Nucleic Acids Research

Isolation of TMP kinase

A crude preparation of Escherichia coli pyrimidine kinase was prepared according to procedures adapted from Lehman et al. (29). This enzyme preparation was used in the production of dNTPs used for synthesis of the DNA triplex and quadruplex oligonucleotides. The other DNA oligonucleotides were synthesized using dNTPs prepared using a highly purified GST-TMP kinase fusion protein.The GST-TMP kinase fusion protein was purified as described by Smith et al. (23) from E.coli strain DH10[beta] containing a recombinant plasmid, GEX-2 (Pharmacia) with the gene for a glutathione-S-transferase/yeast deoxythymidylate kinase fusion protein (GST-TMPK) inserted into its expression site (kindly provided by Fiona Jucker of Nexstar Pharmaceuticals). A 1.25 l culture produced 300 U of GST-dTMP kinase (1 U is defined as the amount of enzyme necessary to convert 1 µmol of dTMP to dTTP in 1 min).

Production of Taq DNA polymerase

A recombinant E.coli clone, DSBI-alpha-1, carrying the plasmid pTAQ was purchased from Display Systems Biotech. The cells were incubated at 37°C in 50 ml 2X-YT broth (30), supplemented with 100 µg/ml ampicillin, until mid-log phase (OD₅₉₀ ~0.8). Expression of the Taq polymerase gene on the pTAQ plasmid was then induced by 0.5 mM IPTG. After 17 h induction, the cells were pelleted by centrifugation at 4000 g at 1°C for 5 min. The pellets were washed once with STE (0.1 M NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0) (30), resuspended in 3 ml STE, and then 3 ml 4× storage buffer (200 mM Tris-HCl, pH 8.0, 400 mM NaCl, 0.4 mM EDTA, 2.0 mM dithiothreitol, 4% Triton X-100) was added. The cells were incubated at 75°C for 1 h with periodic mixing and then lysed by sonication. Denatured proteins were removed by centrifugation for 10 min at 12 000 g at 4°C. The supernatant was decanted, an equal volume of glycerol added, and the solution stored at -20°C. The activity of the recombinant Taq polymerase was assessed by comparison to Amplitaq (Perkin Elmer) and Taq polymerase (Promega) using standard PCR reactions (31). The activity of the Taq was 40 000 U/ml (500 000 U total).

Synthesis and purification of ¹³C,¹⁵N-labeled dNTPs

The procedures for the synthesis and purification of ¹³C,¹⁵N-labeled dNTPs were adapted from Batey et al. (7) and Nikonowicz et al. (8). Methylophilus methylotrophus strain AS-1 was grown as described by Batey et al. (7) and Nikonowicz et al. (8) with the exception that a 5-fold lower concentration of [¹³C]MeOH was included in the growth medium. This decrease in methanol/cell culture results in only 20% lower yield of total nucleic acid but 80% decrease in cost, and thus gives the optimal yield/cost. Subsequent harvesting and purification steps were performed as described. The overall yield was 1.5 g NMPs and 0.5 g dNMPs from 31 l of culture. All dNMPs were initially phosphorylated to dNTPs using the crude kinase preparation (22,29). This resulted in >90% of the dAMP, dGMP and dCMP being converted to dNTPs, but only ~70% of the dTMP was converted to dTTP. In order to get complete conversion of the dNMPs to dNTPs, the products of the reaction mixture above were rephosphorylated using purified GST-TMP kinase as described (23), which resulted in quantitative conversion of all dNMPs to dNTPs.

Separation of dNTPs

The labeled dNTPs were filtered through a 0.2 µm filter at 4°C and then lyophilized. Separation of the dNTPs was accomplished by reverse-phase ion-pairing HPLC (32) on tandem 25 × 10 cm Waters Sep-pack C-18 µbondapack columns. The dNTPs were separated using a linear gradient from 98% 0.1 M triethylamine, 2% methanol to 80% 0.1 M triethylamine, 20% methanol over 59 min at 10 ml/min. Up to 10 mg of nucleotides could be injected without the loss of baseline resolution. Each dNTP was collected, concentrated under vacuum in a Speed-Vac and precipitated with ethanol. Each of the four dNTPs were then resuspended in H₂O and adjusted to ~ pH 7 with HCl.

Production of templates and primers

Template and primer oligodeoxyribonucleotides (Fig. 1) were synthesized on an Applied Biosystems 392 DNA synthesizer using standard phosphoramidite chemistry. The primers were synthesized starting with 1 µmol ribose CPG columns (Glen Research). Templates were deprotected using standard protocols and the primers were deprotected with 75% NH₄OH/25% ethanol. The primers were then dried by vacuum centrifugation (Speed-Vac). Deprotection of the 2[prime]-hydroxyl group was accomplished by incubation in 1 M tetrabutylammoniumflouride in tetrahydrofuran for 30 h. The reaction was quenched by the addition of an equal volume of H₂O. The primers were concentrated by vacuum centrifugation, desalted on a Sephadex G-15 column, reconcentrated, and used without further purification. Template DNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE), electroeluted in a Schleicher & Schuell Elutrap, and precipitated with ethanol.

Enzymatic synthesis of ¹³C,¹⁵N-labeled oligonucleotides

Template DNAs were combined with 10× polymerization buffer (500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100), MgCl₂, H₂O and dNTPs. The ratio of the dNTPs included in each reaction was stoichiometric with respect to the oligonucleotide produced. The concentration of MgCl₂ was 1-4× the dNTP concentration, optimized for each reaction. Each dNTP at 20% excess was included in the reactions to ensure complete primer extension. Taq polymerase (24 000 U/µmol of template) was added, and the mixtures placed in a boiling H₂O bath for 2 min. The reactions were then diluted to a final template concentration of 10 µM with the addition of a 1.1 molar excess of primer. Polymerization was carried out at 72°C for 12-36 h. Progress of each reaction was assessed by native polyacrylamide gel electrophoresis. The reactions were stopped by freezing and thawing twice.

To test for the presence of non-templated additions, a small aliquot of each reaction was combined with an equal volume of 2 M KOH, incubated at 55°C for 2 h, and neutralized with HCl. The length of the product was then compared to that of chemically synthesized oligonucleotides by denaturing PAGE. For reactions where non-templated addition was observed, 200 U of Klenow fragment (Promega) per µmol of template were added and the reactions diluted to 1.2× their original volume. The reactions were incubated at 37°C for 8 h, and then the Klenow fragment was inactivated by heating in a boiling H₂O bath for 5 min. The length of the product was re-tested as described above.

Primers were cleaved from the product oligonucleotides by adjusting the pH of the reactions to 12.5 with KOH and incubating at 55°C for 8 h. The reactions were then neutralized with HCl and analyzed by denaturing PAGE. The reactions were concentrated to ~5 ml and desalted on a Sephadex G-15 column. Following re-concentration in the Speed-Vac, the products, templates and primers were separated by denaturing PAGE. The product bands were electroeluted from the gels and further purified by chromatography on a 50% DEAE sepharose/50% DEAE Sephacel column. The purified product was then concentrated, desalted on a Sephadex G-25 column, and dried by lyophilization.

NMR sample preparation

NMR samples of DNA duplexes composed of one labeled and one unlabeled strand of NHPU and NHPL (sequence given in Fig. 1) were each prepared by dissolving each strand in D₂O, transferring 200 µl of the labeled strand to a Shigemi NMR tube and titrating in the unlabeled strand to form a 1:1 complex. Complex formation was assessed using ¹H-¹³C HSQC spectra. Final sample concentrations were 1 mM for the ¹³C,¹⁵N-labeled NHPU:unlabeled NHPL and 0.2 mM for the ¹³C,¹⁵N-labeled NHPL:unlabeled NHPU. The Triplex (sequence given in Fig. 1) NMR sample was dissolved to 1 mM in 100 mM NaCl, pH 5.3, 5 mM MgCl₂. For spectra acquired in H₂O, the NMR samples were dried in the NMR tube using N₂ gas and redissolved in 90% H₂O/10% D₂O.

NMR spectroscopy of ¹³C,¹⁵N-labeled oligodeoxyribo-nucleotides

All NMR spectra were acquired on Bruker DRX spectrometers at 500 and 600 MHz. For samples in 90% H₂O suppression of the solvent resonance was achieved by using WATERGATE (33) or gradient selection of coherences (34). In the case of D₂O samples, the residual HDO resonance was suppressed by presaturation. Constant time ¹H-¹³C HSQC (34,35), ¹H-¹⁵N HSQC, HCCNH-TOCSY (28) and HCCH-TOCSY (27) experiments were implemented as described. For the ¹³C dimensions the sweep width and position of the carrier were optimized so that aromatics and C2[prime]/C-methyl were folded. Additional acquisition and processing parameters are given in the figure legends. Spectra were processed with XWINNMR 1.3.

RESULTS AND DISCUSSION

Synthesis of ¹³C,¹⁵N-labeled oligodeoxyribonucleotides

We have developed a highly efficient and reliable procedure for the production of milligram quantities of uniformly ¹³C,¹⁵N-labeled oligonucleotides starting from uniformly ¹³C,¹⁵N-labeled dNTPs (Fig. 2). The method is similar to the enzymatic protocol of Zimmer and Crothers (22), but has three major differences: (i) the mixed dNTPs are separated from one another so that stoichiometric amounts can be used in the reaction; (ii) Taq polymerase is used instead of Klenow fragment of DNA Polymerase I in the polymerization step, and (iii) an additional step to remove non-templated additions at the 3[prime] end is used when needed. Quantitative yields are obtained for all steps before purification.

Figure 2. Flow chart diagram of the procedure for the production of ¹³C,¹⁵N-labeled oligodeoxyribonucleotides and efficiency of each step.

The steps for the enzymatic synthesis of the DNA are shown schematically in Figure 3. In the first step, the chemically synthesized DNA template and primer with a 3[prime] ribose are annealed. Separate strands for the primer and template are used, rather than a hairpin primer-template as in Zimmer and Crother's protocol (22). Thus, the same primer can be used for synthesis of any oligonucleotide. In the second step, the primer is extended by Taq polymerase in the presence of only 20% excess ¹³C,¹⁵N-labeled deoxyribonucleotide triphosphates, producing a uniformly labeled oligonucleotide covalently linked to the primer. Optimal yields are obtained by using a ratio of the four dNTPs corresponding to the sequence of the deoxyoligonucleotide being synthesized, as discussed below. In cases where non-templated addition cannot be avoided, the extra 3[prime]A is removed using Klenow fragment of DNA Polymerase I. In the last step, which corresponds to the previously published protocols (22,23), the labeled product oligonucleotide is detached from the primer by base-catalyzed cleavage. The reactant and product deoxyoligonucleotides are purified on denaturing polyacrylamide gels followed by electroelution.

Figure 3. Schematic representation of the protocol for synthesis of ¹³C,¹⁵N-labeled oligodeoxyribonucleotides showing polymerization of dNTPs, trimming of non-templated additions, and cleavage from the primer. The template and primer strands are shown with solid lines and the synthesized DNA is shown with open lines.

Figure 4 shows the results of the polymerization reactions for NHPU and NHPL. Quantitative polymerization on the DNA templates for both NHPU and NHPL deoxyoligonucleotides was achieved with only 20% excess nucleotide triphosphates included in the reaction. Polyacrylamide gel electrophoresis of NHPU after base catalyzed cleavage from the primer shows a single product band with no significant amount of N+1 sequences (Fig. 5). No N-1 sequences were detected for any of the synthesized deoxyoligonucleotides.

Figure 4. Ethidium bromide stained 15% polyacrylamide gel showing the results of polymerization of NHPU and NHPL on template DNA. (A) Lane 1, primer + NHPU template; lane 2, primer/product + NHPU template after polymerization. The polymerization reaction conditions for the full scale reactions contained 10 µM template, 12 µM primer, 0.32 mM MgCl₂, 180 µM dNTPs (46.7% dGTP, 13.3% dATP, 6.7% dCTP, 33.3% dTTP), 48 000 U Taq polymerase in 200 ml. (B) Lane 3, primer + NHPL template; lane 4, primer/NHPL + NHPL template after polymerization. The polymerization reactions for the full scale reactions contained were 10 µM template, 12 µM primer, 0.75 mM MgCl₂, 180 µM dNTPs (46.7% dCTP, 13.3% dTTP, 6.7% dGTP, 33.3% dATP), 40 000 U Taq polymerase in 75 ml.

Non-templated addition

One of the major problems of the previously reported protocols for enzymatic synthesis of DNA oligonucleotides is non-templated addition of an A nucleotide, resulting in heterogeneity at the 3[prime] ends. The potential for non-templated addition was originally discovered with Klenow fragment (36), and has been shown to occur for all DNA polymerases tested, including Taq (37). We found that the amount of non-templated addition depends on the sequence of the deoxyoligonucleotide being synthesized, the relative amounts of the four dNTPs, and on the reaction time. For molecules lacking or with few A nucleotides, non-templated addition can generally be avoided by limiting the amount of dATP in the reaction to 20% excess of stoichiometric amounts of each nucleotide for the desired DNA oligonucleotide, since the non-templated additions are almost always an adenine (37). For example, NHPU, which contains only two adenines out of 15 bases (Fig. 5), showed no non-templated addition under these conditions. Only 2.2 µmol of dATP per µmol of DNA template was present at the start of the reaction, ~80% of which was incorporated into product by templated polymerization when the reaction reached completion. When equimolar rather than stoichiometric amounts of dNTPs were used for NHPU ~10% of the molecules had an extranucleotide on the 3[prime] end (not shown).

Figure 5. Autoradiogram of denaturing 19% polyacrylamide gel of product of NHPU synthesis after cleavage from primer. Lane 1, chemically synthesized NHPU oligonucleotide; lane 2, enzymatically synthesized NHPU oligonucleotide before purification. The DNA was labeled by using [[gamma]-³²P]ATP with T4 polynucleotide kinase. Synthesis conditions are described the legend of Figure 4.

Figure 6 shows the product DNA bands for the synthesis of d(GGGGTTTTGGGG) (Oxy-1.5) (26) in the presence ofequimolar amounts of the four dNTPs as a function of time. Here, non-templated addition is a significant problem, which could be easily avoided by simply leaving dATP (and dCTP) out of the reaction. Since non-templated addition usually occurs more slowly than templated addition (37), the ratio of N+1 to N oligonucleotides produced increases over time. Thus, optimization of polymerization reaction time could be used to avoid non-templated addition, but this has the disadvantage that at the shorter times the reaction is not near completion. We find that it is much more efficient to use the purified dNTPs in the correct ratios to avoid non-templated addition when possible.

Figure 6. Autoradiogram of a 15% polyacrylamide denaturing gel showing the time dependence of non-templated addition during the enzymatic synthesis of Oxy-1.5 [d(GGGGTTTTGGGG]) (10 µM) in the presence of 0.8 mM mixed dNTPs. The time of reaction in hours is given above the lanes. The unpurified samples obtained after base cleavage are run on the gel. Only N and N+1 products are observed. The background bands are due to unpurified primer.

For some sequences, non-templated addition seems to be unavoidable (38,39). This was the case with NHPL, which gives almost exclusively N+1 products even in the presence of stoichiometric amounts of each dNTP (Fig. 7). In order to solve this problem, we have developed a simple protocol for the removal of non-templated additions. After enzymatic synthesis is complete, the extra 3[prime]A residues are removed using the 3[prime]->5[prime] exonuclease activity of the Klenow fragment of DNA Polymerase I. Although both proofreading and non-proofreading polymerases can adenylate a blunt-ended DNA duplex, proofreading polymerases typically are less proficient (40), presumably due to their ability to remove non-templated adenines after their addition. Since Klenow can work in a dilution of the Taq polymerization buffer, this step can be done without intermediate purification steps. The test reactions show quantitative removal of non-templated additions by Klenow (Fig. 7), although lower yields (>80%) of full length product were obtained for the large scale reactions. For both the test reactions and the large scale reactions, no N-1 or smaller products were observed. We note that since the trimming reaction (Klenow) is done in the same solution as the polymerization reaction, only the original excess 20% of each dNTP is present, which helps assure that no net degradation of the correct product sequence occurs.

Figure 7. Autoradiogram of denaturing 19% polyacrylamide gel of NHPL product before and after Klenow treatment. Lane 1: chemically synthesized NHPL oligonucleotide; lane 2: enzymatically synthesized NHPL reaction product before removal of non-templated addition; lane 3: enzymatically synthesized correct NHPL oligonucleotides produced by `trimming' off non-templated additions with Klenow fragment. The DNA was labeled by using [[gamma]-³²P]ATP with T4 polynucleotide kinase. Synthesis conditions are described in the legend of Figure 4.

Overall yields

As discussed above, all steps of the synthesis protocol are nearly quantitative. The major losses occur during the purification steps by PAGE and electroelution. For the NHPU sequence, from a reaction with 2 µmol of template, after polymerization with Taq, cleavage from the primers and purification, 1 µmol of purified product was obtained. For the NHPL sequence, from a reaction with 0.75 µmol of template, after polymerization, `trimming' with Klenow, cleavage and purification, 0.4 µmol of purified, correct product was obtained. Improvements in the purification protocol would result in even higher yields of the products.

HSQC spectra of NHPU and NHPL

¹H-¹³C and ¹H-¹⁵N HSQC spectra of ¹³C,¹⁵N-labeled NHPU annealed to unlabeled NHPL are shown in Figure 8. In the ¹H-¹⁵N HSQC, 11 of the 12 possible imino NH correlations are seen; the imino proton of G1 is exchanging too fast with H₂O to give rise to a crosspeak (Fig. 8A). The full ¹H-¹³C spectrum (Fig. 8B) shows all the expected crosspeaks and no ¹³C impurity peaks. All 15 expected aromatic CH crosspeaks are observed, with no additional crosspeaks present (Fig. 8C), as well as the five expected thymine methyl CH correlations. The ¹H-¹³C and ¹H-¹⁵N HSQC spectra of labeled NHPL annealed to unlabeled complimentary strand give similar results. In particular, all of the expected aromatic CH crosspeaks are observed with no additional crosspeaks present, indicating that no significant amount of molecules with an additional 3[prime] terminal A are present (Fig. 8D).

Figure 8. (A) 500 MHz 1H-15N HSQC correlation of 13C,15N-labeled NHPU/unlabeled NHPL duplex at 274 K in 90% H2O/10% D2O. The spectral widths in F1 and F2 were 1200 and 4500 Hz, respectively. 64 t1 increments were acquired in States-TPPI (52) mode with 128 scans each and 1024 complex points in t2. The spectrum was processed with 1024 × 1024 complex points after apodization with a shifted squared sine bell (shifted by [pi]/4). (B) 600 MHz 1H-13C HSQC spectrum of 13C,15N-labeled NHPU/unlabeled NHPL duplex at 298 K in D2O. The spectral widths in F1 and F2 were 9000 and 6000 Hz, respectively. 128 t1 blocks were acquired in States-TPPI mode with eight scans per block and 1024 complex points in t2. The spectrum was processed with 1024 × 1024 complex points after apodization with a shifted squared sine bell (shifted by [pi]/3 and [pi]/12 in t1 and t2, respectively). (C) Portion of 1H-13C HSQC spectrum showing the correlations between aromatic protons and carbons. Sample and parameters were the same as in (B) except for the spectral width and carrier position for the 13C dimension which were adjusted to 12 000 Hz and 150 p.p.m., respectively, and the spectra were apodized a shifted squared sine bell (shifted by [pi]/12). (D) Portion of 1H-13C HSQC spectrum of 13C,15N-labeled NHPL/unlabeled NHPU duplex at 298 K in D2O spectrum showing the correlations between aromatic protons and carbons. Acquisition and processing parameters are the same as (C) except 128 scans were acquired in t1.

HCCNH-TOCSY and HCCH-TOCSY spectra of the DNA triplex

Assignments of imino proton resonances presents a special problem for DNA and RNA oligonucleotides that do not have a standard double helical structure. The HCCNH-TOCSY experiment (28,42,43) was developed to solve this problem for RNA oligonucleotides, and provides direct correlations between imino and base H8,H6 via the intervening carbons and nitrogen. We have applied this experiment to the DNA triplex oligonucleotide (Fig. 9A). Crosspeaks for 13 of the 16 iminos involved in base triplets are observed. The missing crosspeaks are from the terminal triplets due to the more rapid exchange of the imino protons with water. We have also tested and optimized heteronuclear correlation experiments used in assigning the non-exchangeable proton resonances in labeled RNA to this DNA triplex, including 2D and 3D HCN (44), HCNCH (45), HCCH-TOCSY, HCCH-COSY, NOESY-HSQC and TOCSY-HSQC experiments (11,46,47). One example is the HCCH-TOCSY (27) (Fig. 9B) which allows extension of the deoxyribose 1[prime],2[prime],2[prime][prime] and some 3[prime] assignments obtained from proton only spectra of a related intramolecular triplex (48) to the remaining 3[prime],4[prime],5[prime],5[prime][prime] protons and their directly bound carbons.

Efficiency and comparison to previously reported protocols

In comparison to the previously published protocols using a double mutant exonuclease^- Klenow enzyme to produce labeled oligonucleotides (22,23), our protocol has quantitative and qualitative advantages. Quantitatively, we have obtained incorporation efficiencies of labeled nucleotides in the range of 80% for µmol scale reactions. Up to 100% incorporation (i.e. no excess nucleotides) is achieved in nmol scale test reactions with unlabeled dNTPs. This is a clear improvement over previously published protocols where incorporation efficiencies of [le]50% have been reported for labeled nucleotides. For sequences where non-templated additions occur, up to 100% of the 3[prime] A can easily be removed without degrading the desired product, so that even in these cases the reaction is nearly quantitative. Separation of the dNTPs by HPLC has several advantages for the polymerization step. First, it results in purer dNTPs, which lead to less non-templated addition and higher yields. Second, only the amount of each dNTP needed for a given sequence is used, which decreases waste of labeled dNTPs for sequences with different amounts of the four nucleotides. Third, controlling the amount of dATP in the reaction can decrease the amount of non-templated addition.

Figure 9. (A) 600 MHz 2D HCCNH-TOCSY spectrum of labeled Triplex at 274 K. The spectral widths in F1 and F2 were 1250 and 13 227 Hz, respectively. 128 t₁ blocks were acquired in States-TPPI mode with 1024 scans in t₁ and 2048 complex points in t₂. The spectrum was processed with 1024 × 2048 complex points after apodization with a gaussian multiplication (LB of -0.13 and GB of 0.01) in t₁ and shifted squared sine bell ([pi]/2) in t₂. (B) Strip from HCCH-TOCSY spectrum showing deoxyribose crosspeaks for T21. The spectrum was acquired with spectral widths of 4006, 5030 and 4006 Hz in F1, F2 and F3, respectively. 160 t₁ blocks with 64 and 512 complex points in t₂ and t₃, respectively, were acquired in States-TPPI mode with 16 acquisitions per block. The spectrum was processed with 160 × 64 × 512 complex points after apodization with a shifted squared sine bell (shifted by [pi]/2, [pi]/2 and [pi]/3 in t₁, t₂ and t₃, respectively). The sequence and secondary structure of the intramolecular DNA triplex is shown at the top.

Our protocol has several qualitative advantages over the previously published methods. One of these is the ease of purification of large amounts of Taq polymerase compared to the purification of the double mutant Klenow (49). Using our protocol it is possible to produce 500 000 U of Taq polymerase from a 50 ml culture with only a few hours of work. Another advantage is the high incubation temperature during the polymerization reaction. Incubation at a higher temperature can help prevent mispriming and complications due to secondary structure in the primers or template. Because mispriming is unlikely at high temperature, it is unnecessary for the primers to be attached to the template in a hairpin structure. This fact makes the synthesis of larger oligonucleotides easier, and allows for the same primer sequence to be used to make different oligonucleotides. Furthermore, the separation of primers from template and the thermostability of Taq polymerase possibly allow thermocycling to increase yield. Thermocycling may be useful in circumstances where the amount of available template is limited.

Our protocol has been used to synthesize four different DNA oligonucleotides, of varying lengths, sequence and nucleotide composition. Each of these presented different problems, and solution of these problems led to the generally applicable protocol presented here. While this manuscript was in preparation, two other manuscripts describing methods for enzymatic synthesis of DNA were published (50,51). Chazin and co-workers (51) describe a modification of the Zimmer and Crothers protocol (22) which provides segmentally labeled DNA. The other manuscript (50) describes two approaches: (i) production of ¹⁵N-labeled DNA oligonucleotides by restriction enzyme cleavage from plasmid DNA and (ii) bidirectional PCR with an exponentially expanding template followed by restriction enzyme cleavage. The latter method requires a restriction site as part of the sequence. Each of these methods has advantages and disadvantages depending on the particular DNA sequence of interest, required yield, and desired application, but the overall efficiency of the method described here appears to be the highest.

ACKNOWLEDGEMENTS

This work was supported by NIH grants GM37254 and GM48123 to J.F. The authors thank L. D. Finger for assistance with the HPLC and enzyme purification and F. H. T. Allain for assignments of NHPU and NHPL.

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*To whom correspondence should be addressed. Tel: +1 310 206 6922; Fax: +1 310 825 0982; Email: feigon@ewald.mbi.ucla.edu

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