584
APPLICATIONS
[68]
[68] C h e m i c a l l y C l e a v a b l e B i o t i n - L a b e l e d Nucleotide Analogs
By TIMOTHY M. HERMAN and BARBARAJ. FENN The ability to incorporate biotin-labeled nucleotide analogs into DNA and R N A has stimulated interest in the use of the avidin-biotin affinity system to isolate protein-DNA ~,2 and protein-RNA complexes. 3 However, the high affinity of avidin for biotin has resulted in one major limitation of this approach, namely, the inability to dissociate the biotinylated complex from the avidin affinity column under conditions that preserve the protein-nucleic acid as well as protein-protein interactions present in the complex. Although several approaches have been taken to solve this problem, 4,5 none have been entirely satisfactory. The development of the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP 6 appears to be a general solution to this problem. Bio-12SS-dUTP contains a disulfide bond in the 12-atom linker arm joining biotin to the 5-carbon of uridine. Following binding of biotinylated DNA to an avidin affinity column, a reducing agent such a dithiothreitol is used to cleave the linker arm and release the DNA. Because this cleavage occurs under gentle, nondenaturing conditions, the recovered complexes should be amenable to further investigation of the protein-protein and protein-nucleic acid interactions found within the native complex. We describe here a procedure to synthesize the chemically cleavable biotinylated nucleotide Bio-19-SS-dUTP (Fig. I). This analog is a modification of Bio-12-SS-dUTP in which an additional 7 atoms are present in the linker arm between the disulfide bond and biotin. 7 This modification makes it possible to use Bio-19-SS-dUTP with streptavidin, an acidic protein with no nonspecific DNA-binding properties. The procedure is based on that originally described by Langer et al. 8 The corresponding i M. S. Kasher, D. Pintel, and D. C. Ward, Mol Cell. Biol. 6, 3117 (1986). 2 T. M. Herman, this series, Vol. 170, p. 41. 3 p. j. Grabowski and P. A. Sharp. Science 233, 1294 (1986). 4 R. A. Gravel, K. F. Lam, D. Mahuran, and A. Kronis, Arch. Biochem. Biophys. 201, 669 (1980). 5 K. Hoffmann, S. W. Wood, C. C. Brinton, J. A. Montibeller, and F. M. Finn, Proc. Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 6 M. L. Shimkus, J. Levy, and T. M. Herman, Proc. Natl. Acad. Sci. U.S.A. 82, 2593 (1985). 7 T. M. Herman, E. Lefever, and M. Shimkus, Anal. Biochern. 156, 48 (1986). 8 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[68]
CLEAVABLE BIOTIN-LABELING REAGENTS
585
0
@
H H 0 0 0 0 (~N~ CH=CHCH2NH~;(CHz)2 SS(CH2)2NH~(GH2)5NH~:(CH2)4 -~S .,j o," 9" 9-
,
I..0, CHz-O-P-O-P-O-P-O" It" %
'~......J H
Ii
0
oo
0
.
0
OH
FIG. 1. Structure of Bio-19-SS-dUTP.
ribonucleotide analog can be prepared by the same procedure, substituting UTP for dUTP as the starting material. Synthesis of Bio- 19-SS-dUTP The synthesis of Bio-19-SS-dUTP consists of three distinct steps. First, dUTP is reacted with mercury acetate to modify the 5-carbon of uracil. Second, allylamine is added to this position on the pyrimidine ring in the presence of a palladium catalyst. Third, allylamine-dUTP is reacted with an N-hydroxysuccinimide ester of biotin to produce the final product, Bio-19-SS-dUTP. The entire procedure requires 4 days and involves the use of two ion-exchange columns and a final purification of the product by reversed-phase HPLC. A detailed description of each step of the procedure follows.
Synthesis of 5-Mercurated dUTP (Hg-dUTP) Dissolve 50 mg (91 /zmol) of deoxyuridine 5'-triphosphate (Sigma Chemical Co., St. Louis, MO) in 10 ml of 0.1 M sodium acetate (pH 6.0). Add a 5-fold molar excess (142 mg) of mercury(II) acetate to the dUTP solution. Incubate at 50° for 4 hr. Cool the reaction on ice. Add 34 mg of LiCI (9-fold molar excess over dUTP). Remove HgCI2 by extracting the reaction 6 times with an equal volume of ice-cold ethyl acetate. A brief centrifugation (1,000 g for 3 min at 4°) is used to separate the upper ethyl acetate phase from the lower aqueous phase containing the Hg-dUTP. The aqueous phase remains clear during these extractions only if it is kept at 4 °. On warming, the aqueous phase becomes cloudy. Precipitate the Hg-dUTP by adding 3 volumes of cold absolute ethanol. A white fluffy precipitate is immediately visible. Cool for 1 hr at - 2 0 °. Collect the precipitate by centrifugation (5,000 g for 10 min). Wash the precipitate twice with cold absolute ethanol and once with cold diethyl
586
APPLICATIONS
[68]
ether. Air dry. Dissolve the mercurated nucleotide in 0.1 M sodium acetate (pH 5.0) ( - 2 ml) and adjust to a concentration of 20 m M based on the millimolar extinction coefficient of 10.2 at 260nm. Comments. The above procedure routinely results in the mercuration of at least 90% of the dUTP. Although methods have been reported to assess the efficiency of the ethyl acetate extraction procedure9 and to measure the extent of mercuration, j° such procedures are not normally necessary. Instead, the Hg-dUTP obtained above is used directly in the next step in the synthesis procedure to produce allylamine-dUTP.
Synthesis of 5-(3-Amino)allyldeoxyuridine Triphosphate (AA-dUTP) Prepare a fresh solution of 2.0 M allylamine by adding 1.5 ml of 13.3 M allylamine (Aldrich, Milwaukee, WI) slowly to 8.5 ml of ice-cold 4 N glacial acetic acid. Adjust the solution to pH 7.0 with 5 N NaOH. Add 480 /~1 (960/xmol) of the 2.0 M allylamine solution to 80 tzmol Hg-dUTP (4 ml of the 20 m M solution in 0.1 M sodium acetate, pH 5.0) and 80/~mol of the catalyst K2PdC14 (Aldrich; 0.64 ml of a 125 m M solution in water). Incubate the reaction for 18 hr (overnight) at room temperature. A black precipitate will form during this reaction. Filter the reaction several times through a 0.45-/zm nylon filter to remove the precipitate. Dilute the clear filtrate with 5 volume of water. Load the diluted filtrate onto a 35 ml DEAE-Sephadex A-25 column (1.5 x 20 cm) equilibrated with 0.1 M sodium acetate (pH 5.5). Wash the loaded column with 150 ml of equilibration buffer. Elute the aminoallyl dUTP with a 180-ml linear gradient of 0.1 to 0.6 M sodium acetate (pH 8.5). Collect 3-ml fractions. Monitor the absorbance of the column fractions at 260nm. Aminoallyl-dUTP elutes from this column at approximately 0.4 M sodium acetate. 11,12 Pool the fractions containing the aminoallyl-dUTP and concentrate by addition of 3 volumes of cold absolute ethanol. A heavy white precipitate forms immediately. Cool the solution for 1 hr at - 2 0 °. Collect the precipitate by centrifugation and resuspend in 3 ml of 0.1 M sodium borate (pH 9 A. J. Christopher, Analyst 94, 392 (1969). ~0R. M. K. Dale, D. C. Ward, D. C. Livingston, and E. Martin, Nucleic Acids Res. 2, 915 (1975). u M. L. Shimkus, P. Guaglianone, and T. M. Herman, DNA 5, 247 (1986). 12 Fractions containing aminoallyl-dUTP are easily identified by their absorbance spectrum. The addition of the exocyclic double bond to the pyrimidine ring alters the absorbance properties of the nucleotide such that it now shows an absorbance minimum at 262 nm and absorbance maximums at 240 and 288 nm. The ratio of absorbances at 262 and 288 nm is approximately 1.4. In contrast, both dUTP and Hg-dUTP exhibit a single absorbance maximum at 260 nm.
[68]
CLEAVABLE BIOTIN-LABELING REAGENTS
587
8.5). Determine the concentration of aminoallyl-dUTP spectrophotometrically using a millimolar extinction coefficient of 7.1 at 288 nm. Comments. The yield of aminoallyl-dUTP at this stage in the procedure is normally 15-20% of the starting dUTP.
Synthesis of Bio-19-SS-dUTP Add 14.5 /zmol of sulfo-NHS-LC-SS-biotin (Pierce Chemical Co., Rockford, IL) to 14.5/~mol of aminoallyl-dUTP in 2.7 ml of 0.1 M sodium borate (pH 8.5). Incubate the reaction for 2 hr at room temperature. Load the reaction onto a 35-ml DEAE-Sephadex A-25 column (1.5 x 20cm) equilibrated in 0.1 M triethylamine-carbonate (pH 7.5). 13Following application of the sample, wash the column with 2 volumes of equilibration buffer. Elute the Bio-19-SS-dUTP with a 180-ml linear gradient of 0.1 M triethylamine-carbonate (pH 7.5) to 0.9 M triethylamine-carbonate (pH 8,0). Wash with an additional 100 ml of the 0.9 M buffer to assure the complete elution of the product. Collect 3-ml fractions and monitor the absorbance at 288nm. Pool fractions containing Bio-19-SS-dUTP (last major peak to elute from the column at approximately 0.7 M triethylamine-carbonate). Concentrate the Bio-19-SS-dUTP by rotary evaporation. Following several additions of methanol to the Bio-19-SS-dUTP, the solution is evaporated to dryness. The identity of the pooled DEAE-Sephadex A-25 fractions containing Bio-19-SS-dUTP can be verified by reversed-phase HPLC analysis of an aliquot of each pool. Two-microliter aliquots of each pool, diluted to 200 /zl with 50 m M triethylamine-carbonate (pH 7.5) are analyzed as described below. Dissolve Bio-19-SS-dUTP in 2.0 ml of 50 m M triethylamine-carbonate (pH 7.0) ~3 in preparation for final purification by reversed-phase HPLC. Inject 200-/xl aliquots of Bio-19-SS-dUTP onto a Bio-Sil ODS-5S column (250 x 4 mm; Bio-Rad, Richmond, CA) equilibrated with 50 m M triethylamine-carbonate (pH 7.0)-15% acetonitrile. 14 13 The triethylamine-carbonate buffer is a volatile buffer that facilitates the concentration of the final product by rotary evaporation. The pH of this buffer is adjusted by bubbling CO2 through the solution. This buffer must be prepared just before use and care taken to avoid an increase in pH as the CO2 is lost from the solution. 14 Several precautions must be taken in the preparation of the buffer used in this step. Fifty millimolar triethylamine is first prepared and filtered through a 0.45-tzm filter to remove any particulate impurities. The buffer is then adjusted to pH 7.0 with CO2. Finally, acetonitrile is added to a final concentration of 15%. This final buffer should not be filtered or degassed at this stage. Attempts to do so will alter both the final pH and the acetonitrile concentration of the buffer. This in turn will alter the retention time of the biotinylated nucleotide. In addition, buffers with a pH greater than 7.0 should be avoided when using silica-based HPLC resins.
588
APPLICATIONS
[69]
Bio-19-SS-dUTP is eluted isocratically with a retention time of approximately 10 min (flow rate 1.0 ml/min). 15 Bio-19-SS-dUTP can be identified in two ways. First, it is the most hydrophobic species present in the reaction and therefore is the last peak to elute. Second, simultaneous monitoring of the effluent at both 262 and 288 nm will reveal the characteristic ratio at these two wavelengths of 1.4. Concentrate the Bio-19-SS-dUTP by rotary evaporation to dryness with several additions of methanol. Dissolve in 10 m M Tris-HCi (pH 7.5) and determine the concentration using the millimolar extinction coefficient of 7.1 at 288 nm. Store the nucleotide at - 8 0 °. Bio-19-SS-dUTP can be stored for at least 6 months in this way without any noticeable degradation. Comments. The yield in the last step in the synthesis procedure has ranged from 25 to 75% of the aminoaUyl-dUTP being converted to Bio-19SS-dUTP. This variability is most likely due to differences in the reactivity of the N-hydroxysuccinimide-activated biotin ester. Acknowledgment This work was supported by National Science Foundation Grant DMB 8616956. t5 The retention time of Bio-19-SS-dUTP in this system is critically dependent on the concentration of acetonitrile in the elution buffer. Therefore, the acetonitrile concentration can be easily altered to achieve the desired retention time on individual reversed-phase HPLC columns.
[69] P r e p a r a t i o n a n d U s e s o f P h o t o b i o t i n
By JAMES L. MCINNES, ANTHONY C. FORSTER, DEREK C. SKINGLE, and ROBERT H. SYMONS The development of nonradioisotopic methods for the labeling of nucleic acid probes has created great interest worldwide. The basis for this approach is to introduce into the nucleic acid a modifying group which can subsequently be detected by means of the production of an insoluble or soluble colored product or by some other nonradioactive method. Such modifying groups can be introduced enzymatically 1-6 or chemically. 7-18 A t p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 2 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
APPLICATIONS
[68]
[68] C h e m i c a l l y C l e a v a b l e B i o t i n - L a b e l e d Nucleotide Analogs
By TIMOTHY M. HERMAN and BARBARAJ. FENN The ability to incorporate biotin-labeled nucleotide analogs into DNA and R N A has stimulated interest in the use of the avidin-biotin affinity system to isolate protein-DNA ~,2 and protein-RNA complexes. 3 However, the high affinity of avidin for biotin has resulted in one major limitation of this approach, namely, the inability to dissociate the biotinylated complex from the avidin affinity column under conditions that preserve the protein-nucleic acid as well as protein-protein interactions present in the complex. Although several approaches have been taken to solve this problem, 4,5 none have been entirely satisfactory. The development of the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP 6 appears to be a general solution to this problem. Bio-12SS-dUTP contains a disulfide bond in the 12-atom linker arm joining biotin to the 5-carbon of uridine. Following binding of biotinylated DNA to an avidin affinity column, a reducing agent such a dithiothreitol is used to cleave the linker arm and release the DNA. Because this cleavage occurs under gentle, nondenaturing conditions, the recovered complexes should be amenable to further investigation of the protein-protein and protein-nucleic acid interactions found within the native complex. We describe here a procedure to synthesize the chemically cleavable biotinylated nucleotide Bio-19-SS-dUTP (Fig. I). This analog is a modification of Bio-12-SS-dUTP in which an additional 7 atoms are present in the linker arm between the disulfide bond and biotin. 7 This modification makes it possible to use Bio-19-SS-dUTP with streptavidin, an acidic protein with no nonspecific DNA-binding properties. The procedure is based on that originally described by Langer et al. 8 The corresponding i M. S. Kasher, D. Pintel, and D. C. Ward, Mol Cell. Biol. 6, 3117 (1986). 2 T. M. Herman, this series, Vol. 170, p. 41. 3 p. j. Grabowski and P. A. Sharp. Science 233, 1294 (1986). 4 R. A. Gravel, K. F. Lam, D. Mahuran, and A. Kronis, Arch. Biochem. Biophys. 201, 669 (1980). 5 K. Hoffmann, S. W. Wood, C. C. Brinton, J. A. Montibeller, and F. M. Finn, Proc. Natl. Acad. Sci. U.S.A. 77, 4666 (1980). 6 M. L. Shimkus, J. Levy, and T. M. Herman, Proc. Natl. Acad. Sci. U.S.A. 82, 2593 (1985). 7 T. M. Herman, E. Lefever, and M. Shimkus, Anal. Biochern. 156, 48 (1986). 8 p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[68]
CLEAVABLE BIOTIN-LABELING REAGENTS
585
0
@
H H 0 0 0 0 (~N~ CH=CHCH2NH~;(CHz)2 SS(CH2)2NH~(GH2)5NH~:(CH2)4 -~S .,j o," 9" 9-
,
I..0, CHz-O-P-O-P-O-P-O" It" %
'~......J H
Ii
0
oo
0
.
0
OH
FIG. 1. Structure of Bio-19-SS-dUTP.
ribonucleotide analog can be prepared by the same procedure, substituting UTP for dUTP as the starting material. Synthesis of Bio- 19-SS-dUTP The synthesis of Bio-19-SS-dUTP consists of three distinct steps. First, dUTP is reacted with mercury acetate to modify the 5-carbon of uracil. Second, allylamine is added to this position on the pyrimidine ring in the presence of a palladium catalyst. Third, allylamine-dUTP is reacted with an N-hydroxysuccinimide ester of biotin to produce the final product, Bio-19-SS-dUTP. The entire procedure requires 4 days and involves the use of two ion-exchange columns and a final purification of the product by reversed-phase HPLC. A detailed description of each step of the procedure follows.
Synthesis of 5-Mercurated dUTP (Hg-dUTP) Dissolve 50 mg (91 /zmol) of deoxyuridine 5'-triphosphate (Sigma Chemical Co., St. Louis, MO) in 10 ml of 0.1 M sodium acetate (pH 6.0). Add a 5-fold molar excess (142 mg) of mercury(II) acetate to the dUTP solution. Incubate at 50° for 4 hr. Cool the reaction on ice. Add 34 mg of LiCI (9-fold molar excess over dUTP). Remove HgCI2 by extracting the reaction 6 times with an equal volume of ice-cold ethyl acetate. A brief centrifugation (1,000 g for 3 min at 4°) is used to separate the upper ethyl acetate phase from the lower aqueous phase containing the Hg-dUTP. The aqueous phase remains clear during these extractions only if it is kept at 4 °. On warming, the aqueous phase becomes cloudy. Precipitate the Hg-dUTP by adding 3 volumes of cold absolute ethanol. A white fluffy precipitate is immediately visible. Cool for 1 hr at - 2 0 °. Collect the precipitate by centrifugation (5,000 g for 10 min). Wash the precipitate twice with cold absolute ethanol and once with cold diethyl
586
APPLICATIONS
[68]
ether. Air dry. Dissolve the mercurated nucleotide in 0.1 M sodium acetate (pH 5.0) ( - 2 ml) and adjust to a concentration of 20 m M based on the millimolar extinction coefficient of 10.2 at 260nm. Comments. The above procedure routinely results in the mercuration of at least 90% of the dUTP. Although methods have been reported to assess the efficiency of the ethyl acetate extraction procedure9 and to measure the extent of mercuration, j° such procedures are not normally necessary. Instead, the Hg-dUTP obtained above is used directly in the next step in the synthesis procedure to produce allylamine-dUTP.
Synthesis of 5-(3-Amino)allyldeoxyuridine Triphosphate (AA-dUTP) Prepare a fresh solution of 2.0 M allylamine by adding 1.5 ml of 13.3 M allylamine (Aldrich, Milwaukee, WI) slowly to 8.5 ml of ice-cold 4 N glacial acetic acid. Adjust the solution to pH 7.0 with 5 N NaOH. Add 480 /~1 (960/xmol) of the 2.0 M allylamine solution to 80 tzmol Hg-dUTP (4 ml of the 20 m M solution in 0.1 M sodium acetate, pH 5.0) and 80/~mol of the catalyst K2PdC14 (Aldrich; 0.64 ml of a 125 m M solution in water). Incubate the reaction for 18 hr (overnight) at room temperature. A black precipitate will form during this reaction. Filter the reaction several times through a 0.45-/zm nylon filter to remove the precipitate. Dilute the clear filtrate with 5 volume of water. Load the diluted filtrate onto a 35 ml DEAE-Sephadex A-25 column (1.5 x 20 cm) equilibrated with 0.1 M sodium acetate (pH 5.5). Wash the loaded column with 150 ml of equilibration buffer. Elute the aminoallyl dUTP with a 180-ml linear gradient of 0.1 to 0.6 M sodium acetate (pH 8.5). Collect 3-ml fractions. Monitor the absorbance of the column fractions at 260nm. Aminoallyl-dUTP elutes from this column at approximately 0.4 M sodium acetate. 11,12 Pool the fractions containing the aminoallyl-dUTP and concentrate by addition of 3 volumes of cold absolute ethanol. A heavy white precipitate forms immediately. Cool the solution for 1 hr at - 2 0 °. Collect the precipitate by centrifugation and resuspend in 3 ml of 0.1 M sodium borate (pH 9 A. J. Christopher, Analyst 94, 392 (1969). ~0R. M. K. Dale, D. C. Ward, D. C. Livingston, and E. Martin, Nucleic Acids Res. 2, 915 (1975). u M. L. Shimkus, P. Guaglianone, and T. M. Herman, DNA 5, 247 (1986). 12 Fractions containing aminoallyl-dUTP are easily identified by their absorbance spectrum. The addition of the exocyclic double bond to the pyrimidine ring alters the absorbance properties of the nucleotide such that it now shows an absorbance minimum at 262 nm and absorbance maximums at 240 and 288 nm. The ratio of absorbances at 262 and 288 nm is approximately 1.4. In contrast, both dUTP and Hg-dUTP exhibit a single absorbance maximum at 260 nm.
[68]
CLEAVABLE BIOTIN-LABELING REAGENTS
587
8.5). Determine the concentration of aminoallyl-dUTP spectrophotometrically using a millimolar extinction coefficient of 7.1 at 288 nm. Comments. The yield of aminoallyl-dUTP at this stage in the procedure is normally 15-20% of the starting dUTP.
Synthesis of Bio-19-SS-dUTP Add 14.5 /zmol of sulfo-NHS-LC-SS-biotin (Pierce Chemical Co., Rockford, IL) to 14.5/~mol of aminoallyl-dUTP in 2.7 ml of 0.1 M sodium borate (pH 8.5). Incubate the reaction for 2 hr at room temperature. Load the reaction onto a 35-ml DEAE-Sephadex A-25 column (1.5 x 20cm) equilibrated in 0.1 M triethylamine-carbonate (pH 7.5). 13Following application of the sample, wash the column with 2 volumes of equilibration buffer. Elute the Bio-19-SS-dUTP with a 180-ml linear gradient of 0.1 M triethylamine-carbonate (pH 7.5) to 0.9 M triethylamine-carbonate (pH 8,0). Wash with an additional 100 ml of the 0.9 M buffer to assure the complete elution of the product. Collect 3-ml fractions and monitor the absorbance at 288nm. Pool fractions containing Bio-19-SS-dUTP (last major peak to elute from the column at approximately 0.7 M triethylamine-carbonate). Concentrate the Bio-19-SS-dUTP by rotary evaporation. Following several additions of methanol to the Bio-19-SS-dUTP, the solution is evaporated to dryness. The identity of the pooled DEAE-Sephadex A-25 fractions containing Bio-19-SS-dUTP can be verified by reversed-phase HPLC analysis of an aliquot of each pool. Two-microliter aliquots of each pool, diluted to 200 /zl with 50 m M triethylamine-carbonate (pH 7.5) are analyzed as described below. Dissolve Bio-19-SS-dUTP in 2.0 ml of 50 m M triethylamine-carbonate (pH 7.0) ~3 in preparation for final purification by reversed-phase HPLC. Inject 200-/xl aliquots of Bio-19-SS-dUTP onto a Bio-Sil ODS-5S column (250 x 4 mm; Bio-Rad, Richmond, CA) equilibrated with 50 m M triethylamine-carbonate (pH 7.0)-15% acetonitrile. 14 13 The triethylamine-carbonate buffer is a volatile buffer that facilitates the concentration of the final product by rotary evaporation. The pH of this buffer is adjusted by bubbling CO2 through the solution. This buffer must be prepared just before use and care taken to avoid an increase in pH as the CO2 is lost from the solution. 14 Several precautions must be taken in the preparation of the buffer used in this step. Fifty millimolar triethylamine is first prepared and filtered through a 0.45-tzm filter to remove any particulate impurities. The buffer is then adjusted to pH 7.0 with CO2. Finally, acetonitrile is added to a final concentration of 15%. This final buffer should not be filtered or degassed at this stage. Attempts to do so will alter both the final pH and the acetonitrile concentration of the buffer. This in turn will alter the retention time of the biotinylated nucleotide. In addition, buffers with a pH greater than 7.0 should be avoided when using silica-based HPLC resins.
588
APPLICATIONS
[69]
Bio-19-SS-dUTP is eluted isocratically with a retention time of approximately 10 min (flow rate 1.0 ml/min). 15 Bio-19-SS-dUTP can be identified in two ways. First, it is the most hydrophobic species present in the reaction and therefore is the last peak to elute. Second, simultaneous monitoring of the effluent at both 262 and 288 nm will reveal the characteristic ratio at these two wavelengths of 1.4. Concentrate the Bio-19-SS-dUTP by rotary evaporation to dryness with several additions of methanol. Dissolve in 10 m M Tris-HCi (pH 7.5) and determine the concentration using the millimolar extinction coefficient of 7.1 at 288 nm. Store the nucleotide at - 8 0 °. Bio-19-SS-dUTP can be stored for at least 6 months in this way without any noticeable degradation. Comments. The yield in the last step in the synthesis procedure has ranged from 25 to 75% of the aminoaUyl-dUTP being converted to Bio-19SS-dUTP. This variability is most likely due to differences in the reactivity of the N-hydroxysuccinimide-activated biotin ester. Acknowledgment This work was supported by National Science Foundation Grant DMB 8616956. t5 The retention time of Bio-19-SS-dUTP in this system is critically dependent on the concentration of acetonitrile in the elution buffer. Therefore, the acetonitrile concentration can be easily altered to achieve the desired retention time on individual reversed-phase HPLC columns.
[69] P r e p a r a t i o n a n d U s e s o f P h o t o b i o t i n
By JAMES L. MCINNES, ANTHONY C. FORSTER, DEREK C. SKINGLE, and ROBERT H. SYMONS The development of nonradioisotopic methods for the labeling of nucleic acid probes has created great interest worldwide. The basis for this approach is to introduce into the nucleic acid a modifying group which can subsequently be detected by means of the production of an insoluble or soluble colored product or by some other nonradioactive method. Such modifying groups can be introduced enzymatically 1-6 or chemically. 7-18 A t p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 2 j. j. Leary, D. J. Brigati, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 80, 4045 (1983).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
