Patent Application
20150247186
1. Technical Field
The present invention relates to a nucleic acid amplification method.
2. Related Art
In recent years, as a result of the development of technologies utilizing genes, medical treatments utilizing genes such as genetic diagnosis or genetic therapy are drawing attention, and in addition, many methods utilizing genes in the determination of breed varieties or breed improvements have also been developed in agricultural and livestock industries. As technologies for utilizing genes, nucleic acid amplification methods such as a PCR (Polymerase Chain Reaction) method are widely used. Nowadays, the PCR method has become an indispensable technology in the elucidation of information regarding biological materials.
The PCR method is a method of amplifying a target nucleic acid by subjecting a reaction mixture containing a target nucleic acid to be amplified and a reagent to a thermal cycle. The thermal cycle is a process of subjecting a reaction mixture to two or more stages of temperatures periodically. In the PCR method, a method of performing a two- or three-stage thermal cycle is generally used.
In recent years, a method of simultaneously amplifying two types of target DNAs such as influenza virus DNAs has been effectively developed (see Ozdemir M., Yavru S., Baysal B. “Comparison of the detection of influenza A and B viruses by different methods.” J. Int. Med. Res. 2012; 40(6): 2401-8). In this case, two types of target DNAs are amplified in one tube, and therefore, a reaction is generally performed by adding two types of target DNAs and two types of PCR reagents such as primers to one solution placed in the tube.
An advantage of some aspects of the invention is to provide a novel nucleic acid amplification method capable of efficiently performing multiple types of nucleic acid amplification reactions in one vessel.
An aspect of the invention is directed to a method of performing multiple nucleic acid amplification reactions in a single common vessel including: forming a first liquid droplet containing a first reaction reagent for amplifying a first nucleic acid in one vessel containing a silicone oil to which a fluoro-modified silicone resin is added; forming a second liquid droplet containing a second reaction reagent for amplifying a second nucleic acid in the one vessel; and performing a nucleic acid amplification reaction in the one vessel by fitting the vessel in a nucleic acid amplification reaction apparatus while maintaining the first liquid droplet independent of the second liquid droplet and then operating the nucleic acid amplification reaction apparatus. The first liquid droplet and the second liquid droplet may contain the same type of nucleic acid sample or may contain different types of nucleic acid samples. The fluoro-modified silicone resin may be XS66-C1191. Further, the first liquid droplet may contain a first fluorescent dye, and the second liquid droplet may contain a second fluorescent dye which is different from the first fluorescent dye. Further, the addition amount of the fluoro-modified silicone resin may be 1% by mass or more and 50% by mass or less.
Another aspect of the invention is directed to a nucleic acid amplification reaction vessel, which is for performing multiple nucleic acid amplification reactions, and contains: a silicone oil to which a fluoro-modified silicone resin is added; a first liquid droplet containing a reaction reagent for amplifying a first nucleic acid; and a second liquid droplet containing a reaction reagent for amplifying a second nucleic acid. The fluoro-modified silicone resin may be XS66-C1191. Further, the first liquid droplet may contain a first fluorescent dye, and the second liquid droplet may contain a second fluorescent dye which is different from the first fluorescent dye. Further, the addition amount of the fluoro-modified silicone resin may be 1% by mass or more and 50% by mass or less.
Still another aspect of the invention is directed to a nucleic acid amplification apparatus fitted with any of the vessels described above.
According to the aspects of the invention, a novel nucleic acid amplification method capable of efficiently performing multiple types of nucleic acid amplification reactions in one vessel.
Embodiments of the invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements.
The object, characteristics, and advantages of the invention as well as the idea thereof will be apparent to those skilled in the art from the description given herein, and the invention can be easily reproduced by those skilled in the art based on the description given herein. It is to be understood that the embodiments, specific examples, and the like of the invention described below are to be taken as preferred embodiments of the invention, and are presented for illustrative or explanatory purposes and are not intended to limit the invention. It is further apparent to those skilled in the art that various changes and modifications may be made based on the description given herein within the intent and scope of the invention disclosed herein.
When there is no particular description in the embodiments or examples, methods described in standard protocols such as J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified or altered methods thereof are used. When using a commercially available reagent kit or a measuring apparatus, unless otherwise particularly indicated, a protocol attached thereto is used.
The method according to an embodiment of the invention includes: forming a first liquid droplet containing a first reaction reagent for amplifying a first nucleic acid in one vessel containing a silicone oil to which a fluoro-modified silicone resin is added; forming a second liquid droplet containing a second reaction reagent for amplifying a second nucleic acid in the vessel; and performing a nucleic acid amplification reaction in the vessel by fitting the vessel in a nucleic acid amplification reaction apparatus while independently maintaining the first liquid droplet and the second liquid droplet and then operating the nucleic acid amplification reaction apparatus. According to this method, multiple nucleic acid amplification reactions can be performed in one vessel. In this method, the nucleic acid amplification reaction may be performed using a reaction reagent for amplifying a nucleic acid, and it is not always necessary to amplify the nucleic acid after the reaction.
The concentration of the fluoro-modified silicone resin is not particularly limited, but is preferably 1% by mass or more and 50% by mass or less, more preferably 1% by mass or more and 20% by mass or less, and further more preferably 1% by mass or more and 5% by mass or less. The type of the fluoro-modified silicone resin is not particularly limited, but is preferably XS66-C1191 (Momentive, Inc.). The type of the silicone oil is not particularly limited, and examples thereof include dimethyl silicone oil, methyl hydrogen silicone oil, methylphenyl silicone oil, and cyclic dimethyl silicone oil. The nucleic acid amplification method is not particularly limited and may be any method as long as the method is commonly used by those skilled in the art, but it is preferably an elevating-type PCR apparatus (see, for example, JP-A-2012-115208). By incorporating a fluorescent dye having a different emission wavelength in each liquid droplet, even when emitted fluorescence is measured simultaneously, each nucleic acid can be distinguished, and thus, so-called multiplex PCR or the like can be achieved.
Here, the first nucleic acid and the second nucleic acid which can be amplified with the respective reaction reagents may be the same or different. In the case where the first nucleic acid and the second nucleic acid are the same type of nucleic acid and the first reaction reagent and the second reaction reagent are the same reaction reagent, for example, by incorporating a sample containing the nucleic acid in both liquid droplets and performing an amplification reaction, it is possible to obtain a reaction product in a larger amount than in the case where a reaction is performed with one liquid droplet. In the case where the first nucleic acid and the second nucleic acid are the same type of nucleic acid and the first reaction reagent and the second reaction reagent are different from each other, for example, by incorporating a sample containing the nucleic acid in both liquid droplets and performing an amplification reaction, it is possible to examine which reaction reagent can more efficiently achieve amplification or the like. According to this, for example, by preparing primers having a partially different sequence for the same gene, it is possible to examine which primer functions more efficiently.
In the case where the first nucleic acid and the second nucleic acid are different from each other and the first reaction reagent and the second reaction reagent are the same reaction reagent, that is, in the case where the reaction reagent can amplify multiple nucleic acids, for example, it is possible to simultaneously amplify two types of nucleic acids in one vessel and moreover with one type of reaction reagent.
In the case where the first nucleic acid and the second nucleic acid are different from each other and preferred conditions for the first reaction reagent and the second reaction reagent are different with respect to each of the nucleic acids, for example, in the case where a sample in which it is unknown whether a nucleic acid contained therein is the first nucleic acid or the second nucleic acid is incorporated in both liquid droplets, it is possible to examine whether the nucleic acid is the first nucleic acid or the second nucleic acid. According to this, for example, it is possible to use them for identifying the type of influenza virus or the like. Alternatively, by incorporating a sample containing the first nucleic acid and a sample containing the second nucleic acid in liquid droplets separately, it is possible to simultaneously amplify two types of nucleic acids in one vessel.
In order to examine whether background amplification occurs due to a primer dimer or a nucleic acid or the like mixed in the reaction reagent in one liquid droplet or both liquid droplets, a reaction may be performed without incorporating a nucleic acid sample.
A reagent contained in the reaction reagent is not particularly limited, and examples thereof include reagents commonly used in nucleic acid amplification such as a buffer, a DNA polymerase, dNTPs, a primer, and depending on the amplification reaction, a probe. A primer or a probe may be fluorescently labeled.
The vessel used here is preferably a tube, and may be a micro-centrifuge tube or a tube designed for PCR.
According to one embodiment of the invention, a nucleic acid amplification reaction vessel contains: a silicone oil to which a fluoro-modified silicone resin is added; a first liquid droplet containing a reaction reagent for amplifying a first nucleic acid; and a second liquid droplet containing a reaction reagent for amplifying a second nucleic acid. By using this vessel, as described in (1), various nucleic acid amplification reactions can be performed for one or multiple nucleic acid samples. In the same manner as in (1), this vessel is preferably a tube, and may be a micro-centrifuge tube or a tube designed for PCR.
Hereinafter, one example of an elevating-type PCR apparatus will be described in detail.
This elevating-type PCR apparatus 1 includes the main body 10 and a driving mechanism 20 as shown in
The fitting section 11 is configured to fit the nucleic acid amplification reaction tube 100, which will be described later. As shown in
This elevating-type PCR apparatus 1 includes a structure in which the nucleic acid amplification reaction tube 100 is held at a predetermined position with respect to the first heating section 12 and the second heating section 13. More specifically, as shown in
When the nucleic acid amplification reaction tube 100 is fitted in the fitting section 11, the first heating section 12 heats the first region 111 of the nucleic acid amplification reaction tube 100, which will be described later, to a first temperature. In the example shown in
The first heating section 12 may include a mechanism that generates heat and a member that transfers the generated heat to the nucleic acid amplification reaction tube 100. In the example shown in
When the nucleic acid amplification reaction tube 100 is fitted in the fitting section 11, the second heating section 13 heats the second region 112 of the nucleic acid amplification reaction tube 100 to a second temperature that is different from the first temperature. In the example shown in
In this embodiment, the temperatures of the first heating section 12 and the second heating section 13 are controlled by a temperature sensor (not shown) and a control section (not shown), which will be described later. The temperatures of the first heating section 12 and the second heating section 13 are preferably set so as to heat the nucleic acid amplification reaction tube 100 to a desired temperature. In this embodiment, by controlling the first heating section 12 at the first temperature and the second heating section 13 at the second temperature, the first region 111 of the nucleic acid amplification reaction tube 100 can be heated to the first temperature, and the second region 112 can be heated to the second temperature. The temperature sensor in this embodiment is a thermocouple.
The driving mechanism 20 is a mechanism that drives the fitting section 11, the first heating section 12, and the second heating section 13. In this embodiment, the driving mechanism 20 includes a motor (not shown) and a drive shaft (not shown), and the drive shaft is connected to the flange 16 of the main body 10. The drive shaft in this embodiment is provided perpendicular to the longitudinal direction of the fitting section 11, and when the motor is activated, the main body 10 is rotated about the drive shaft as the axis of rotation.
The elevating-type PCR apparatus 1 of this embodiment includes the control section (not shown). The control section controls at least one of the first temperature, the second temperature, the first period, the second period, and the number of thermal cycles, which will be described later. In the case where the control section controls the first period or the second period, the control section controls the operation of the driving mechanism 20, thereby controlling the period in which the fitting section 11, the first heating section 12, and the second heating section 13 are held in a predetermined arrangement. The control section may have mechanisms that independently control different items, or may control all items collectively. However, the control section in the elevating-type PCR apparatus 1 of this embodiment is an electronic control system and controls all of the above-described items. The control section of this embodiment includes a processor such as CPU (not shown) and a storage device such as an ROM (Read Only Memory) or an RAM (Random Access Memory). In the storage device, various programs, data, and the like for controlling the above-described respective items are stored. Further, the storage device has a work area for temporarily storing data in processing, processing results, and the like of various processes.
As shown in the example of
The main body 10 of this embodiment includes the fixing plate 19. The fixing plate 19 is a member that holds the fitting section 11, the first heating section 12, and the second heating section 13. In the example shown in
The elevating-type PCR apparatus 1 of this embodiment includes the lid 50. In the example shown in
It is preferred that the fixing plate 19, the bottom plate 17, the lid 50, and the flange 16 are formed using a thermally insulating material.
As shown in the example of
The flow channel 110 is formed such that the reaction mixture 140 moves in close proximity to opposed inner walls. Here, the term “opposed inner walls” of the flow channel 110 refers to two regions of a wall surface of the flow channel 110 having an opposed positional relationship. The phrase “in close proximity to” refers to a state in which the distance between the reaction mixture 140 and the wall surface of the flow channel 110 is close, and includes a case where the reaction mixture 140 is in contact with the wall surface of the flow channel 110. Therefore, the phrase “the reaction mixture 140 moves in close proximity to opposed inner walls” refers to “the reaction mixture 140 moves in a state of being close in distance to both of the two regions of a wall surface of the flow channel 110 having an opposed positional relationship”, for example, the reaction mixture 140 moves along the opposed inner walls.
In the example shown in
The first region 111 of the nucleic acid amplification reaction tube 100 is a partial region of the flow channel 110 which is heated to the first temperature by the first heating section 12. The second region 112 is a partial region of the flow channel 110 which is different from the first region 111 and is heated to the second temperature by the second heating section 13. In the nucleic acid amplification reaction tube 100 of this embodiment, the first region 111 is a region including one end portion in the longitudinal direction of the flow channel 110, and the second region 112 is a region including the other end portion in the longitudinal direction of the flow channel 110. In the example shown in
The flow channel 110 contains the silicone oil 130 and multiple liquid droplets of the reaction mixture 140. Since the silicone oil 130 is immiscible with the reaction mixture, that is, has a property that it is not mixed with the reaction mixture, the reaction mixture 140 is held in a state of a liquid droplet in the silicone oil 130 as shown in
Hereinafter, with reference to
The shuttle PCR is a method of amplifying a nucleic acid in a reaction mixture by subjecting the reaction mixture to a two-stage temperature process at a high temperature and a low temperature repeatedly. In the process at a high temperature, denaturation of a double-stranded DNA occurs and in the process at a low temperature, annealing (a reaction in which a primer is bound to a single-stranded DNA) and elongation (a reaction in which a complementary strand to the DNA is formed by using the primer as a starting point) occur.
In general, in the shuttle PCR, the high temperature is a temperature between 80° C. and 100° C. and the low temperature is a temperature between 50° C. and 70° C. The processes at the respective temperatures are performed for a predetermined period, and a period of maintaining the reaction mixture at a high temperature is generally shorter than a period of maintaining the reaction mixture at a low temperature. For example, the period for the process at a high temperature may be about 1 to 10 seconds, and the period for the process at a low temperature may be about 10 to 60 seconds, or a period longer than this range may be adopted depending on the condition of the reaction.
Since the appropriate period, temperature, number of cycles (number of times of repetition of the process at a high temperature and the process at a low temperature) varies depending on the type or amount of a reagent to be used, it is preferred to determine an appropriate protocol in consideration of the type of a reagent or the amount of the reaction mixture 140 before performing the reaction.
First, the nucleic acid amplification reaction tube 100 is fitted in the fitting section 11. In this embodiment, the nucleic acid amplification reaction tube 100, in which the reaction mixture 140 is introduced into the flow channel 110 previously filled with the silicone oil 130, and thereafter the flow channel 110 is sealed with the sealing section 120, is fitted in the fitting section 11. The introduction of the reaction mixture 140 can be performed using a micropipette, an ink-jet dispenser, or the like. In a state in which the nucleic acid amplification reaction tube 100 is fitted in the fitting section 11, the first heating section 12 is in contact with the nucleic acid amplification reaction tube 100 at a place including the first region 111 and the second heating section 13 is in contact with the nucleic acid amplification reaction tube 100 at a place including the second region 112.
Here, the arrangement of the fitting section 11, the first heating section 12, and the second heating section 13 is the first arrangement. As shown in
Subsequently, the nucleic acid amplification reaction tube 100 is heated by the first heating section 12 and the second heating section 13. The first heating section 12 and the second heating section 13 heat different regions of the nucleic acid amplification reaction tube 100 to different temperatures. That is, the first heating section 12 heats the first region 111 to the first temperature, and the second heating section 13 heats the second region 112 to the second temperature. Accordingly, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 111 and the second region 112 of the flow channel 110. Here, a temperature gradient in which the temperature is decreased from the first region 111 to the second region 112 is formed. The thermal cycling process of this embodiment is the shuttle PCR, and therefore, the first temperature is set to a temperature suitable for the denaturation of a double-stranded DNA, and the second temperature is set to a temperature suitable for the annealing and elongation.
Since the arrangement of the fitting section 11, the first heating section 12, and the second heating section 13 is the first arrangement, when the nucleic acid amplification reaction tube 100 is heated, the reaction mixture 140 is heated to the first temperature. When the first period has elapsed, the main body 10 is driven by the driving mechanism 20, and the arrangement of the fitting section 11, the first heating section 12, and the second heating section 13 is switched over from the first arrangement to the second arrangement. The second arrangement is an arrangement in which the second region 112 is located in a lowermost portion of the flow channel 110 with respect to the gravitational direction. In other words, the second region 112 is a region located in a lowermost portion of the flow channel 110 with respect to the gravitational direction when the fitting section 11, the first heating section 12, and the second heating section 13 are in a predetermined arrangement that is different from the first arrangement. In the elevating-type PCR apparatus 1 of this embodiment, by the control of the control section, the driving mechanism 20 rotatably drives the main body 10. When the flanges 16 are rotatably driven by the motor by using the drive shaft as the axis of rotation, the fitting section 11, the first heating section 12, and the second heating section 13 which are fixed to the flanges 16 are rotated. Since the drive shaft is a shaft extending in the direction perpendicular to the longitudinal direction of the fitting section 11, when the drive shaft is rotated by the activation of the motor, the fitting section 11, the first heating section 12, and the second heating section 13 are rotated. In the example shown in
Here, the positional relationship between the first region 111 and the second region 112 with respect to the gravitational direction is opposite from that of the first arrangement, and therefore, the reaction mixture 140 moves from the first region 111 to the second region 112 by the gravitational force. After the arrangement of the fitting section 11, the first heating section 12, and the second heating section 13 has reached the second arrangement, when the operation of the driving mechanism 20 is stopped, the fitting section 11, the first heating section 12, and the second heating section 13 are held in the second arrangement. When the second period has elapsed in the second arrangement, the main body 10 is rotated again. A nucleic acid amplification reaction is performed by rotating the main body 10 while switching over between the first arrangement and the second arrangement in this manner until the number of thermal cycles has reached a predetermined number of cycles.
According to another embodiment of the invention, it is possible to independently form water-soluble liquid droplets in one tube containing a silicone oil to which XS66-C1191 is added in an amount of 1% by mass or more and 50% by mass or less. As described in the Examples, even after the tube is subjected to a harsh reaction such as a nucleic acid amplification reaction, the independence of the liquid droplets is maintained. Therefore, it is also considered that in a reaction other than the nucleic acid amplification reaction, by forming multiple liquid droplets in a silicon oil, and subjecting different samples to the reaction in each liquid droplet, a multiplex reaction can be efficiently performed. Here, the conditions for XS66-C1191 and the silicone oil are as described above.
The nucleic acid amplification reaction tube of this Example has a cylindrical outer shape, and has a flow channel in the shape of a cylinder with an inner diameter of 2 mm and a length of 25 mm. First, the nucleic acid amplification reaction tube was filled with 130 μm of a solution obtained by adding a fluoro-modified silicone resin (XS66-C1191, manufactured by Momentive, Inc.) in an amount of 1, 5, 20, 50, 80, or 100% by mass to dimethyl silicone oil (KF-96L-2cs, manufactured by Shin-Etsu Silicone Co., Ltd.). Then, by using a micropipette, three droplets (1 μL each) of water colored with a color marker (loading buffer, manufactured by Takara Bio, Inc.) were introduced into the flow channel. As a result, as shown in
(2) PCR with Two Liquid Droplets
Subsequently, PCR was performed by using the same tube and introducing reaction mixtures having the following composition into KF-96L-2cs containing 20% by mass of XS66-C1191 in an amount of 1 μL each. Incidentally, an amplification reaction was performed with respect to the case where InfA plasmid was added as the plasmid to both liquid droplets (graph in
As a control experiment, PCR was performed with one liquid droplet. A reaction mixture used has the following composition (reaction mixture 3). Incidentally, an amplification reaction was performed with respect to the case where InfA plasmid was added as the plasmid (graph in
The sequences of the primer and the probe are as follows.
After the reaction mixture was treated at 98° C. for 10 seconds, 50 thermal cycles were performed under the following conditions: 98° C. for 5 seconds, and 52° C. for 20 seconds.
Even after the 50 thermal cycles were performed, the two liquid droplets did not mix with each other and existed independently. The measurement results of fluorescence during the reaction are shown in
As apparent from the comparison between
The entire disclosure of Japanese Patent Application No. 2014-038659 filed Feb. 28, 2014 is expressly incorporated by reference herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-038659 | Feb 2014 | JP | national |
