MULTICOMPONENT TABLET FOR MAKING AGAROSE GEL AND METHODS THEREOF

Abstract

The present invention relates to direct compressed compositions for use in electrophoretic analysis comprising polymer matrix particles and lyophilized fluorescent DNA-binding dye, and/or direct compressed compositions for use in electrophoretic analysis comprising a blend of polymer matrix particle(s) and electrophoretic conductive medium particle(s) having a uniform particle size and lyophilized fluorescent DNA-binding dye.

Claims

1. A compressed composition comprising polymer matrix particles, and a lyophilized fluorescent DNA-binding dye.

2-8. (canceled)

9. A compressed composition comprising a blend of polymer matrix particle(s) and electrophoretic conductive medium particle(s) having a uniform particle size, and a lyophilized fluorescent DNA-binding dye.

10. The compressed composition according to claim 9, wherein the blend has a uniform particle size between 20-300 m.

11. The compressed composition according to claim 1, wherein the composition consists of the polymer matrix particles, the lyophilized fluorescent DNA-binding dye and/or the electrophoretic conductive medium particles.

12. The compressed composition according to claim 2, wherein the blend is between: 40 to 75 wt % agarose powder; 25 to 60 wt % DNA electrophoretic conductive medium; less than 0.01 wt % lyophilized fluorescent DNA-binding dye or 99 wt % agarose powder; less than 1 wt % lyophilized fluorescent DNA-binding dye.

13. A method for preparing a compressed composition for use in electrophoretic analysis comprising: (a) providing a blend of a polymer matrix particle(s) and a lyophilized fluorescent DNA-binding dye, and (b) compressing the blend obtained in step (a).

14. A method for preparing a compressed composition for use in electrophoretic analysis comprising: (a) providing a blend having a uniform particle size of a polymer matrix particle(s), an electrophoretic conductive medium particle(s), and a lyophilized fluorescent DNA-binding dye, and (b) compressing the blend obtained in step (a).

15. A method for preparing a lyophilized dye for use in a compressed composition according to claim 1: (a) providing a fluorescent DNA-binding dye in a liquid form and a cryoprotectant, and (b) lyophilizing the fluorescent DNA-binding dye and cryoprotectant by applying low temperature, pressure and sublimation.

16. A method of electrophoretic analysis comprising: (a) contacting the compressed composition of claim 1 with water; (b) heating the mixture in (a); (c) pouring the heated mixture in (b) into a form and allow to cool to a gel; (d) applying a biological sample to said gel; and (e) conducting electrophoresis on the biological sample in the gel of (d).

17. A method of making a gel suitable for electrophoresis comprising: (a) contacting the compressed composition of claim 1 with water; (b) heating the mixture of (a); and (c) cooling the heated mixture of (b) until a gel is formed.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0319] FIG. 1

[0320] Shows the easy way of how to make 1% agarose gel for gel electrophoresis from compressed composition (tablet) with an electrophoretic conductive medium TBE or TAE. To make 1 agarose gel, simply take an empty bottle or flask, pour 50 ml of room temperature water into the bottle, take one compressed composition out of the blister pack and place it in the bottle and let the compressed composition soak for 1-3 min prior heating. Pour the gel into the gel casting tray.

[0321] FIG. 2

[0322] Compressed composition in uniform size particles and different particle sizes. As seen on the lower tablet, which is the uniform sized tablet of the invention, it is a complete tablet without cracks and missing parts, thus having the right size/weight. In contrast to the upper tablet, which shows sign of breaking, thus it no longer has the right size/weight.

[0323] FIG. 3

[0324] Trehalose was tested as a cryoprotectant in different concentration range (5-50% w/v). Regardless of the trehalose concentration, trehalose and fluorescent DNA binding dye always formed a paste-like, stretchy and glassy product during the lyophilization process. This kind of lyophilized fluorescent DNA binding dye was impossible to mill and mix evenly in a compressed composition. Thus, trehalose was considered less suitable as a cryoprotectant for optimal compressed compositions.

[0325] FIG. 4

[0326] Dextran was tested as a cryoprotectant in different concentration range (1,2-25% w/v). Regardless of the dextran concentration, dextran and fluorescent DNA binding dye always formed a very hard and insoluble mass product during the lyophilization process, suggesting that the combination of dextran and used fluorescent DNA binding dye is not chemically suitable for the lyophilization or the chosen physical conditions (pressure, temperature) caused the structural collapse of the mixture. That kind of lyophilized fluorescent DNA binding dye is impossible to mill and mix evenly in the compressed composition mixture. Thus, dextran was considered less suitable as a cryoprotectant for optimal compressed compositions.

[0327] FIG. 5

[0328] Mannitol was tested as a cryoprotectant in at 10% w/v in a small amount of fluorescent DNA binding dye (200 l). Mannitol and fluorescent DNA binding dye formed a powdery, easy to mill and well dissolving product during the lyophilization process, thus in the chosen physical conditions (pressure, temperature) water was removed from the fluorescent DNA binding dye without destructing its structure. This combination of lyophilized fluorescent DNA binding dye and mannitol resulted in products that was easy to mill and mix evenly in the blend and prepare suitable compressed compositions.

[0329] FIG. 6

[0330] Mannitol was tested as a cryoprotectant in at 10% w/v in a larger amount of fluorescent DNA binding dye (5000 l). Mannitol and fluorescent DNA binding dye still formed a powdery, easy to mill and well dissolving product during the lyophilization process. thus in the chosen physical conditions (pressure, temperature) water was removed from the fluorescent DNA binding dye without destructing its structure. This combination of lyophilized fluorescent DNA binding dye and mannitol resulted in products that was easy to mill and mix evenly in the blend and prepare suitable compressed compositions.

[0331] FIG. 7

[0332] Show the results of a photosensitivity test. Tablets were stored at room temperature for 8 months protected from light. Tablet is used for making agarose gel at the parameters 1 agarose gel and running parameters: 150 V, 45 min and in 1TBE buffer. The image was taken using a The Blue Light Transilluminator. The gel was loaded with DNA ladder1 kb Solis DNA ladderfrom left to right 2, 4, 6 or 8 l respectfully. The quality of the gel and the image is high and the migrated DNA bands are strong and sharp, and the background intensity lowdespite the long-term storage of the tablet for 8 months.

[0333] FIG. 8

[0334] Show the results of a photosensitivity test. Tablets were stored at room temperature for 8 months without protection from light (placed in direct sunlight). Tablet is used for making agarose gel at the parameters 1 agarose gel and running parameters: 150 V, 45 min and in 1TBE buffer. The image was taken using a The Blue Light Transilluminator. The gel was loaded with DNA ladder 1 kb Solis DNA ladderfrom left to right 2, 4, 6 or 8 l respectfully. The quality of the gel and the image is high and the migrated DNA bands are strong and sharp, and the background intensity lowdespite the long-term storage of the tablet for 8 months without protection from sunlight. This shows that the compressed composition in itself provides protection for the photosensitive lyophilized fluorescent DNA binding dye.

[0335] FIG. 9

[0336] FIGS. 9-11 show the effect if the lyophilized fluorescent DNA-binding dye is not milled/grinded, where FIG. 9 is a positive control or well-stained gel with milled lyophilized fluorescent DNA-binding dye.

[0337] The images 9-11 was made using tablets according the invention with or without milling/grinding and the following parameters were used for making the agarose gel: 1% agarose gel and running parameters: 150 V, 45 min and in 1TBE buffer. The images was taken using a The Blue Light Transilluminator. The gels were loaded with DNA ladder1 kb Solis DNA ladderfrom left to right 2, 4, 6 or 8 l respectfully.

[0338] FIG. 10

[0339] FIG. 10 is an under stained negative gel with lyophilized fluorescent DNA-binding dye without a milling or a grinding process.

[0340] FIG. 11

[0341] FIG. 11 is an over stained negative gel with lyophilized fluorescent DNA-binding dye without a milling or grinding process.

EXAMPLES

Example 1TBE Buffer Components

[0342]

TABLE-US-00003 Mass of the Used substance substance expressed as % Powder DNA binding dye 0.004% Agarose I (mixed with DNA-binding agent) 0.8% Grinded TBE buffer .sup.50% Agarose II 49.2%

[0343] Description of the Exercise: [0344] 1) In the binding of the DNA, cryopreservation agents were added in the range of 5-10% w/v. The resulting solution was frozen 80 C. and dry-frozen at 0.07-0.0011 mbar for 17 hours. [0345] 2) The resulting solid was mixed with Agarose I powder (0.8% of final volume) and peeled off for 2 min until the granule size ranged from 20-200 m. [0346] 3) The components of the TBE buffer were triturated for 3 seconds until the granule size ranged from 20-200 m. [0347] 4) Powder DNA binding binder 5 and agarose II mixture (0.8%) were mixed with the rest of the agarose (49.2%) and stirred for 5 min using the appropriate stirrer. The finely divided TBE powder was then added to the mixture of remaining substances and stirred for an additional 30 minutes (counting 30 min for 1 kg of mixed components) using a suitable stirrer (e.g., using a roller mixer) [0348] 5) Pressing the tablet

[0349] Tablet Characteristics:

TABLE-US-00004 Size 10 20 6 mm Shape Oval, without folding Weight 1 g Strength (force used by the press) 15 kg Solubility Less than 3 min

Example 2TAE Buffer Components

[0350]

TABLE-US-00005 Mass of the Used substance substance expressed as % Powder DNA binding dye 0.004% Agarose I (mixed with DNA-binding agent) 0.8% Grinded TBA buffer .sup.35% Agarose II 64.2%

[0351] Description of the Exercise:

[0352] 1) In the binding of the DNA dye, cryopreservation agents were added in the range of 5-10% w/v. The resulting solution was frozen 80 C. and subsequently dry-frozen at 0.07-0.0011 mbar for 17 hours.

[0353] 2) The resulting solid was mixed with Agarose I powder (0.8% of final volume) and grinded for 2 min until the granule size ranged from 20-200 m.

[0354] 3) The components of the TAE buffer were grinded for 3 seconds until the pellet size ranged from 20-200 m.

[0355] 4) Powder DNA-binding dye and Agarose II mixture (0.8%) were mixed with the rest of the agarose (64.2%) and stirred for 5 min using the appropriate stirrer. The peeled TAE powder was then added to a mixture of the remaining substances and stirred for an additional 30 minutes (counting 30 min per 1 kg of mixed components) using a suitable mixer (e.g., using a roller mixer).

[0356] 5) Pressing the tablet

[0357] 6) Tablet characteristics:

TABLE-US-00006 Size 10 20 6 mm Shape Oval, without folding Weight 1 g Strength (force used by the press) 15 kg Solubility Less than 3 min

[0358] According to this method, the electrophoresis agarose gel is formed from the tablet prepared according to the present method as follows: In the gel preparation vessel, the required number of tablets and the corresponding amount of warm water used to make the gel are added. The tablet(s) is/are dissolved in 3 minutes. Visually inspect the whole powder of the tablet to dissolve in the water before heating the gel. An example of making a gel for DNA/RNA analysis is described in the table.

TABLE-US-00007 Gel % 1 tablet 2 tablets .sup.1% 50 ml room temp water 100 ml room temp water 1.5% 33 ml room temp water 67 ml room temp water

[0359] The solution is heated until it has become transparent and all visible particles are dissolved. Then cool the gel solution to 60 C. before pouring it on the base. The gel is then run in the appropriate buffer with the desired parameters.

Example 3Atlas ClearSight Tablet Use with TBE

[0360] To make 1% agarose gel for gel electrophoresis, take one compressed composition (tablet) with electrophoretic conductive medium TBE out of the blister pack. Place it in the bottle or flask and add 50 ml of room temperature water. Soak the compressed composition for 1-3 minutes. Heat solution until all particles are dissolved. Pour the gel out into the gel casting tray, see FIG. 1.

[0361] As only water needs to be added to the compressed composition (tablet) to make the agarose gel for electrophoresis, it makes it much faster and easier to make the agarose gel compared to preparing agarose gel out of separate components, which need to be weighed.

Example 4Atlas ClearSight Tablet Use with TAE

[0362] To make 1.3% agarose gel for gel electrophoresis, take one compressed composition (tablet) with electrophoretic conductive medium TAE out of the blister pack. Place it in the bottle or flask and add 50 ml of room temperature water. Soak the compressed composition for 1-3 minutes. Heat solution until all particles are dissolved. Pour the gel out into the gel casting tray, see FIG. 1. As only water needs to be added to the compressed composition (tablet) to make the agarose gel for electrophoresis, it makes it much faster and easier to make the agarose gel compared to preparing agarose gel out of separate components, which need to be weighed.

Example 5Atlas ClearSight Agarose Tablets without Electrophoretic Conductive Medium

[0363] To make 1% agarose gel for gel electrophoresis take one compressed composition (tablet) without electrophoretic conductive medium out of the blister pack, place it in the bottle or flask and add 25 ml of electrophoretic conductive medium solution. Soak the compressed composition for 1-3 minutes. Heat solution until all particles are dissolved. Pour the gel out into the gel casting tray.

Example 6Uniform Ingredients in the Blend

[0364] Two types of tablet where produced by direct compression with the following blends: [0365] 40 to 75 wt % agarose powder; [0366] 25 to 60 wt % DNA electrophoretic conductive medium; [0367] less than 0.05 wt % lyophilized fluorescent DNA-binding dye
with or with-out grinding the blends.

[0368] The compressed composition comprising the grinded blend is intact, and its shape is preserved. In the subsequent use, the buffer properties are maintained since all the components are in the right proportions. Also, the tablet weight is maintained, since all the components are in the right proportions.

[0369] The compressed composition comprising the blend, which was not grinded shows signs of cracks, and its shape is not preserved. In the subsequent use, the buffer properties were not maintained, since all the components were not in the right proportions.

[0370] Therefore, the size of the polymer matrix particle component in the compressed composition according to the invention should be similar or uniform to that of the electrophoretic conductive medium particle, since it is otherwise difficult to obtain a homogeneous composition. A non-uniform size of the components in the compressed composition affects the efficacy of tablet formation. For example, if the blend is not non-uniform during the compression, the form of compressed composition (tablet) can be broken or it may not be formed to the correct shape at all, see FIG. 2.

Example 7Cryoprotectants

[0371] Tablets produced by direct compression with the following blends: [0372] 40 to 75 wt % agarose powder; [0373] 25 to 60 wt % DNA electrophoretic conductive medium; [0374] less than 0.05 wt % lyophilized fluorescent DNA-binding dye
with different cryoprotectants.

[0375] Here is the list and the results of those cryoprotectants which were tested: [0376] A) Glycerol does not work in any concentration range because it is impossible to lyophilize due to its oily-like chemical structure, [0377] B) Trehalose (tested in the range of 5-50% w/v). Despite the trehalose concentration, trehalose and fluorescent DNA binding dye always formed a paste-like, stretchy and glassy product during the lyophilization process. This means that either the combination of trehalose and used fluorescent DNA binding dye is not chemically suitable for the lyophilization or the chosen physical conditions (pressure, temperature) caused the structural collapse of the mixture. Such a lyophilized fluorescent DNA binding dye is impossible to mill and mix evenly in the compressed composition mixture (See for example FIG. 3). [0378] C) Dextran (tested in the range of 1.2-25% w/v). Despite the dextran concentration, dextran and fluorescent DNA binding dye always formed a very hard and insoluble mass product during the lyophilization process and thus were impossible to mill and mix evenly in the compressed composition mixture. [0379] (See for example FIG. 4), [0380] D) Trehalose+sucrose (tested in the range of 10-25% w/v+3-10% w/v) does not work, stretchy and glassy product (data not shown), and [0381] E) Mannitol (tested in the range of 10-18.18% w/v). Mannitol and fluorescent DNA binding dye formed a powdery, easy to mill and well dissolving product during the lyophilization process despite of the amount of the fluorescent DNA binding dye used in lyophilization process. Thus, this tested cryoprotectant in range of 10-18.18% w/v was considered extremely suitable (See FIGS. 5 and 6).

Example 8Efficiency Gain in Lab Time

[0382] It is easier and faster to make the agarose gel for electrophoresis if only water needs to be added to the compressed composition (tablet) compared to preparing agarose gel out of separate components, which need to be weighed.

TABLE-US-00008 The compressed composition With separate Activities (tablet) components Polymer matrix particle 2 min 35 sec component weighting Electrophoretic conductive 2 min 25 sec medium weighting Adding water 30 sec 30 sec Dissolving time 1 min Melting time 3 min 3 min Time to cool No need 2 min Adding fluorescent 1 min DNA binding dye Total 4 min 30 sec 11 min 30 sec

Example 9Dust Wipe

[0383] Two gels were prepared, one by use of a compressed composition in 4 min and 30 sec, as shown in Example 8, and a second gel by use of separate components in 11 min and 30 sec. After the preparation, the work desk was wiped with a dust wipe and inspected for visual dust. The wiping was made in one direction only, not back and forth.

[0384] The work desk used for both preparations was cleaned prior to the test with a High Efficiency Particulate Air (HEPA) vacuum cleaner.

[0385] The work table used for preparing an agarose gel from with a compressed composition (tablet), did not show any dust in the visual inspection.

[0386] The work table used for preparing an agarose gel from separate components, showed significant dust in the visual inspection (data not shown).

Example 10Precision of the Compressed Compositions

[0387] For making gel out of a powdery polymer matrix particle component and electrophoretic conductive medium, two separate weighs must be performed. This leads to different weighing errors. At first there are 2 scale errors taking place due to the 2-step weighing (usually the scale error is 0.01% per weighing). And then some part of the weighed material (usually 0.5-1%) remain(s) in the weighing container, causing an additional error to occur.

[0388] Thus 2 scale errors may take place. A human error coming from wrong weighing or rounding the numbers is approximately 2%. The additional error coming from preparing the electrophoretic conductive media solution (error from beaker/measuring glass, human error, rounding error) is around 1-4%.

[0389] Adding fluorescent DNA-binding dye to the electrophoretic conductive media solution causes another error, of which the pippet error is 0.001% and pipetting error may be up to 5%.

[0390] 2 Weighing Errors May Take Place:

[0391] Human errorwrong weighting or rounding error (2%)

[0392] For making electrophoretic conductive mediafluid error (error from beaker/measuring glass, human error, rounding error) 1-4%.

[0393] Adding fluorescent DNA binding dyePipette error (0.001%) and pipetting error (up to 5%).

Example 11Easy to Use Protocol TBE

[0394] Use the bottle or flask that is at least 3 times of the volume of the solution being prepared. For tablet dissolving use water which is at room temperature, do not use hot water.

[0395] Add an appropriate number of agarose tablets in the water and do not add any buffer. See the table below to achieve needed gel percentage.

TABLE-US-00009 Gel % 1 tablet 2 tablets .sup.1% 50 ml water 100 ml water 1.5% 33 ml water 67 ml water

[0396] Soak the tablet in the water for 3 minutes (or until it is dissolved) before heating.

[0397] Heat the solution until it is clear and visually all particles are dissolved.

[0398] Cool the solution to approximately 60 C. before pouring it out.

[0399] Run the gel in the TBE running buffer.

Example 12Easy to Use Protocol TAE

[0400] Use the bottle or flask that is at least 3 times of the volume of the solution being prepared.

[0401] For tablet dissolving use water which is at room temperature, DO NOT use hot water.

[0402] Add an appropriate number of tablets in the water and do NOT add any buffer. See the table below to achieve the needed gel percentage.

TABLE-US-00010 Gel % 1 tablet 2 tablets .sup.1% 65 ml water 130 ml water 1.5% 43 ml water 86 ml water

[0403] Soak the tablet in the water for 3 minutes (or until it is dissolved) before heating.

[0404] Heat the solution until it is clear and visually all the particles are dissolved.

[0405] Cool the solution to approximately 60 C. before pouring it out.

[0406] Run the gel in the TAE running buffer.

Example 13Photosensitivity

[0407] Fluorescent DNA-binding dye is typically photosensitive, and thus must be stored in non-permeable packaging. However, the in the compressed composition, the polymer matrix particles and the electrophoretic conductive medium particles cover the lyophillized fluorescent DNA-binding dye and thus the need for such a packaging is less relevant. The tablet covers the dye molecules from direct light. Consequently, these types of compressed compositions are more stable over time. The compressed compositions can be stored at room temperature (FIGS. 7 and 8).

Example 14Lyophillized Fluorescent DNA-Binding Dye Milling/Grinding

[0408] For pre-compressing the compressed composition, one important step is milling the lyophillized fluorescent DNA-binding dye. Otherwise the lyophillized fluorescent DNA-binding dye will not be evenly distributed in that compressed composition, which results in an uneven quality of agarose gel electrophoreses (some agarose gels are over stained, and some are under stained due to a non-optimal concentration of fluorescent DNA-binding dye) (see FIGS. 9, 10 and 11).

[0409] Items

[0410] 1. A multicomponent tablet for agarose gel for use in DNA/RNA analysis comprising fluorescent lyophilized DNA-binding dye, agarose, DNA-binding agarose, a cryoprotective agent, and an acrylic buffer (in alternative embodiments of the invention, for example, TBE or TAE).

[0411] 2. The multicomponent tablet according to claim 1, wherein the TBE buffer is used as a powder buffer.

[0412] 3. A multicomponent tablet according to claim 1, wherein a TAE buffer is used as a powder buffer.

[0413] 4. A process for the preparation of a multicomponent tablet according to claim 1 to form an agarose gel for use in a DNA/RNA assay, comprising the steps of: [0414] Freeze-dry DNA-binding dye [0415] Mixing the resulting solid with agarose powder (0.4% of the final volume) and finely grind [0416] Finely grinding TBE and TAE buffers suitable for pressing into tablets for physical substance modification of existing substances [0417] Mixing together the three components of the tablet on the basis of the respective ratio and compressing them into a tablet, and [0418] Preparing the tablet by adding water to the agarose gel electrophoresis solution.

[0419] 5. The method of claim 4, wherein the preparation of the powder comprises the steps of: [0420] Adding in the binding of the DNA, cryoprotectants were added in the range of 5-10% w/v and the resulting solution was frozen 80 C. and subsequently freeze-dried at 0.07-0.0011 mbar for 17 hours, and [0421] Mixing the resulting solid with agarose powder (0.4% of the final volume) and grinding it for 1 min.

[0422] 6. The method of claim 4, wherein the preparation of the buffer comprises the steps of: [0423] Grinding TBE powder for 3 seconds in a shredder, and [0424] Mixing the grinded TBE powder with agarose and ground pulp containing a lyophilized dye mixture with an agarose mixture.

[0425] 7. The method of claim 1, wherein the preparation of the gel solution comprises the steps of: [0426] For the preparation of gel electrophoresis agarose gel, it is only necessary to add water that is used to make the gel.

[0427] 8. A mixture of gel electrophoresis agarose gel for DNA/RNA analysis according to the tablet according to claim 1, characterized in that it comprises the following components: [0428] lyophilized DNA-binding dye of 0.004% of the final volume with a cryo-preservative of 5-10% DNA-binding dye and solvent residues in the range of 0.1-1.5% of the final binding volume of DNA [0429] crushed acrylic buffer 35 to 50%, and [0430] agarose 50 to 65%.

[0431] 9. The mixture according to claim 8, wherein the mixture comprises a TBE buffer.

[0432] 10. The mixture according to claim 8, wherein the mixture comprises a TAE buffer.