Patent Publication Number: US-2013248366-A1

Title: Methods and apparatus for amplifying nucleic acids

Description:
The invention relates to methods and microfluidic devices for amplifying nucleic acids, to an improved microfluidic device and a method of operating a microfluidic device, and to a system comprising detection apparatus and a microfluidic device. 
     WO 2010/041088 describes a microfluidic device and a method of operating the microfluidic device. The microfluidic device has a reaction chamber in which a PCR reaction is performed. The reaction chamber is filled with a gel and the reagents necessary for performing the PCR reaction are held within the matrix of the gel until the microfluidic device is used. The microfluidic device can be stored with the reagents in a stable form prior to use. The microfluidic device is also suitable for automated use. The microfluidic device is provided with a separation chamber for separation of DNA to be amplified from other cell components. 
     According to a first aspect of the invention, there is provided, a method of amplifying nucleic acid, comprising: providing a microfluidic device having a space therein, the space being filled with a gel medium, at least one reagent for carrying out a PCR reaction being supported within the matrix of the gel medium within the space; bringing a nucleic acid containing sample into contact with the gel medium; performing PCR amplification of nucleic acid from the sample in the space using the at least one reagent; and wherein the sample brought into contact with the gel medium comprises whole cells or cell lysate without any prior separation of the nucleic acid from other components of the cells. 
     Preferably the method also includes analysing nucleic acid products of the PCR amplification within the microfluidic device. For example, the nucleic acid products may be analysed by performing electrophoretic separation of the PCR products. 
     The sample may be a buccal swab sample or a blood sample. 
     In accordance with a second aspect of the invention, there is provided a microfluidic device for amplification of nucleic acid, comprising: a space filled with a gel medium; the space also containing at least one reagent for carrying out a PCR reaction, the at least one reagent being supported within the matrix of the gel medium; an opening extending from an exterior surface of the microfluidic device to the gel medium in the space; and wherein the opening is devoid of any means for separating nucleic acid from other cellular components. 
     Preferably, the microfluidic device comprises a channel in fluid communication with the space, the channel containing a separation medium for separating nucleic acid products of a PCR reaction. 
     For both the first and second aspects of the invention, the gel medium may contain at least one of nucleoside triphosphates, primer nucleic acid and polymerase enzyme within the gel matrix. Preferably all of these reagents are supported within the matrix of the gel medium. 
     For both the first and second aspects of the invention, the gel medium preferably has a homogenous composition. In addition the PCR reagents within the matrix of the gel medium are preferably homogenously distributed within the gel medium. 
     In an especially preferred embodiment of the second aspect of the invention, the microfluidic device comprises a channel and a well extending from the exterior surface to the channel, a gel being provided in the channel and optionally extending partially into the well, a liquid space devoid of the gel for receiving a liquid, the liquid space lying at least partially in the well and extending to the gel so that a liquid filling the liquid space contacts the gel, and an electrode receivable in the well so as to lie at least partially in the liquid space. 
     In accordance with a third aspect of the invention, there is provided a microfluidic device comprising a channel and a well extending from an exterior surface of the device to the channel, a gel being provided in the channel and optionally extending partially into the well, a liquid space devoid of the gel for receiving a liquid, the liquid space lying at least partially in the well and extending to the gel so that a liquid filling the liquid space contacts the gel, and an electrode receivable in the well so as to lie at least partially in the liquid space. 
     In accordance with a fourth aspect of the invention, there is provided a method of operating a microfluidic device comprising: providing a microfluidic device having a channel containing a gel; applying a voltage using an electrode to cause electrokinetic movement along the channel; and the electrode being in contact with an electrically conductive liquid in a space devoid of the gel and the liquid contacting the gel. The electrokinetic movement may comprise electroosmotic movement or electrophoretic movement or a mixture of the two. 
     In accordance with a fifth aspect of the invention there is provided a system comprising detection apparatus and a microfluidic device, the detection apparatus having detection means and the microfluidic device having an analysis region, the detection apparatus having at least one fixed locator and at least one locator moveable against a resilient bias, the microfluidic device being holdable by the detection apparatus with the locators contacting the microfluidic device and the at least one biased locator urging the microfluidic device against the at least one fixed locator so as to locate the microfluidic device in a predetermined position relative to the detection apparatus, and wherein when the microfluidic device is so held the detection means and the analysis region are mutually positioned for operation of the detection means on the analysis region. 
     Preferably, the microfluidic device has a base surface and the detection apparatus has a supporting surface, the base surface lying against the supporting surface when the microfluidic device is held. 
     In a preferred embodiment, the microfluidic device has four side edges arranged in a rectangle. The detection apparatus has at least two of the fixed locators and at least two of the resiliently biased locators. The arrangement is such so that when the microfluidic device is held by the detection apparatus, the at least two fixed locators contact two adjacent ones of the side edges and the at least two resiliently biased locators contact a different two adjacent ones of the four side edges. 
     The analysis region may include a channel in which an analyte is detected using a beam emitted by the detection means. In this case, the width of the beam is greater than the width of the channel. When the microfluidic device is held by the detection apparatus, the variation in position of the mierofluidic device relative to the detection apparatus is sufficiently small in relation to the relative widths of the beam and the channel so that the position variation does not affect the detection of the analyte in the channel. 
    
    
     
       The following is a more detailed description of embodiments of the invention, by way of example, reference being made to the appended schematic drawings in which: 
         FIG. 1  is a plan view from above of a microfluidic device; 
         FIG. 2  is a cross-sectional view of the microfluidic device of  FIG. 1 , taken on the line A-A in  FIG. 1 ; and 
         FIG. 3  is a plan view from above showing the microfluidic device of  FIGS. 1 and 2  while the microfluidic device is held by detection apparatus. 
     
    
    
     As seen in  FIG. 1 , the microfluidic device  10  is rectangular and may, by way of example, have a length of about 120 mm and a width of about 60 mm. Referring to  FIG. 2 , the microfluidic device  10  is formed from an upper rectangular glass plate  12  and a lower rectangular glass plate  14 . The lower surface  16  of the upper glass plate  12  is bonded to the upper surface  18  of the lower glass plate  14  by a suitable known method. Thermal bonding is the preferred method. The upper surface  20  of the upper plate  12  forms an upper external surface of the microfluidic device  10  and the lower surface  22  of the lower glass plate  14  forms a lower external surface of the microfluidic device  10 . 
     As seen in  FIG. 2 , the thickness (height as shown in  FIG. 2 ) of the upper glass plate  12  is greater than the thickness of the lower glass plate  14 . By way of example, the upper glass plate may have a thickness of  3 mm and the lower glass plate may have a thickness of 1 mm. 
     The microfluidic device  10  is provided with first, second, third and fourth wells  24 ,  26 ,  28 ,  30 . Each one of the wells  24 ,  26 ,  28 ,  30  serves to receive a respective electrode as will be discussed in more detail below. The first well  24  is shown in longitudinal (vertical) section in  FIG. 2 . As best seen in  FIG. 2 , each well takes the form of the cylindrical hole that has been drilled through the upper plate  12  between the upper and lower surfaces  20 ,  16  of the upper plate  12 . Each one of the first, second, third and fourth wells,  24 ,  26 ,  28 ,  30  may have a diameter of about 2 to 3 mm. 
     The microfluidic device  10  also has an internal chamber  32  in which a polymerase chain reaction (PCR) amplification process is performed, as will be discussed below in more detail. The PCR chamber  32  is cylindrical in shape and has been formed by drilling a cylindrical well part of the way into the upper glass plate  12 , starting from the lower surface  16  of the upper glass plate  12 , before binding together of the upper glass plate  12  and the lower glass plate  14 . 
     The microfluidic device  10  also has a sample collection well  34 . The sample collection well  34  has an upper cylindrical portion  36  with a greater diameter and a lower cylindrical portion  38  with a lesser diameter. The lower portion  38  of the sample collection well  34  extends between the upper portion  36  and the PCR chamber  32 , as shown in  FIG. 2 . The sample collection well  34  is formed by firstly drilling into the upper glass plate  12  from the upper surface  20  with a larger diameter drill. This forms the upper portion  36 . A smaller diameter drill is then used to drill between the upper portion  36  and the PCR chamber  32  so as to form the lower portion  38 . 
     The microfluidic device  10  also has a sample transfer channel  40  and a separation channel  42 . 
     As best seen in  FIG. 1 , the sample transfer channel  40  extends from the first well  24  to the PCR chamber  32  and from the PCR chamber  32  to the second well  26 . As seen in  FIG. 2 , the sample transfer channel  40  opens directly into the first well  24  and also opens directly into the PCR chamber  32 . Although not shown in the drawings, the sample transfer channel  40  opens directly into the second well  26 . The cross-sectional dimensions of the sample transfer channel  40  will generally be less than 500 micrometers (although larger dimensions may be used). For example, the sample transfer channel  40  may have a width of about 100 micrometers and a depth of about 20 micrometers. The sample transfer channel  40  is formed by forming a groove of appropriate cross-section and dimensions in the upper surface  18  of the lower plate  14 , before the upper plate  12  and the lower plate  14  are bonded together. On bonding of the two plates  12 ,  14 , the lower surface  16  of the upper plate  12  closes the groove to form the sample transfer channel  40 . 
     As seen in  FIG. 1 , the separation channel  42  extends from the third well  28  to the fourth well  30 . The separation channel  42  opens directly into both the third and fourth wells  28 ,  30 . The separation channel  42  has similar dimensions, and is formed in a similar manner, to the sample transfer channel  40 . 
     The sample transfer channel  40  and the separation channel  42  are in fluid communication with one another at an intersection  43  between the two channels  40 ,  42 . 
     In addition, the microfluidic device  10  is provided with four electrode plugs  44 , one of which is shown in  FIG. 2 . The four electrode plugs  44  are preferably identical to one another, but need not be so. Each electrode plug  44  is formed from a cap  46  made of electrically non conductive material and an electrode  48  which passes through the cap  46 . As shown in  FIG. 2 , the cap  46  is sized so that it can be inserted into one of the wells  24 ,  26 ,  28 ,  30  so as to form a tight seal. 
     The surfaces of the first, second, third and fourth wells  24 ,  26 ,  28 ,  30 , the PCR chamber  32 , the sample collection well  34  and the sample transfer and separation channels  40 ,  42  are silanised to reduce binding of nucleic acid to the glass during use. Silanisation is performed after the upper and lower glass plates  12 ,  14  have been connected together. The microfluidic device  10  is cleaned with water and dried with air before being left to dry in an oven at 90° C. overnight. The device  10  is then kept in a dessicator until silanisation is performed. (Similar measures are taken to treat the glassware used to hold the silanisation reagents.) Iso-octane (1000 microlitres) is mixed with trichloro (1H, 1H, 2H, 2H) perfluorooctyl silane (145 microlitres). The mixture is pumped into the microfluidic device  10  and moved through the channels  40 ,  42  and the wells/chambers  24 ,  26 ,  28 ,  30 ,  32 ,  34 . After leaving for 5 minutes, the mixture is removed by pumping air through the microfluidic device. The microfluidic device  10  is then washed with iso-octane followed by drying with air. Finally, acetone, then air, then water are pumped through the microfluidic device  10 . The microfluidic device  10  is dried in an oven for an hour before being filled with various gels as described below. 
     Firstly, the sample transfer channel  40  is filled with an agarose gel  50 . In order to form the agarose gel  50 , low melting point agarose is dissolved in nucleic acid free water and the solution is heated at 75° C. for ten minutes. The final concentration of the agarose is 1.5% (weight:weight). The agarose gel  50  is inserted into the sample transfer channel  40  as follows. Firstly, the third and fourth wells  28 ,  30  and also the upper portion  36  of the sample collection well  34 , are plugged with tight fitting plugs which occupy most of the space of the wells  28 ,  30 ,  36 . Then, when the gel has formed, but whilst the gel is still in molten form, the gel is injected under pressure into the sample transfer channel  40  through the first well  24 . During this process, the agarose gel  50  passes through the sample transfer channel  40  to the second well  26 . In view of the fact that the sample collection well  34  is plugged, the agarose gel  50  does not enter, or enters only to a small degree, into the PCR chamber  32  (which is in fluid communication with the sample transfer channel  40 ). Once the agarose gel  50  reaches the base of the second well  26  injection is stopped. The gel is then allowed to solidify within the sample transfer channel  40  and the first and second wells  24 ,  26  are cleaned of gel. 
     The separation channel  42  is then filled with polyethylene oxide gel  52 . The polyethylene oxide gel  52  is made by mixing polyethylene oxide to a concentration of 2.5% (weight:weight) in nucleic acid free Tris-EDTA buffer, by a prolonged stirring method. The gel  52  is introduced into the separation chamber  42  before it sets. In order to introduce the polyethylene oxide gel  52 , the first and second wells  24 ,  26  and the sample collection well  34  are plugged. The molten gel is then introduced under pressure into the separation channel  42  via the third well  28 . This process is continued until the polyethylene oxide gel reaches the base of the fourth well  30 . After the polyethylene oxide gel  52  has set, gel is removed from the third and fourth wells  28 ,  30 . 
     During the introduction of the polyethylene oxide gel  52  into the separation channel  42 , a small amount of agarose gel  50  is dislodged from the intersection  43  of the sample transfer channel  40  and the separation channel  42 . This portion of agarose gel  50  is carried to the fourth well  30  and does not serve any purpose. 
     In this example, the PCR chamber  32  is filled with a gel  54  which contains all the reagents necessary for performing PCR. The reagents are held within the matrix of the gel  54  within the PCR chamber  32 . The reagents are the normal reagents used for performing PCR amplification, as is well known. Hence, the PCR reagents include nucleic acid sequences that act as primers in order to amplify predetermined portions of sample nucleic acid that is introduced into the PCR chamber  32 . The reagents also include nucleoside triphosphates and a polymerase enzyme. 
     Preferably, the reagents will include a dye that binds to nucleic acid fragments that are produced in the PCR reaction. The dye may be, for example, a fluorescent dye or a dye having an intense absorption peak for colorimetric detection. The dye is used to aid detection of DNA fragments produced in the PCR reaction during subsequent separation of the DNA fragments, as discussed below. 
     By way of specific example, the PCR reagents may include the following components. The concentrations in the right hand column are the concentrations before dilution 1:1 with the gel, as described below. 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 1. autoclaved ultra-filtered water (pH 7.0) 
                 20.7 μL  
                 — 
               
               
                 2. 10x PCR Buffer* 
                 2.5 μL 
                 1x 
               
               
                 3. dNTPs mix (25 mM each nucleotide) 
                 0.2 μL 
                 200 μM 
               
               
                   
                   
                 (each nucleotide) 
               
               
                 4. primer mix (25 pmoles/μL each primer) 
                 0.4 μL 
                 0.4 μM 
               
               
                   
                   
                 (each primer) 
               
               
                 5. Taq DNA polymerase (native enzyme) 
                 0.2 μL 
                 1 Unit/25 μL 
               
               
                 6. genomic DNA template (100 ng/μL) 
                 1.0 μL 
                 100 ng/25 μL 
               
               
                   
               
            
           
         
       
     
     The gel containing the PCR reagents is formed as follows. Low melting point agarose is dissolved in nucleic acid free water at a concentration of 3% (weight:weight) and then heated at 75° C. for ten minutes. When the gel has formed and whilst the gel is still in molten form, the gel is mixed with an equal volume of a solution containing all of the reagents that are required in the PCR chamber  32  (the reagents being present in the solution at double the final required concentration). After thorough mixing, the still molten gel, together with the PCR reagents, is injected through the lower portion  38  of the sample collection well  34  into the PCR chamber  32 . The PCR gel  54  comes into contact with the agarose gel  50  contained in the sample transfer channel  40 . The PCR gel  54  is then allowed to set in the PCR chamber  32 . 
     As will be appreciated, the final concentration of the agarose in the PCR gel  54  is 1.5% (weight:weight). 
     The sample collection well  34 , and the first, second, third and fourth wells  24 ,  26 ,  28 ,  30  remain empty. 
     Once the microfluidic device  10  has been loaded with the gels and reagents as discussed above, it can be kept refrigerated at 4° C. for about four weeks before use. 
     The following is a description, by way of example, of one potential use of the microfluidic device  10 . In this example, the microfluidic device  10  is used to amplify DNA contained in human cells obtained by way of buccal swab for the purposes of DNA profiling. The primers contained in the PCR gel  54  are chosen to amplify predetermined loci of human DNA used in DNA profiling. After amplification, the DNA fragments produced by the PCR reaction are analysed by electrophoretic separation. 
     Firstly, the first, second, third and fourth wells  24 ,  26 ,  28 ,  30  are filled with a suitable electrically conductive buffer. An electrode plug  44  is then inserted into each well  24 ,  26 ,  28 ,  30  so that the electrode  48  of the plug lies within the buffer and the cap  46  of the plug seals the well as shown in  FIG. 2 . The electrodes  48  are used to apply voltages between the wells  24 ,  26 ,  28 ,  30  as discussed below. 
     A human cell sample is then taken by buccal swab. The sample bearing end of the swab is cut off and inserted into the sample collection well  34 . Hence, there will be whole human cells on the end of the swab in the sample collection well  34 . A suitable cell lysing solution is then introduced into the sample collection well  34 . For example, the lysing solution may be a conventional guanidine lysing solution. The lysing solution lyses cells on the end of the swab and the lysing solution, together with the cell lysate migrates through the lower portion  38  of the sample collection well  34  into the PCR gel  54  in the PCR chamber  32 . A cap may be inserted into the sample collection well  34 , both to seal the well  34  and also to press the solution and cell lysate into the PCR gel  54 . 
     During this process, the PCR gel  54  may act as a filter, preventing larger cell fragments from passing into the matrix of the PCR gel  54 . However, this is not essential and will depend on the physical characteristics of the PCR gel  54 . 
     Once the cell lysate has passed into the PCR gel  54 , the PCR reaction may be commenced. As is well known, the PCR reaction involves cycling between two or three different temperatures. In the current method, the temperature within the PCR chamber  32  is cycled by a suitable known method. Suitable methods are described, for example, in WO2010/041088. The temperature cycling may be by way of a Peltier heater or by way of microwave heating, for example. 
     After the desired number of PCR temperature cycles have been completed, the desired loci of the human DNA will have been amplified. The PCR product DNA fragments are held within the PCR chamber  32 . 
     A small amount of the PCR product DNA fragments is then loaded onto the polyethylene oxide gel  52  in the separation channel  42 . In order to achieve this, the DNA fragments produced by the PCR reaction are moved to the intersection  43  between the sample transfer channel  40  and the separation channel  42  electrophoretically, by applying appropriate voltages to the electrodes  48  inserted into the first, second, third and fourth wells  24 ,  26 ,  28 ,  30 . 
     For example, a positive voltage of 1,000 v may be applied to the second well  26  for 15 seconds while the first, third and fourth wells  24 ,  28 ,  30  are held constant at 0 v. 
     The DNA fragments are then electrophoretically separated in the polyethylene oxide gel  52  within the separation channel  42 . In order to achieve this, a positive voltage of 8,500 v is applied to the fourth well  30  for 25 minutes while the third well  28  is held at 0 v, the second well  26  at 500V and the first well  24  at 1000 v. 
     The DNA fragments move from the intersection  43  towards the fourth well  30 . The DNA fragments can be detected by known methods, for example either by fluorimetry or colorimetry, as they pass a predefined point in the separation channel  42 . For example, as shown in  FIG. 3 , the DNA fragments may be detected as they pass through an analysis region  56  provided towards the an end of the separation channel  42  located adjacent the fourth well  30 . 
     The method described above may be carried out readily in an automated manner, for example using the detection apparatus  58  shown in  FIG. 3 . The detection apparatus  58  includes suitable electronics (not shown) for applying, in a known manner, the required voltages (as described above) to the electrodes  48 . The detection apparatus  58  also includes a microcontroller (not shown) for controlling, in a known manner, the timing of the application of the required voltages to the electrodes  48 . In addition, the detection apparatus  58  contains detection means, such as a fluorimeter or a colorimeter, for detecting the DNA fragments (bound to dye if applicable) as they pass the analysis region  56  of the microfluidic device  10 . 
     As shown in  FIG. 3 , the detection apparatus  58  has an upper surface  60  on which is placed the lower surface  22  of the microfluidic device  10 . 
     In addition, the detection apparatus  58  has first, second, third and fourth fixed pegs  62 ,  63 ,  64 ,  65  and first and second moveable pegs  66 ,  68 . The first moveable peg  66  is moveable towards and away from the first and second fixed pegs  62 ,  63  and the second moveable peg  68  is moveable towards and away from the third and fourth fixed pegs  64 ,  65 . The first moveable peg  66  is spring loaded in the direction of the first and second fixed pegs  62 ,  63 . The second moveable peg  68  is spring loaded in the direction of the third and fourth fixed pegs  64 ,  65 . The pegs  62 ,  63 ,  64 ,  65 ,  66  and  68  are spaced so that the microfluidic device  10  can be located between the pegs as shown in  FIG. 3 . The first moveable peg  66  urges the microfluidic device  10  towards the first and second fixed pegs  62 ,  63 . The second moveable peg  68  urges the microfluidic device  10  towards the third and fourth fixed pegs  64 ,  65 . 
     In this way, the microfluidic device  10  is held precisely in a predetermined position on the upper surface  60  of the detection apparatus  58 . Only a very small degree of variation in the position of the microfluidic device  10  can occur. 
     The detection means (not shown) is positioned on the detection apparatus  58 , so as to be aligned with the analysis region  56  of the microfluidic device  10  when the microfluidic device  10  is located between the pegs  62 ,  63 ,  64 ,  65 ,  66 ,  68  as described above. 
     The detection means emits a beam of light (or other electromagnetic radiation) through the analysis region  56  in order to detect the DNA fragments. The beam is wider than the width of the separation channel  42 . In this way, the very small degree of variation in the position of the microfluidic device  10 , when the microfluidic device  10  is located between the pegs  62 ,  63 ,  64 ,  65 ,  66 ,  68 , will not influence detection of DNA fragments passing through the analysis region  56 . 
     A number of advantages ensue from the microfluidic device  10  and the amplification method described above. 
     Firstly, the microfluidic device  10  and the method of operation do not require separation of nucleic acid from other cell components prior to DNA amplification in the PCR chamber  32  (although some separation may optionally occur if the matrix of the PCR gel  54  filters out larger cell fragments). Various known microfluidic devices used for DNA amplification use a distinct DNA separation step to separate DNA from other cellular components. The elimination of such a separation step simplifies both the design of the microfluidic device and also the amplification method, making the method more suitable for automated use by less skilled operators. 
     Secondly, in the microfluidic device  10  described above, the electrodes  48  are immersed in a conducting buffer which is in turn in contact with the gels  50  and  52  in the sample transfer and separation channels  40 ,  42 . This is advantageous compared to inserting electrodes directly into the gels themselves—as it improves the electrical connection. When an electrode is inserted into a gel, the electrode is partially in contact with the gel itself (which is generally non-conductive) and partially in contact with a conducting liquid in the matrix of the gel. The overall electrical connection of such an arrangement may not be sufficient. 
     It will be appreciated that numerous changes may be made to the example given above without departing from the scope of the invention as defined in the claims. 
     Firstly, instead of adding a lysing solution into the sample collection well  34  as described above, the PCR gel  54  may contain a lysing agent. In this case whole cells enter into the PCR gel  54  and become lysed in the gel  54 . 
     The sample need not be a buccal swab sample. The sample could be, for example, a blood sample. 
     The geometry and/or structure of the microfluidic device  10  need not be as described above. Any microfluidic device capable of performing the invention as claimed may be used. 
     Any suitable PCR reagents may be used. 
     In the example described above, all of the reagents needed for the PCR reaction are included in the matrix of the PCR gel  54 . However, this need not be the case. For example, some of the reagents required for the PCR reaction may be incorporated in the PCR gel  54  and others may be added at the time of use. Any reagents to be added could be added in a lysing solution or in a wash solution used to wash cells into the PCR gel  54 .