Does Your Application Require Extremely Pure Nucleotides? Only you can answer this question. Most applications have supporters and detractors for the use of extremely pure nucleotides How Can You Monitor Nucleotide Purity and Degradation? Nucleotides produce very specific spectroscopic absorbance data Absorbance ratios not within predicted ranges(Table 10.2 indicate a contaminated deoxy- or ribonucleotide, such as if dATP and dctP were accidentally mixed together. This technique is dequate to quickly determine if a large contamination problem exists, but a high-performance liquid chromatography approach is required to detect minor levels of impurities. The absorbance ratio will not indicate when the triphosphate form of a nucleotide breaks down into the di-and tetraphosphate forms. This form of degradation can be monitored most effectivel Table 0.2 Nucleotide absorbtion maxima Am(pH 7.0)me Nucleotide Lambda Maximum(pH 7.0) xtinction coeffic 2′-dATP 2′-dCTP 13.1×10 2′-dGTP 253nm 13.7×10 2′dITP 2′-dTTP 267nm3 2′-dUTP 262nm 10.2×103 c7-2′ATP 7-2′dGTP 257nm 23′- ddATP 15.2×10 2.3-ddcTP 280nn 13.1×103a 2. 3-ddGTP 253n 2.3-ddTTP CTP 280nn 13.0×103a GTP 252nm 13.7 UTP 262nm 10.2×10 ote: The spectral terms and definitions used are those recommended by the national Bureau of Standards Circular LCD 857, May 19, 1947 pectral analysis done at pH 6. Value determined at Amer Pharmacia biotech 42 dAMP NRC referenc constants employed 2-dCMP NRC reference spectral constants employe 12-dGMP NRC reference spectral constants employed 2-dTMP NRC reference spectral constants employed 2-dIMP NRC reference spectral constants employed. 2-dU NRC reference spectral constants employed. /Leela and Kehne (1983 272 Gerstein
Does Your Application Require Extremely Pure Nucleotides? Only you can answer this question. Most applications have supporters and detractors for the use of extremely pure nucleotides. How Can You Monitor Nucleotide Purity and Degradation? Nucleotides produce very specific spectroscopic absorbance data. Absorbance ratios not within predicted ranges (Table 10.2) indicate a contaminated deoxy- or ribonucleotide, such as if dATP and dCTP were accidentally mixed together. This technique is adequate to quickly determine if a large contamination problem exists, but a high-performance liquid chromatography approach is required to detect minor levels of impurities. The absorbance ratio will not indicate when the triphosphate form of a nucleotide breaks down into the di- and tetraphosphate forms.This form of degradation can be monitored most effectively 272 Gerstein Table 10.2 Nucleotide Absorbtion Maxima Am (pH 7.0) molar Nucleotide Lambda Maximum (pH 7.0) extinction coefficient 2¢-dATP 259nm 15.2 ¥ 103d 2¢-dCTP 280nma 13.1 ¥ 103a,e 2¢-dGTP 253nm 13.7 ¥ 103f 2¢-dITP 249nm 12.2 ¥ 103b,h 2¢-dTTP 267nmb 9.6 ¥ 103g 2¢-dUTP 262nm 10.2 ¥ 103i c7-2¢-ATP 270nm 12.3 ¥ 103j c7-2¢-dGTP 257nm 10.5 ¥ 103c 2¢,3¢-ddATP 259nm 15.2 ¥ 103d 2¢,3¢-ddCTP 280nma 13.1 ¥ 103a,e 2¢,3¢-ddGTP 253nm 13.7 ¥ 103f 2¢,3¢-ddTTP 267nm 9.6 ¥ 103g ATP 259nm 15.4 ¥ 103 CTP 280nma 13.0 ¥ 103a GTP 252nm 13.7 ¥ 103 UTP 262nm 10.2 ¥ 103 Note: The spectral terms and definitions used are those recommended by the National Bureau of Standards Circular LCD 857, May 19, 1947. a Spectral analysis done at pH 2.0. b Spectral analysis done at pH 6.0. cValue determined at Amersham Pharmacia Biotech. d 2¢-dAMP NRC reference spectral constants employed. e 2¢-dCMP NRC reference spectral constants employed. f 2¢-dGMP NRC reference spectral constants employed. g 2¢-dTMP NRC reference spectral constants employed. h 2¢-dIMP NRC reference spectral constants employed. i 2¢-dU NRC reference spectral constants employed. j Leela and Kehne (1983)
by high-performance chromatography, but when such equipment is unavailable, thin layer chromatography can provide qualitative data(Table 10.3) How Should You Prepare, Quantitate, and Adjust the ph of Small and Large Volumes of nucleotides? o The following procedure can be used to prepare solutions of oxynucleotides, ribonucleotides, and dideoxynucleotides pr vided that the different formula weights are taken into account A 100 mM solution of a solid nucleotide triphosphate is pre pared by dissolving about 60mg per ml in purified H,O The exact weight will depend on the formula weight, which will vary by nucleotide, supplier, and salt form. As solid nucleotide triphos phates are very unstable at room temperature, they should be stored frozen until immediately before preparing a solution pectroscopy The most accurate method of quantifying a solution is to measure the absorbance by UV spectrophotometry. a dilution should be made to obtain a sample within the linear range of the spectrophotometer. The sample should be analyzed at the specifi Amax for the nucleotide being used. The concentration can then be obtained by multiplying the UV absorbance reading by the dilution factor, and dividing by the characteristic Am for that nucleotide. These data are provided in Table 10.2 Table 0.3 TLc conditions to monitor dntP Degradation RA Principal R, Trac 0.35(dADP) dcTP 0.21(dCDP) dGTP .34(dGDP) B dTTP 0.14 . 21(dTDP) Note: Solvent System A: Isobutyric acid/concentrated NH,OH/water, 66/1/33; PH 3.7. Add 10ml of concentrated NhOH to 329 ml of water and mix with 661 ml of isobu. uric acid. Solvent System B: Isobutyric acid/concentrated NH OHA water, 57/4/39: pH 43. Add 38ml of concentrated NH,OH o 385 ml of water and mix with 577 ml of isobutyric acid. TLC Plates: Eastman chromagram sheets (#13181 silica gel and #13254 cellulose). Nucleotides, Oligonucle and Polynucleotides 273
by high-performance chromatography, but when such equipment is unavailable, thin layer chromatography can provide qualitative data (Table 10.3). How Should You Prepare, Quantitate, and Adjust the pH of Small and Large Volumes of Nucleotides? The following procedure can be used to prepare solutions of deoxynucleotides, ribonucleotides, and dideoxynucleotides provided that the different formula weights are taken into account. A 100 mM solution of a solid nucleotide triphosphate is prepared by dissolving about 60mg per ml in purified H2O. The exact weight will depend on the formula weight, which will vary by nucleotide, supplier, and salt form. As solid nucleotide triphosphates are very unstable at room temperature, they should be stored frozen until immediately before preparing a solution. Quantitation Spectroscopy The most accurate method of quantifying a solution is to measure the absorbance by UV spectrophotometry. A dilution should be made to obtain a sample within the linear range of the spectrophotometer. The sample should be analyzed at the specific lmax for the nucleotide being used. The concentration can then be obtained by multiplying the UV absorbance reading by the dilution factor, and dividing by the characteristic Am for that nucleotide. These data are provided in Table 10.2. Nucleotides, Oligonucleotides, and Polynucleotides 273 Table 10.3 TLC Conditions to Monitor dNTP Degradation Solvent dNTP Rf, Principal Rf, Trace System dATP 0.25 0.35 (dADP) A dCTP 0.15 0.21 (dCDP) A dGTP 0.27 0.34 (dGDP) B dTTP 0.14 0.21 (dTDP) A Note: Solvent System A: Isobutyric acid/concentrated NH4OH/water, 66/1/33; pH 3.7. Add 10 ml of concentrated NH4OH to 329 ml of water and mix with 661 ml of isobutyric acid. Solvent System B: Isobutyric acid/concentrated NH4OH/ water, 57/4/39; pH 4.3. Add 38 ml of concentrated NH4OH to 385 ml of water and mix with 577 ml of isobutyric acid. TLC Plates: Eastman chromagram sheets (#13181 silica gel and #13254 cellulose)
One would think that the mass of an extremely pure nucleotide could be reliably determined on a laboratory balance. Not so, because during the manufacturing process, nucleotide prepara- tions typically accumulate molecules of water(via hydration) and counter-ions (lithium or sodium, depending on the manufacturer) which signficantly contribute to the total molecular weight of the nucleotide preparation. Unless you consider the salt form and the presence of hydrates, you're adding less nucleotide to the solution than you think. The presence of salts and water also contribute o the molecular weights of oligo-and polynucleotides, which are also most reliably quantitated by spectroscopy H Adjustment The pH of a solution prepared by dissolving a nucleotide in water will vary, depending on the ph at which the nucleotide triphosphate was dried. An aqueous solution of nucleotide triphosphate prepared at Amersham Pharmacia Biotech will have a pH of approximately pH 4.5. The ph may be raised by addition of Naoh (0. 1n NaOH for small volumes, up to 5n NaOH for larger volumes). Approximately 0.002 mmol NaoH per mg nucleotide triphosphate is required to raise the pH from 4.5 to neutral pH. If the pH needs to be lowered, addition of a H* cation exchanger to the nucleotide solution will lower the pH without adding a counter-ion. The amount of cation-exchanger resin per volume of 100 mM nucleotide solution varies greatly depending on the starting and ending pH For very small volumes(<5 ml) of icleotide solutions, a 50% slurry of SP Sephadex can be added dropwise. For larger volumes(>5ml), solid cation exchanger can be added directly in approximately 0.2cm'increments The cation exchanger can be removed by filtration when the desired ph is obtained The triphosphate group gives the solution considerable buffer ng capacity. If an additional buffer is added, the ph should be checked to ensure that the buffer is adequate. The ph should be adjusted when the solution is at or near the final concentration. A significant change in the concentration will change the pH.An increase in concentration will lower the pH, and dilution will raise he ph, if no other buffer is present Similar results will be obtained for all of the nucleotide triphos- phates. Monitor the pH of the solutions as a precaution; purines are particularly unstable under pH 4.5, and all will degrade at acid ph 274 Gerstein
Weighing One would think that the mass of an extremely pure nucleotide could be reliably determined on a laboratory balance. Not so, because during the manufacturing process, nucleotide preparations typically accumulate molecules of water (via hydration) and counter-ions (lithium or sodium, depending on the manufacturer), which signficantly contribute to the total molecular weight of the nucleotide preparation. Unless you consider the salt form and the presence of hydrates, you’re adding less nucleotide to the solution than you think. The presence of salts and water also contribute to the molecular weights of oligo- and polynucleotides, which are also most reliably quantitated by spectroscopy. pH Adjustment The pH of a solution prepared by dissolving a nucleotide in water will vary, depending on the pH at which the nucleotide triphosphate was dried. An aqueous solution of nucleotide triphosphate prepared at Amersham Pharmacia Biotech will have a pH of approximately pH 4.5. The pH may be raised by addition of NaOH (0.1 N NaOH for small volumes, up to 5 N NaOH for larger volumes). Approximately 0.002mmol NaOH per mg nucleotide triphosphate is required to raise the pH from 4.5 to neutral pH. If the pH needs to be lowered, addition of a H+ cation exchanger to the nucleotide solution will lower the pH without adding a counter-ion. The amount of cation-exchanger resin per volume of 100 mM nucleotide solution varies greatly depending on the starting and ending pH. For very small volumes (<5 ml) of nucleotide solutions, a 50% slurry of SP Sephadex can be added dropwise. For larger volumes (>5ml), solid cation exchanger can be added directly in approximately 0.2 cm3 increments. The cation exchanger can be removed by filtration when the desired pH is obtained. The triphosphate group gives the solution considerable buffering capacity. If an additional buffer is added, the pH should be checked to ensure that the buffer is adequate. The pH should be adjusted when the solution is at or near the final concentration. A significant change in the concentration will change the pH. An increase in concentration will lower the pH, and dilution will raise the pH, if no other buffer is present. Similar results will be obtained for all of the nucleotide triphosphates. Monitor the pH of the solutions as a precaution; purines are particularly unstable under pH 4.5, and all will degrade at acid pH. 274 Gerstein