PIPETTABLE REPLACEMENT FLUID FOR MIMICKING PIPETTABILITY OF PCR MASTERMIX AND RELATED METHOD

Abstract

A replacement fluid suitable to replace a PCR Mastermix, wherein the replacement fluid is different from the PCR Mastermix. The replacement fluid has pipetting characteristics substantially equivalent to the PCR Mastermix, wherein for a desired pipetted volume of the target fluid delivered by a pipette, a pipetted volume of the replacement fluid delivered by the pipette is substantially equivalent to the desired pipetted volume of the PCR Mastermix. The replacement fluid is a primary equivalent fluid having substantially similar pipetting characteristics. One or more additives may be added to the primary equivalent fluid. The one or more additives may include pipettability modifying additives and non pipettability modifying additives.

Claims

1. A replacement fluid suitable to replace PCR Mastermix, wherein the replacement fluid is different from the PCR Mastermix, the replacement fluid comprising: a fluid having pipetting characteristics substantially equivalent to PCR Mastermix, wherein for a desired pipetted volume of the PCR Mastermix delivered by a pipette, a pipetted volume of the replacement fluid delivered by the pipette is substantially similar to the desired pipetted volume of the PCR Mastermix.

2. The replacement fluid as claimed in claim 1, wherein the pipetted volume of the replacement fluid is within about +/−20% of the desired pipetted volume of the PCT Mastermix.

3. The replacement fluid as claimed in claim 1, wherein the replacement fluid has rheological and other properties substantially equivalent to rheological properties of PCR Mastermix.

4. The replacement fluid as claimed in claim 3, wherein the rheological properties and other properties include viscosity, shear rate, surface tension, and contact angle.

5. The replacement fluid as claimed in claim 4, wherein the replacement fluid includes a primary equivalent fluid and one or more additives.

6. The replacement fluid as claimed in claim 5, wherein the one or more additives include one or more pipetting-modifying additives selected to modify one or more of the rheological and other properties of the primary equivalent fluid.

7. The replacement fluid as claimed in claim 6, wherein the one or more pipetting-modifying additives are selected from the group consisting of water, a water-soluble polymer, buffering salt, glycerol, sucrose, protein, lipid, and surfactant.

8. The replacement fluid as claimed in claim 5, wherein the one or more additives include one or more non-pipetting-modifying additives selected not to modify one or more of the rheological and other properties of the primary equivalent fluid.

9. The replacement fluid as claimed in claim 8, wherein the one or more non-pipetting-modifying additives are selected from the group consisting of stabilizing buffer, chelator, and preservative agent.

10. The replacement fluid as claimed in claim 7, wherein the primary equivalent fluid is a mix of water and 20% glycerol and the pipetting-modifying additive is a surfactant.

11. The replacement fluid as claimed in claim 10, wherein the surfactant is a nonionic surfactant.

12. The replacement fluid as claimed in claim 11, wherein the nonionic surfactant is octylphenol ethoxylate.

13. The replacement fluid as claimed in claim 1, further comprising one or more analysis-aiding components, wherein the one or more analysis-aiding components are selected to enable analysis of the replacement fluid as though it were the PCR Mastermix.

14. The replacement fluid as claimed in claim 31, wherein the one or more analysis-aiding components are one or more absorbance or fluorescence dyes selected to enable spectroscopic analysis of the replacement fluid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a graph of viscosity as a function of shear rate for PCR Mastermix and a plurality of replacement solutions.

[0024] FIG. 2 is a graphical representation comparing the pipettability of an example replacement fluid of the present invention to a plurality of PCR Mastermix solutions at 2 μL pipette volume.

[0025] FIG. 3 is a graphical representation comparing the pipettability of an example replacement fluid of the present invention to a plurality of PCR Mastermix solutions at 5 μL pipette volume.

[0026] FIG. 4 is a graphical representation comparing the pipettability of an example replacement fluid of the present invention to a plurality of PCR Mastermix solutions at 10 μL pipette volume.

[0027] FIG. 5 is a graphical representation comparing the pipettability of an example replacement fluid of the present invention to a PCR Mastermix solutions at 20 μL pipette volume.

DETAILED DESCRIPTION OF THE INVENTION

[0028] A replacement fluid of the present invention for mimicking the pipetting characteristics of PCR Mastermix is a primary equivalent fluid having substantially similar pipettability without being the PCR Mastermix. The primary equivalent fluid may be combined with one or more pipetting modifying additives that impart chemical and physical properties of pipettability interest. There may be additional optional non pipetting modifying additives forming part of the replacement fluid. The modifying additives and the ratio of such additives in the replacement fluid are selected to generate in the replacement fluid a pipettability that is substantially the same as the pipettability of the PCR Mastermix.

[0029] A first example of a fluid replacement for PCR Mastermix includes water as the primary fluid, and about 210 g/L of glycerol, about 4 g/L of KHP and about 5.3 g/L of octylphenol ethoxylate as pipettability modifying additives. Further non-pipettability modifying additives include about 4 g/L of the chelating agent ethylenediaminetetraacetic acid (EDTA) and about 0.6 g/L of the preservative Mergal (Troy Chemical, part number 100421). Yet further non-pipettability modifying additives include about 1.1 g/L of copper chloride dihydrate as one spectroscopic dye, and about 0.33 g/L of Ponceau S as a second spectroscopic dye (the amount of Ponceau S depends on the spectroscopic properties needed). Further additions of hydrochloric acid and/or sodium hydroxide are made to bring the fluid to a pH of 6.

[0030] A second example of a fluid replacement for PCR Mastermix includes water as the primary fluid, and about 210 g/L of glycerol, about 4 g/L of KHP and about 5.3 g/L of octylphenol ethoxylate as pipettability modifying additives. Further non-pipettability modifying additives include about 4 g/L of the chelating agent ethylenediaminetetraacetic acid (EDTA) and about 0.6 g/L of the preservative Mergal (Troy Chemical, part number 100421). Yet further non-pipettability modifying additives include about 1.1 g/L of copper chloride dihydrate as one spectroscopic dye, and about 1.6 g/L of Ponceau S as a second spectroscopic dye (the amount of Ponceau S depends on the spectroscopic properties needed). Further additions of hydrochloric acid and/or sodium hydroxide are made to bring the fluid to a pH of 6.

[0031] Test results for the first and second examples of a fluid replacement for PCR Mastermix include testing of rheological properties as shown in FIG. 1 and Table 1 (below). FIG. 1 is a graph showing the viscosity as a function of shear rate for three commercial PCR Mastermix solutions, a first example PCR Mastermix substitute (called PCRMix A) and a second example PCR Mastermix substitute (called PCRMix B). An important observation from this data is how different the viscosity of water is as compared to the PCR Mastermix and the substitute fluids. The viscosity of water remains flat and is not strongly impacted by applied shear stress. However, each PCR Mastermix and substitute fluid experiences a significant decrease in viscosity as shear stress is applied (shear thinning). This shear thinning results in solution that flows more freely. The amount of shear stress that a solution experiences at the orifice of a pipette tip can be roughly modeled but is often unknown to the user. For a PCR Mastermix with a flow rate of 150 μL/s, flowing from a tip orifice of 0.74 mm, the shear rate of that fluid upon exiting the tip orifice is on the order of 4000 Hz. It can be observed from this data that the viscosity is strongly dependent upon the amount of glycerol or sucrose present. Commercial Mastermix #1 contains 10-30% glycerol and has the lowest viscosity. Commercial Mastermix #2 contains 40-70% glycerol and Commercial Mastermix #3 contains 30% sucrose. The fluid replacements demonstrated in FIG. 1 both contain 20% glycerol, plus other additives, which provide the viscosity profile displayed. Addition of the glycerol to water significantly changes the viscosity and viscosity profile as compared to water, making the fluid replacements behave more like PCR Mastermix than like water. Table 2 further demonstrates how additives can make rheological properties less like water, and more like PCR Mastermix. Table 2 provides the three commercial PCR Mastermix solutions, each with a surface tension of 32-37 mN/m. Addition of only about 20% glycerol and red dye to water results in very little change to the surface tension (72 mN/m) as compared to that of water (75 mN/m). However, adding octylphenol ethoxylate (non-ionic surfactant) to the 20% glycerol and red dye mix significantly reduces the surface tension and gives the fluid replacements (PCRMix B and PCRMix C) a surface tension much more similar to the Commercial Mastermix than water.

TABLE-US-00001 TABLE 2 Surface tension at 5° C. for three commercial Master Mixes, a first example of a Master Mix replacement fluid, and a second example of a Master Mix replacement fluid. Commercial Commercial Commercial (20% Glycerol (PCRMix B) (PCRMix C) (Water) Master Mix 1 Master Mix 2 Master Mix 3 and Red Dye) at 5° C. at 5° C. at 5° C. (10-30% Glycerol) (40-70% Glycerol) (30% Sucrose) at 5° C. Surface Surface Surface at 5° C. Surface at 5° C. Surface at 5° C. Surface Surface Tension Tension Tension Tension (mN/m) Tension (mN/m) Tension (mN/m) Tension (mN/m) (mN/m) (mN/m) (mN/m) 35.04 37.49 32.86 71.97 38.55 36.28 75
The test data in FIG. 1 and Table 1 are useful in guiding which additives might be introduced to improve the rheological properties. This data proves a useful guide. However, the more important parameter to match is the pipettability of the replacement fluids. FIGS. 2-5 demonstrate pipetted volumes of various fluids including four commercial PCR Mastermix solutions. Also included are the pipetting data for a first example of a replacement PCRMix A fluid and for a second example of a replacement PCRMix B. Adjusting the rheological properties is important, but only to the extent that the desired pipettability is achieved. For example, the viscosity as a function of shear rate of the PCRMix A and PCRMix B, shown in FIG. 1, are not identical to any of the commercial Master Mixes. However, they are close enough to provide a good replacement fluid for mimicking pipetting properties. This can be observed in FIGS. 2-6 by how closely the pipetted volumes of PCRMix A and PCRMix B match those same volumes for the various sera.

[0032] The present invention further includes a related method for replacing a target fluid for pipetting activities with a replacement fluid that functions substantially the same as the target fluid in terms of pipetting characteristics. A first step of the method is to determine pipetting characteristics for the PCR Mastermix. A second step is to identify one or more primary equivalent fluids that may substantially match the pipettability of the PCR Mastermix based on the identified pipetting characteristics of the target fluid. A third step is to analyze one or more rheological and/or other properties of the one or more identified primary equivalent fluids and compare them to the corresponding properties of the PCR Mastermix. The identified primary equivalent fluid(s) should be less expensive and/or more readily available as compared to the PCR Mastermix. A fourth step is optionally to introduce one or more additives to the primary equivalent fluid or fluids and evaluate adjustments made to the pipettability of the primary equivalent fluid(s) to determine mimicking of PCR Mastermix pipettability. The one or more additives may include pipettability-modifying and non-modifying additives. A fifth step of the method is to replace the PCR Mastermix with one or more selected ones of the identified primary equivalent fluids. An optional step is to carry out analysis of the primary equivalent fluid, which may include one or more spectroscopic analyses, wherein the primary equivalent fluid may include a non-modifying additive for that purpose.

[0033] While the invention has been described with respect to specific example embodiments, it is not intended to be limited to those specific embodiments. Instead, the invention covers those embodiments and all reasonable equivalents.