mtDNA in total amount of extracted mtDNA in the sample.
The real-time PCR machine of Comparative Example 1 was used in the present comparative example to detect mtDNA with 0, 30, 60, 90, 100% mutation degree.
As shown in
The results of Examples 1-2 and Comparative Examples 1-2 will show that the method, the device and the system for detection mtDNA alterations of Embodiment 1 can incur similar effect to the conventional device.
In conclusion, the method, the device and the system for detection mtDNA alterations of the present invention indeed can work to detect mtDNA alterations. More particularly, the device provided by the present invention can perform cell lysis, enzyme digestion and optical detection on a single device, so that complex treatment and detection can be simplified. In addition, the device of the present invention is a cheap, micro-sized and disposable device, so detection on mtDNA alterations can be performed rapidly and easily accessible to the public. Hence, the device of the present invention can further be applied in clinical settings to determine whether a subject is at risk of contracting diseases related to mitochondria gene alterations.
Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20100196881 | Callison et al. | Aug 2010 | A1 |
| Entry |
|---|
| Maricic. PLoS One. 2010. 5(11): e14004. |
| Wallace. PNAS. 1994. 91(19): 8739-8746. |
| Guttman et al. Clinical Chemistry. 2001. 47(8): 1469-1472. |
| Chen-Min Chang,Dar-Bin Shieh, Li-Fang Chiu and Gwo-Bin Lee, The 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Oct. 2-6, 2011,Washington State Convention Center 800 Convention Place Seattle, USA. |
| Number | Date | Country | |
|---|---|---|---|
| 20140093871 A1 | Apr 2014 | US |
