Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-47 (Electronic) Nucleotides, Oligonucleotides, and Polynucleotides Alan S Gerstein 268 Nomenclature: De facto and Du jour What Makes a Nucleotide pure? 269 Are Solution Nucleotides Always More Pure Than Lyophilized Nucleotides 269 Are Solution Nucleotides More Stable Than Lyophilized Nucleotides? 270 Does Your Application Require Extremely Pure Nucleotides? 272 How Can You Monitor Nucleotide Purity and Degradation 272 The author would like to thank Anita Gradowski of Pierce Milwaukee for contributing such thorough and helpful information regarding the preparation of nucleotide solutions. Special thanks also to Cica Minetti and David Remeta of Rutgers University for discussing a method to calculate the extinction coefficient of an oligonucleotide. The contributions to this chapter by Howard Coyer and Thomas Tyre, also of Pierce Milwaukee are too numerous to list 267
267 10 Nucleotides, Oligonucleotides, and Polynucleotides Alan S. Gerstein Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Nomenclature: De facto and Du jour . . . . . . . . . . . . . . . . . . 268 What Makes a Nucleotide Pure? . . . . . . . . . . . . . . . . . . . . . . . 269 Are Solution Nucleotides Always More Pure Than Lyophilized Nucleotides?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Are Solution Nucleotides More Stable Than Lyophilized Nucleotides? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Does Your Application Require Extremely Pure Nucleotides? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 How Can You Monitor Nucleotide Purity and Degradation?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 The author would like to thank Anita Gradowski of Pierce Milwaukee for contributing such thorough and helpful information regarding the preparation of nucleotide solutions. Special thanks also to Cica Minetti and David Remeta of Rutgers University for discussing a method to calculate the extinction coefficient of an oligonucleotide. The contributions to this chapter by Howard Coyer and Thomas Tyre, also of Pierce Milwaukee, are too numerous to list. Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
How Should You Prepare, Quantitate, and Adjust the PH of Small and Large volumes of Nucleotides? 273 What ls the Effect of Thermocycling on Nucleotide Is There a difference between absorbance, A260, and Optical Density? 275 Why Do A260 Unit Values for Single-Stranded DNA and Oligonucleotides Vary in the Research Literature? 278 279 How Pure an ol Application? 279 What Are the Options for Quantitating Oligonucleotides 279 What ls the Storage Stability of Oligonucleotides? Your Vial of Oligonucleotide ls Empty, or ls It? 28 Synthetic Polynucleotides Is a Polynucleotide ldentical to an Oligonucleotide 28 How Are Polynucleotides Manufactured and How Might This Affect your research? 282 Would the World Be a Better Place If Polymer Length Never varied? Oligonucleotides Don't Suffer from Batch to Batch Size Variation. Why Not ow Many Micrograms of Polynucleotide Are in Your Vial? Is It possible to determine the Molecular Weight of a Polynucleotide? 285 What Are the Strategies for Preparing Polymer Solutions of Known concentration 285 Your Cuvette has a 10mm Path Length what Absorbance values Would be observed for the same Why Not Weigh out a portion of the polymer Instead..286 Solution If Your Cuvette Had a 5mm Path Length of Dissolving the Entire Contents of the Vial Is a Phosphate Group Present at the 5 End of ynthetic Nucleic Acid Polym ner What Are the Options for Preparing and Storing Solutions of Nucleic Acid Polymers? Bibliography NUCLEOTIDES Nomenclature: De facto and Du jour Lehninger (1975) provides a thorough discussion of prope nucleotide nomenclature and abbreviations. Unfortunately, 268 Gerstein
How Should You Prepare, Quantitate, and Adjust the pH of Small and Large Volumes of Nucleotides? . . . . . . . . . . . 273 What Is the Effect of Thermocycling on Nucleotide Stability?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Is There a Difference between Absorbance, A260, and Optical Density? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Why Do A260 Unit Values for Single-Stranded DNA and Oligonucleotides Vary in the Research Literature? . . . . . . 278 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 How Pure an Oligonucleotide Is Required for Your Application?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 What Are the Options for Quantitating Oligonucleotides? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 What Is the Storage Stability of Oligonucleotides?. . . . . . . . 280 Your Vial of Oligonucleotide Is Empty, or Is It? . . . . . . . . . . . 281 Synthetic Polynucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Is a Polynucleotide Identical to an Oligonucleotide?. . . . . . . 281 How Are Polynucleotides Manufactured and How Might This Affect Your Research? . . . . . . . . . . . . . . . . . . . . . . . . . . 282 Would the World Be a Better Place If Polymer Length Never Varied? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 Oligonucleotides Don’t Suffer from Batch to Batch Size Variation. Why Not? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 How Many Micrograms of Polynucleotide Are in Your Vial? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 Is It Possible to Determine the Molecular Weight of a Polynucleotide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 What Are the Strategies for Preparing Polymer Solutions of Known Concentration? . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Your Cuvette Has a 10mm Path Length. What Absorbance Values Would Be Observed for the Same Solution If Your Cuvette Had a 5mm Path Length? . . . . . 286 Why Not Weigh out a Portion of the Polymer Instead of Dissolving the Entire Contents of the Vial?. . . . . . . . . . 287 Is a Phosphate Group Present at the 5¢ End of a Synthetic Nucleic Acid Polymer? . . . . . . . . . . . . . . . . . . . . . 287 What Are the Options for Preparing and Storing Solutions of Nucleic Acid Polymers? . . . . . . . . . . . . . . . . . . 287 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 NUCLEOTIDES Nomenclature: De facto and Du jour Lehninger (1975) provides a thorough discussion of proper nucleotide nomenclature and abbreviations. Unfortunately, 268 Gerstein
commercial catalogs and occasionally the research literature troduce different notations. Some consider"NTP"a general term for deoxynucleotides, but the absence of the letter"d"indicates a ribonucleotide to others Commercial literature also describes ribonucleotides as“"RTP's” If the letter“d” Is present, the name describes a deoxynucleotide. If"d"is absent, check the literature piece closely to avoid a common purchasing error. Dideoxynu cleotides are generically referred to as"ddNTPs. What Makes a Nucleotide pure? Using dATP as an example, what categories of impurities could be present? One potential contaminant is a nucleotide other than dATP, such as dCTP. A second class of impurity could be the mono-, di-, or tetraphosphate form of the deoxyadenosine nucleotide. Since most if not all commercial nucleotides are chem. ically synthesized from highly analyzed precursors, contamination with a nucleotide not based on deoxyadenosine is very unlikely a third class of impurities is the non-UV-absorbing organic and inorganic salts accumulated during the synthesis and purification p ocedures While essentially all commercial nucleotides are chemically synthesized, the final products are not necessarily identical Manufacturing processes vary; raw materials and intermediates of the nucleotide synthesis reactions are subjected to different purification strategies and processes. It is these intermediate eps, and the scrutiny of the products' final specifications, that allow manufacturers to legitimately claim that nucleotides are extremely pure A formal definition of extremely pure does not exist, but com- mercial preparations of such products typically contain greater than 99% of the desired nucleotide in the triphosphate form Contaminating nucleotides are rarely detected in commercial preparations, even using exceedingly stringent high-performance chromatography procedures, but some contaminants escape HPLC detection. Freedom from non-UV-absorbing materials is typically judged by comparison of a measured molar extinction (Am) coefficient to published extinction coefficients (E)values Nuclear magnetic resonance(NMR) may also be used to monitor for contaminants such as pyrophosphate Are Solution Nucleotides always More Pure Than Lyophilized Nucleotides? Nucleotides were first made commercially available as solvent precipitated powders. The lyophilized and extremely pure solution Nucleotides, Oligonucleotides, and Polynucleotides 269
commercial catalogs and occasionally the research literature introduce different notations. Some consider “NTP” a general term for deoxynucleotides, but the absence of the letter “d” indicates a ribonucleotide to others. Commercial literature also describes ribonucleotides as “RTP’s.” If the letter “d” is present, the name describes a deoxynucleotide. If “d” is absent, check the literature piece closely to avoid a common purchasing error. Dideoxynucleotides are generically referred to as “ddNTP’s.” What Makes a Nucleotide Pure? Using dATP as an example, what categories of impurities could be present? One potential contaminant is a nucleotide other than dATP, such as dCTP. A second class of impurity could be the mono-, di-, or tetraphosphate form of the deoxyadenosine nucleotide. Since most if not all commercial nucleotides are chemically synthesized from highly analyzed precursors, contamination with a nucleotide not based on deoxyadenosine is very unlikely. A third class of impurities is the non-UV-absorbing organic and inorganic salts accumulated during the synthesis and purification procedures. While essentially all commercial nucleotides are chemically synthesized, the final products are not necessarily identical. Manufacturing processes vary; raw materials and intermediates of the nucleotide synthesis reactions are subjected to different purification strategies and processes. It is these intermediate steps, and the scrutiny of the products’ final specifications, that allow manufacturers to legitimately claim that nucleotides are extremely pure. A formal definition of extremely pure does not exist, but commercial preparations of such products typically contain greater than 99% of the desired nucleotide in the triphosphate form. Contaminating nucleotides are rarely detected in commercial preparations, even using exceedingly stringent high-performance chromatography procedures, but some contaminants escape HPLC detection. Freedom from non-UV-absorbing materials is typically judged by comparison of a measured molar extinction (Am) coefficient to published extinction coefficients (e)values. Nuclear magnetic resonance (NMR) may also be used to monitor for contaminants such as pyrophosphate. Are Solution Nucleotides Always More Pure Than Lyophilized Nucleotides? Nucleotides were first made commercially available as solventprecipitated powders.The lyophilized and extremely pure solution Nucleotides, Oligonucleotides, and Polynucleotides 269
forms appeared in the early 1980s. Some lyophilized preparations approach 98% purity or more but rarely match the >99% achieved by extremely pure solutions. Generally, solution nucleotides are purer than the lyophilized version, but unless supporting quality control data are provided, it should not be concluded that a solution nucleotide is extremely pure or even more pure than a yophilized preparation Are Solution Nucleotides More Stable Than Lyophilized Nucleotides? Peparations of deoxynucleoside triphosphates decompose into nucleoside di- and tetraphosphates via a disproportionation reaction. This reaction is concentration and temperature dependent. At temperatures above 4oC, lyophilized prepara- tions of deoxynucleotides undergo disproportionation faster than nucleotides in solution. In contrast, the rate of degradation for both forms is less than 1% per year at -20oC and below(Table 10.1). Solutions of dideoxynucleotides and ribonucleotides are similarly stable for many months at temperatures of -20C and below. Most, but not all, dideoxy- and ribonucleotides are stable for many months at4°C Table 10. I Storage Stability of Nucleotides Triphosphate Form Months 70°C 2 dATP 99.14 9747 95.46 3945 (275) (1.75) dTTP 97.2 9428 3945 (30mo) (2.75mo) duTP NA NA NA Solution (100mM) dATP 98.75 91.8 (2mo) 37.07 dCTP 9938 (2mo) 21.25 270 Gerstein
forms appeared in the early 1980s. Some lyophilized preparations approach 98% purity or more but rarely match the >99% achieved by extremely pure solutions. Generally, solution nucleotides are purer than the lyophilized version, but unless supporting quality control data are provided, it should not be concluded that a solution nucleotide is extremely pure or even more pure than a lyophilized preparation. Are Solution Nucleotides More Stable Than Lyophilized Nucleotides? Peparations of deoxynucleoside triphosphates decompose into nucleoside di- and tetraphosphates via a disproportionation reaction. This reaction is concentration and temperature dependent. At temperatures above 4°C, lyophilized preparations of deoxynucleotides undergo disproportionation faster than nucleotides in solution. In contrast, the rate of degradation for both forms is less than 1% per year at -20°C and below (Table 10.1). Solutions of dideoxynucleotides and ribonucleotides are similarly stable for many months at temperatures of -20°C and below. Most, but not all, dideoxy- and ribonucleotides are stable for many months at 4°C. 270 Gerstein Table 10.1 Storage Stability of Nucleotides % Triphosphate Form Months -70°C -20°C 4°C 21°C Powder dATP 54 99.44 99.14 97.47 93.93 (48 mo) 97.78 (3 mo) dCTP 54 98.46 95.46 39.3 39.45 (33 mo) (2.75) dGTP 54 96.95 95.37 25.74 34.4 (27 mo) (1.75) dTTP 54 97.29 94.28 27.4 39.45 (30 mo) (2.75mo) dUTP N.A. N.A. N.A. N.A. N.A. Solution (100 mM) dATP 54 99.2 98.75 95.3 91.8 (2 mo) 37.07 (39 mo) dCTP 54 99.38 99.15 96.98 95.2 (2 mo) 21.25 (42 mo)
Table 10.(Continued) %o Triphosphate Form Months Powder dgTP 9883 95.47 0.5 dTTP 98.87 duTP 98.02 71.55 90.1 40.13(6mo) Solution(10mM) dATP 9968 dcTP 982 9885(2mo 98.6 9951 dTTP 93.57 9929 dUTP 93.8 9945 (12mo) 98.5(2mo Solution ddNTP(10mM) ddATP 99.69 94.52 ddCTP 984 94. 9936 Solution ddNTP(5mM) ddATP 68.56 9963 ddcTP 100 ddGTP 34343 96.67 ddTTP RTP Solutions(100mM) ATP 9857 98.18 95.39 CTP 333 99 98.43 GTP 9846 98.44 96.82 UTP 9971 Source: Data aration of nucleotide species via high perfor on an Amersham Pharmacia Biotech FPLCe Syster Notes: Each umoles(0. 2 ml of a 1 mM solution) was injected onto a Mono QB Ion Exchange column Using the following buffers: Buffer A, 5mM sodium phosphate, PH 7.0 Buffer B, 5mM sodium phosphate, IM NaCl PH 7.0. trification was achieved via a gradient of 5-35% NaCl over 15 minutes using a flow rate Nucleotides, Oligonucleotides, and Polynucleotides 271
Nucleotides, Oligonucleotides, and Polynucleotides 271 Table 10.1 (Continued) % Triphosphate Form Months -70°C -20°C 4°C 21°C Powder dGTP 54 99.63 98.83 95.47 90.5 (2 mo) 19.7 (42 mo) dTTP 54 99.44 98.87 93.54 95.6 (2 mo) 0.07 (42) dUTP 54 99.23 98.02 71.55 90.1 (1.2mo) 40.13 (6mo) Solution (10 mM) dATP 15 99.68 99.59 88.6 (12 mo) 98.5 (2 mo) dCTP 15 98.2 99.56 86.11 (12 mo) 98.85 (2mo) dGTP 15 98.6 99.51 89.47 (12 mo) 98.35 (2mo) dTTP 15 93.57 99.29 81.05 (12 mo) 98.86 (2mo) dUTP 15 93.8 99.45 84.95 (12 mo) 98.5 (2mo) Solution ddNTP (10 mM) ddATP 3 99.69 99.49 94.52 ddCTP 3 100 98.51 97.38 ddGTP 3 98.4 98.08 94.23 ddTTP 3 99.36 99.13 87.06 Solution ddNTP (5 mM) ddATP 3 99.77 98.12 68.56 4 99.63 96.31 2 ddCTP 3 98.77 100 98.4 4 99.27 99.46 93.72 ddGTP 3 95.61 98 96.67 4 98.25 97.9 93.68 ddTTP 3 93.1 55.09 49.03 4 94.25 63.23 3.6 RTP Solutions (100 mM) ATP 3 98.57 98.18 95.39 CTP 3 99.25 99.43 98.43 GTP 3 98.46 98.44 96.82 UTP 3 99.71 99.69 97.99 Source: Data based on chromatographic separation of nucleotide species via high performance chromatography on an Amersham Pharmacia Biotech FPLC® System. Notes: Each sample, 0.2mmoles (0.2 ml of a 1 mM solution) was injected onto a Mono Q® Ion Exchange column. Using the following buffers: Buffer A, 5mM sodium phosphate, pH 7.0. Buffer B, 5mM sodium phosphate, 1M NaCl, pH 7.0. purification was achieved via a gradient of 5–35% NaCl over 15 minutes using a flow rate of 1 ml/min. Nucleotide peaks were detected at of 254 nm. (Data from Amersham Pharmacia Biotech, 1993a.)
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(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 (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
E To prepare a 10mM solution from a 250mg package of dGTP, the dGTP may be dissolved in about 40ml of purified H,O. The pH may then be adjusted from a pH of about 4.5 to the desired pH with 1N NaOH, carefully added dropwise with stirring. About 05ml of 1N NaOH will be needed for this example. A dilution of 1: 200 will give a reading in the linear range of most spectropho- tometers. Spectroscopy should be performed at the nucleotide absorbance maximum, which is 253 nm for dGTP. In this example an absorbance of about 0.700 is expected. The formula for deter mining the concentration is: Absorbance atλmax× dilution factor = molar concentration A Using the Am for dgtP of 13, 700. the concentration in this xample is found to be 0.700×200 13,700=0.0102M,or10.2 mm dGTP What Is the Effect of Thermocycling on Nucleotide stability? Properly stored, lyophilized and solution nucleotides are stable for years. The data in Table 10.4(Amersham Pharmacia Biotech 1993b)describe the destruction of nucleotides under common thermocycling conditions. Fortunately, due to the excess presence of nucleotides, thermal degradation does not typically impede a PCR reaction Is There a Difference between Absorbance, A260 and Optical Density? Readers are strongly urged to review Efiok (1993)for a thorough and clearly written discussion on the spectrophoto- metric quantitation of nucleotides and nucleic acids. Absorbance(A) Absorbance(A), also referred to as optical density(OD),is a unitless measure of the amount of light a solution traps, as measured on a spectrophotometer. The Beer-Lambert equation (Efiok, 1993) defines absorbance in terms of the concentration of the solution in moles per liter(C), the path length the light travels through the solution in centimeters(D), and the extinction coeffi cient in liter per moles times centimeters (E) Nucleotides, Oligonucleotides, and Polynucleotides 275
Example To prepare a 10mM solution from a 250mg package of dGTP, the dGTP may be dissolved in about 40 ml of purified H2O. The pH may then be adjusted from a pH of about 4.5 to the desired pH with 1 N NaOH, carefully added dropwise with stirring. About 0.5 ml of 1 N NaOH will be needed for this example. A dilution of 1 :200 will give a reading in the linear range of most spectrophotometers. Spectroscopy should be performed at the nucleotide’s absorbance maximum, which is 253 nm for dGTP. In this example an absorbance of about 0.700 is expected. The formula for determining the concentration is: Using the Am for dGTP of 13,700, the concentration in this example is found to be What Is the Effect of Thermocycling on Nucleotide Stability? Properly stored, lyophilized and solution nucleotides are stable for years. The data in Table 10.4 (Amersham Pharmacia Biotech, 1993b) describe the destruction of nucleotides under common thermocycling conditions. Fortunately, due to the excess presence of nucleotides, thermal degradation does not typically impede a PCR reaction. Is There a Difference between Absorbance,A260, and Optical Density? Readers are strongly urged to review Efiok (1993) for a thorough and clearly written discussion on the spectrophotometric quantitation of nucleotides and nucleic acids. Absorbance (A) Absorbance (A), also referred to as optical density (OD), is a unitless measure of the amount of light a solution traps, as measured on a spectrophotometer. The Beer-Lambert equation (Efiok, 1993) defines absorbance in terms of the concentration of the solution in moles per liter (C), the path length the light travels through the solution in centimeters (l), and the extinction coeffi- cient in liter per moles times centimeters (E): 0 700 200 13 700 0 0102 10 2 . , ., . ¥ = M mM or dGTP Absorbance at dilution factor molar concentration m lmax¥ = A Nucleotides, Oligonucleotides, and Polynucleotides 275
Table 10.4 Breakdown of Nucleotides under Thermocycling Conditions 0 PCR Cycles 25 PCR Cycles dATP 9241 dCTP dGTP 99.14 dTTP dATP dCTP dgTP dTTP 94.17 dATP dCTP 93.84 dGTP 9939 dTTP nt 4 dATP 92.77 dCTP dGTP dTTP 99.19 ource: Data from Amerhsam Pharmacia Biotech(1993b) lote: Each nucleotide was mixed with 10x PCR buffer from the Gene Amp@ PCR Reagent Kit(Perking Elmer catalogue number N801-0055)to give a final nucleotide co buffer. Noncycled control samples(0 cycle ately assayed Test samples were cycled for 25 rounds in a Perkin Elmer Gene Amp@ PC System 9600 using the cycling program of 94 C for 10 seconds. 55 C for 10 seconds, and For analysis, samples were diluted to give a nucleotide concentration of 0. 133 mM. The luted samples were then assayed on FPLCO System using a MonoQ( column. The assay ime for a sample was 10 minutes using a sodium chloride gradient (50-400 mM)in 20mM Tris-HCl at pH.0. Nucleotide peaks were detect using a wavelength of 254 nm. A= CIE Since the units of c. l. and e all cancel. a is unitless. Absorbance Unit Also referred to as an optical density(oD)unit, an absorbance unit (AU) is the concentration of a material that gives an absorbance of one and therefore is also a unitless measure. Typi cally, when working with nucleic acids, we express the extinction coefficient in ml per mg times cm E mg xcm USing an extinction coefficient expressed in these terms, one Azo unit of double-stranded dNA has a concentration of DNA of 50 ug/mL For practical reasons, suppliers typically define the total volume of material to be one milliliter when selling their nucleic acids 276 Gerstein
A = ClE Since the units of C, l, and E all cancel, A is unitless. Absorbance Unit Also referred to as an optical density (OD) unit, an absorbance unit (AU) is the concentration of a material that gives an absorbance of one and therefore is also a unitless measure. Typically, when working with nucleic acids, we express the extinction coefficient in ml per mg times cm: Using an extinction coefficient expressed in these terms, one A260 unit of double-stranded DNA has a concentration of DNA of 50mg/ml. For practical reasons, suppliers typically define the total volume of material to be one milliliter when selling their nucleic acids. E = ¥ ml mg cm 276 Gerstein Table 10.4 Breakdown of Nucleotides under Thermocycling Conditions % Purity of Triphosphate Nucleotides 0 PCR Cycles 25 PCR Cycles Experiment 1 dATP 99.31 92.41 dCTP 99.47 93.64 dGTP 99.14 92.43 dTTP 99.06 93.38 Experiment 2 dATP 99.56 94.17 dCTP 99.80 95.36 dGTP 99.78 94.02 dTTP 99.60 94.17 Experiment 3 dATP 99.40 92.02 dCTP 99.66 93.84 dGTP 99.39 92.68 dTTP 99.15 93.69 Experiment 4 dATP 99.44 92.77 dCTP 99.59 93.89 dGTP 99.43 92.88 dTTP 99.19 93.65 Source: Data from Amerhsam Pharmacia Biotech (1993b). Note: Each nucleotide was mixed with 10¥ PCR buffer from the GeneAmp® PCR Reagent Kit (Perking Elmer catalogue number N801-0055)to give a final nucleotide concentration of 0.2 mM in 1¥ PCR buffer. Noncycled control samples (0 cycles) were immediately assayed. Test samples were cycled for 25 rounds in a Perkin Elmer GeneAmp® PC System 9600 using the cycling program of 94°C for 10 seconds, 55°C for 10 seconds, and 72°C for 10 seconds. After cycling, the samples were stored on ice until assayed. For analysis, samples were diluted to give a nucleotide concentration of 0.133 mM. The diluted samples were then assayed on FPLC® System using a MonoQ® column. The assay time for a sample was 10 minutes using a sodium chloride gradient (50–400 mM) in 20 mM Tris-HCl at pH 9.0. Nucleotide peaks were detect using a wavelength of 254 nm