Synthesis of Double-stranded DNA Complementary to Lysozyme, Ovomucoid, and Ovalbumin mRNAs OPTIMIZATION FOR FULL LENGTH SECOND STRAND SYNTHESIS BY ESCHERICHIA COLI DNA POLYMERASE

Sequential reverse transcriptase, DNA polymerase, and Sl nuclease reactions can he employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, mono-valent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing

Procedures." Avian myeloblastosis virus reverse transcriptase is used to synthesize first strands from sucrose gradient-fractionated messenger RNAs under conditions shown previously to yield a high proportion of full length copying (9). The total reverse transcriptase product is boiled briefly in order to free first strands from the messenger template to which they are hybridized (9). The boiled first strand mixture is added directly to an equal volume of DNA polymerase I reaction mixture containing fresh deoxynucleoside triphosphates, a new buffer selected to minimize exonucleolysis (111, and E. coli DNA polymerase I. After a 2-h, 15" incubation, the reaction is stopped, RNA carrier added, and the entire mixture extracted with chloroform and passed over Sephadex G-150 in 20 mM NaCl. The pooled void volume is then adjusted to appropriate salt and buffer concentrations and digested with Aspergillus single strand-specific Sl nuclease. For simplicity we refer to the products of the reverse transcriptase reaction as first strands and to material synthesized by DNA polymerase I as second strands. The products of the combined reactions are called double-stranded cDNA. Sequential reverse transcriptase and DNA polymerase I reactions in which only first strands are radiolabeled are called first strand labelings; when only second strands are radiolabeled, the combined reactions are called second strand labelings.
Various omissions were made from a complete reaction protocol using ovalbumin mRNA and the net incorporation of either [3H]dCTP or [3H]dATP during second strand synthesis determined as a per cent of a complete reaction control (Table  I). DNA polymerase I does not copy the mRNA (even though oligo (dT) still is present at 50 pg/ml), since without reverse transcriptase one finds less than 0.5% of control incorporation. It also does not copy the oligo(dT) itself, since no labeling with [3H]dATP is found in the absence of mRNA. Both reverse transcriptase and DNA polymerase I are required for second strand synthesis.  The extent to which first strands are copied by DNA polymerase I can be monitored by the Sl nuclease resistance of labeled first strands. For this and subsequent experiments in which radioactivity was to be incorporated into first strands only, our standard protocol was modified so that radioactive precursor remaining from the reverse transcriptase reaction was only negligibly incorporated into second strands by DNA polymerase I (see "Reactions using Radioactive Deoxynucleoside Triphosphate Precursors" under "Experimental Procedures"). The specific radioactivity during second strand syn- Aliquots of a single first strand labeling were removed at various times during second strand synthesis and electrophoresed on 1.5% agarose, 20 mM methylmercury hydroxide denaturing gels ( Fig. 2A). Full length first strands disappear rapidly during the incubation, accompanied by the appearance of higher molecular weight products; very little remains at the unreacted position after 2 h. Between 1 and 2 h a prominent band emerges at the position predicted for full length second strand synthesis (3730 bases, average of nine experiments). Combined with Fig. 1, this suggests that after 1 h, most second strands terminate at different distances near but not -ov cDNA yet at the 5' end of the first strand. It seemed possible that some of the 2-h products between about 2500 bases and the complete duplex band might also ultimately be converted to full length with prolonged incubation. In a second experiment, we examined first strand labelings at much longer incubation times.
No significant change in size distribution ( Fig. 2B) was seen between 2 and 24 h.
All deoxynucleoside triphosphates polymerized by DNA polymerase I are Sl-resistant.
In the experiment shown in Fig. 3, L3HldCTP was introduced at the start of second strand synthesis and aliquots removed at various times thereafter. Greater than 95% of the radioactivity in second strands is protected from Sl nuclease digestion at each time point. Ovomucoid and lysozyme second strands synthesized under standard conditions also are more than 95% resistant. The kinetics of incorporation in Fig. 3  and how much was needed to obtain full length ovalbumin second strand synthesis, Conalbumin, ovalbumin, ovomucoid, and lysozyme mRNAs were each used as templates for first strand synthesis by reverse transcriptase. After boiling, each reaction was added to an equal volume of DNA polymerase I reaction mixture containing radioactive precursor, and divided into several aliquots. Different amounts of DNA polymerase I were added to catalyze the second reaction and the total incorporation into second strands determined.
The results are summarized in Fig. 4. For each template, with increasing enzyme, net synthesis approaches a plateau and then declines slightly. The ratios of enzyme required to saturate first strand products made from equal masses of mRNA are approximately 0.3 (conalbumin):l.O (ovalbuminM.7 (ovomucoid):2.7 (lysozyme). Reverse transcriptase synthesizes roughly the same mass of first strands using ovalbumin, ovomucoid, or lysozyme mRNA fractions under our conditions; the reaction is about 4-fold less productive with the conalbumin mRNA fraction (9). Correcting for relative sizes of the messengers (9), the calculated molar ratios of first strands produced from identical RNA inputs are 0.2 (cona1bumin):l.O (ovalbumin):2.3 (ovomucoid):3.0 (lysozyme).4 The correlation between the relative molar yields of first strand and the amount of DNA polymerase I required for saturation suggests that the molar ratio of enzyme to reverse transcriptase products required for saturation is simi-4 No correction has been made for differences in distribution of partial reverse transcriptase products among the mRNA templates, since no quantitative data are available. However, the accompanying paper (9) reports that, relative to the ovalbumin distribution, ovomucoid and lysozyme first strands may contain slightly fewer partial copies, and conalbumin more. These corrections would tend to improve the agreement between the amount of enzyme required to saturate and the molar yield of first strand. lar with each template. (In each case, that ratio is roughly 5 to 1.) It might be anticipated that subsaturating enzyme levels would leave most first strands unreacted while producing a small proportion of completed second strands. This was not the case with ovalbumin (Fig. 5). To obtain complete second strand synthesis, enzyme had to be present near the saturating level determined in Fig. 4. Below this amount, very little full length was seen. The addition of enzyme beyond the amount required for saturation (Track 5) produced no detectable difference on denaturing gels. were synthesized in the absence of salt. First strands made in 0 and 140 mM KC1 appear identical upon denaturing gel electrophoresis and display no material larger than the full length band. After-boiling, aliquots were removed and added to DNA polymerase I reaction mixtures containing [32P1dGTP and various KC1 concentrations.
After a Z-h incubation with saturating polymerase (based on Figs. 4 and 5 at 70 mM KCl), the products were analyzed on denaturing gels (Fig. 6). Without added salt, material longer than double-stranded full length was found on long autoradiographic exposures. Approaching 70 mM, the size of this product progressively decreased, until between 70 and 125 mM KCl, the full length band was the slowest migrating species. Beyond 125 mM KCl, no complete second strands are made. (In repeating this experiment several times we found that 125 mM KC1 was too close to the point at which no full length synthesis was found to be used routinely. We therefore used 70 mM KC1 in standard reactions.) Data in the accompanying paper make it unlikely that the ovalbumin mRNA used is significantly contaminated by messengers of higher molecular weight. Therefore, the slowly migrating material produced in low salt concentrations is likely to represent aberrant ovalbumin second strand synthesis. NaCl versus KC1 -DNA polymerase I products significantly different in size are synthesized if NaCl is used instead of KC1 during second strand synthesis. Ovalbumin-directed first strand synthesis was carried out using either KC1 or NaCl at 140 mM. The standard protocol was followed thereafter, so that 70 mM salt (judged optimal from Fig. 6) was present during second strand synthesis. With either strand labeled, no material of full length was found upon electrophoresis of the NaCl product on denaturing gels (Fig. 7). Instead the largest species made was not a discrete band, but a distribution of lengths between about 2800 and 3300 bases. The addition of more enzyme (up to 4 times the level needed for full length synthesis in standard KC1 reactions) did not yield larger products. The monovalent cation effect is probably on the DNA polymerase I reaction per se, since first strands made in either salt at 140 mM do not differ substantially in yield, Sl resistance, or size distribution on denaturing gels. We have not determined whether there is an NaCl concentration which would enhance full length second strand synthesis.
Incubation Temperature-A single ovalbumin reverse transcriptase reaction (in the absence of radioactive precursors) was added to an equal volume of DNA polymerase I reaction mixture containing 13'P1dGTP. After the addition of DNA polymerase I, the mixture was divided into four aliquots and incubated of 4", 15", 25", and 42" for 2 h. The ratios of total  7. Comparison of DNA polymerase I products synthesized in NaCl and KCI. A, first strand labelings. A reverse transcriptase reaction mixture without salt, containing 50 ~8 of ovalbumin mRNA/ml and [32PldGTP at 2.6 Cilmmol, was divided into two parts. One received concentrated KC1 to a final concentration of 140 mM, the other NaCl to the same concentration. The protocol for first strand labelings under "Experimental Procedures" was then followed exactly. (RTM mixtures contained either KC1 or NaCI.) DNA polymerase I was used at 13.2 units/pg of mRNA. Samples were processed, prepared for electrophoresis, and electrophoresed on 1.5% agarose, 20 rnM CH,HgOH gels as described under "Experimental Procedures." A track not shown contained SV40 DNA restriction fragments and was viewed by ethidium bromide fluorescence; the positions of the Rl fragment and the largest Hind111 fragments are indicated. Truck 1, a mixture of eaual aliauots (40.000 cpm each) of the material displayed in Tracks Z-and 3; hack 2, products made in NaCl (80,000 corn); Truck 3, products made in KC1 (80.000 cam). B. second strand labeling. Two-second strand labelings were carried out essentially as described under "Experimental Procedures." One used 140 mM KC1 during first strand svnthesis. the other 140 mM NaCI. Both used 20 pg-of ovalbumin "mRNA/ml during the first reaction, a DNA polymerase I mixture containing 13LPldGTP at 3.8 Ci/mmol, and DNA polymerase I at 12.8 units/pg ;fmRNA. Samples were processed, prepared for electrophoresis, and electrophoresed on 1.5% agarose, 20 mM CH,HgOH gels as described under "Experimental Procedures." Tracks on the same gel contained the same markers as in A. Another track contained unreacted first strand (ou cDNA): the position of the full length band is shown. Both tracks received 125,000 cpm. Track 1, products made in NaC1; Track 2, products made in KCl.
incorporation were roughly 0.5:1.0:2.0:20, respectively. Since about 100% of the input first strand mass is synthesized during a 15" polymerase I reaction, a 42" synthesis probably generates products not directly copied from input first strands. When the products were electrophoresed on denaturing gels, a dramatic trend toward increased length with elevated temperature was found (Fig. 8). At 4", no full length second strand synthesis occurs, as corroborated by first strand labelings in which most first strand is found unreacted (data not shown). At 25" material co-migrating with the 4000base-long 15" product is superimposed on a wide distribution of sizes extending almost to the top of the gel. Even this residual 4000-base-long band disappears at 42"; most of the product barely enters the gel. Analogous second strand labeled ovomucoid (Fig. 8B (Truck 1))-and lysozyme (Fig. 8B (Truck   3))-directed 42" products were electrophoresed on denaturing gels; they too migrated very slowly. Upon native gel electrophoresis all three behaved as if either very long or extensively single-stranded, not entering 1.5% agarose gels (data not shown). We considered the possibility that the 42" products might be polyphosphate, although it seemed unlikely since no such products are found under nearly identical reaction conditions during the reverse transcriptase incubation. The ovalbumin product is not, since it is 101% phosphatase-resistant, 100% trichloroacetic acid-precipitable before and after boiling, binds ethidium bromide, and does not behave like the non-DNA material described by Efstratiadis et al. (6) in that it is excluded on Sephadex G-150 and is acid-precipitable.
The slowly migrating DNA polymerase I products may contain hairpins as judged by their behavior upon digestion by Sl nuclease. Each second strand labeled 42" product is 80 to 100% Sl-resistant; boiling prior to digestion decreases the Sl resistance by only 10%. After digestion, however, each enters a denaturing gel as a broad size distribution near the size of the appropriate Sl-cleaved 15" product. Fig. 8B (Tracks 2 and 4) displays these results with ovomucoid and lysozyme. Procedures," then divided in half. One part was kept on ice, while the other was denatured in a boiling water bath as usual. An equal volume of DNA polymerase I reaction mixture containing [32PldGTP was then added to each tube, and the reactions begun by the addition of DNA polymerase I.
When the boiling step was omitted from an otherwise standard protocol, DNA polymerase I directed the incorporation of 13'P1dGTP into acid-precipitable material. Net synthesis was approximately 50% of that found in a standard reaction. However, when analyzed on methylmercury-containing agarose gels, no labeled second strand products longer than full length first strands (1900 bases) were found. Instead a broad distribution of radioactivity was observed, with an average length of roughly 500 bases, extending nearly up to 1900 bases. The length distribution suggests that in an unboiled reaction, DNA polymerase I does not extend the first strand from a hairpin primer, but instead copies the messenger itself. Messenger RNA-oligo(dT) hybrids would not have been denatured; any such hybrids which had not been used as templates by reverse transcriptase might provide suitable template. primer complexes for DNA polymerase copying of mRNA. When reverse transcriptase reactions are boiled prior to second strand synthesis to separate mRNA-oligo(dT) and mRNA-first strand hybrids, DNA polymerase I does not copy the messenger significantly (see Table I). These data indicate that the denaturation step is necessary for efficient second strand synthesis.
We also investigated the effects of not boiling on the second strand labeled ovalbumin products synthesized by DNA polymerase I at 42". Total incorporation of [3*P1dGTP into second strands in a 42" reaction which had not been boiled was 3 times higher than that found with boiling. Upon methylmercury-agarose electrophoresis, second strands from the unboiled reaction were indistinguishable from those of a boiled reaction (see Fig. 8 then determined over a range of nuclease concentrations. Without denaturation, Sl resistance approaches a plateau of 80% for the first strand and 100% for the second (as shown in Figs. 2 and 4). After denaturation the level of resistance of each strand, while remaining high, drops by about 30%. Thus roughly 70% of the double-stranded molecules after DNA polymerase I reaction are indeed the expected hairpin structures, while the remainder do not snap back after denaturation. Non-hairpin duplexes may be derived from priming by messenger fragments or by oligo(dT) (see "Discussion").
In preparing material for transformation, the addition of exogenous DNA is undesirable.
Therefore, in experiments analogous to those described in Fig. 9, 10, and 12C, we carried out Sl nuclease digestions in the absence of exogenous DNA carrier using lysozyme and ovalbumin double-stranded cDNAs as substrates. The plateau Sl resistance of ovalbumin first strands was 78% and of second strands 93% (as opposed to The incubation was stopped after 2 h at 15", inactivated, extracted, and chromatographed over Sephadex G-150 in 20 mM NaCl as described under "Experimental Procedures." Half the pooled void volume was then denatured in a boiling water bath for 5 min; the other half was not treated. Each portion was added to 25 volumes (6.2 ml) of Sl buffer containing 10 pg of native and 10 pg of denatured salmon sperm DNA/ml and divided into six l-ml aliquots.
Each was assayed for Sl resistance using a different amount of Sl nuclease as described under "Experimental Procedures" (see Sl Nuclease Digestion").
The input radioactivity in each digestion was 12,000 32P cpm and 1300 3H cpm. 0, 3H-labeled Sl resistance, sample not denatured prior to nuclease digestion; A, 32Plabeled Sl resistance, sample not denatured prior to nuclease digestion; 0, 3H-labeled Sl resistance, sample denatured prior to nuclease digestion; A, 32P-labeled Sl resistance, sample denatured prior to nuclease digestion. 81% and 97%, respectively, in parallel reactions containing salmon sperm DNA). The denaturing gel distributions of double-stranded cDNAs digested in the presence or absence of salmon sperm DNA were indistinguishable over the range of nuclease concentrations tested (up to 5 units of Sl/ml).
If second strand synthesis is primed by a hairpin loop at the 3' end of the first strand, then Sl cleavage full length doublestranded cDNA should reduce its single-stranded length by half while leaving the duplex length nearly unchanged. Either first or second ovalbumin strands were labeled, digested with different amounts of Sl, and electrophoresed under denaturing conditions (Fig. 10). As Sl is added the band at about 3800 bases is converted to one-half that size co-migrating with full length ovalbumin first strands. When the same samples are electrophoresed on native gels (Fig. 111, the 1900 base pair band and faster moving species do not change significantly upon Sl treatment; slower moving material progressively disappears. The most obvious interpretation of these data is that the band itself and most lower molecular weight material are completely double-stranded before digestion (probably full length second strands synthesized on partial first strands) and relatively unaffected by cleavage of their hairpins. The slower moving material on native gels which disappears with nu-ov cDNA - clease is likely to be derived from incomplete second strand synthesis, since single-stranded DNA probably migrates more slowly than double-stranded DNA on this gel system (16). We estimate that with either strand labeled about 25% of the total radioactivity on native gels is found in full length ovalbumin double-stranded cDNA after Sl cleavage (Fig. 11, A (Truck 4) and B (Track 4)).
Ovomucoid messenger RNA is about 850 bases long. Full length ovomucoid DNA polymerase I products should therefore migrate at about 1600 bases long on denaturing gel electrophoresis.
Similarly, lysozyme (about 650 bases long) should yield a full length DNA polymerase I product 1200 bases long. When first strand labeled ovomucoid and lysozyme products were electrophoresed on denaturing gels, two prominent bands were observed in each case (Fig. 12, A and C). One co-migrated with full length first strand; the other was found at approximately the predicted full length second strand position. (Second strand labelings did not display a band at the unreacted position.) Higher amounts of enzyme did not alter the distribution.
When treated with Sl nuclease, the higher molecular weight band disappeared and an increase in messenger-sized material was observed, as predicted from hairpin cleavage. When these same ovomucoid and lysozyme products were electrophoresed on native gels (Fig. 12, B and D), an overall pattern similar to that observed with ovalbumin ( Fig. 11)  strand-labeled DNA polymerase I products were prepared using an enzyme concentration estimated to be sufficient for the synthesis of full length second strands. A portion was digested with Sl nuclease. The reverse transcriptase, DNA polymerase, and Sl-treated DNA polymerase products were electrophoresed under denaturing conditions (Fig. 13). The pattern of Sl-treated double-stranded cDNA is very similar to that of the unreacted reverse transcriptase products themselves. With the exception of conalbumin, which serves as a poor template for the synthesis of full length first strand, each messenger RNA is efficiently converted to opened doublestranded DNA.

DISCUSSION
Some of the drawbacks of previous protocols for sequential reverse transcriptase, DNA polymerase, and Sl nuclease reactions were presented in the introduction.
To limit difflculties due to E. coli DNA polymerase I inefficiency, we have examined in detail many reaction conditions with respect to the synthesis of second strands. Each parameter investigated had a significant effect on product length. A suboptimal choice in any one variable reduced the proportion of complete products substantially.
In addition, the method we have used considerably reduces the handling of the in vitro products and thereby minimizes losses.
A rough estimate of the yield of full length double-stranded ovalbumin DNA synthesized under our conditions can be made.5 In a reaction using 1 pg of ovalbumin mRNA we estimate that about 0.1 pg of full length duplex is synthesized. (Some full length duplexes may be nicked or contain short gaps as judged by a comparison of native and denaturing gels of the same products.) This estimate was derived from reactions using as little as 50 ng of mRNA and so is applicable to small amounts of template.
When hen oviduct total poly(A)-containing RNA was used as a template for the three reactions, the distribution of Slcleaved double-stranded products on denaturing gels was very similar to that of first strands themselves (Fig. 13). The only obvious, reproducible difference between the two is that the bands in the double-stranded products (especially lysozyme and ovomucoid) are less sharp than those in unreacted first strands, probably the result of variable digestion of duplex ends by Sl. Their similarity suggests that the optimized DNA polymerase I and Sl nuclease reactions are efficient using total mRNA and may be applied to obtain full length copies in 5 From a starting reverse transcriptase reaction using 1 pg of ovalbumin mRNA, roughly 0.25 pg of first strand is synthesized (9). After the DNA polymerase reaction, first strands are 80% resistant; thus 0.4 pg of double-stranded cDNA is present after Sl cleavage. When electrophoresed on native gels, we estimate that 10 to 50% of this material is full length (Fig. 11, and  many systems in which only crude or slightly purified mRNAs are available. Several lines of evidence indicate that most of the synthesis seen during the DNA polymerase I reaction is primed from a 3'-terminal hairpin on the first strand: (a) a large proportion of the duplex formed is capable of snapping back immediately after denaturation (Fig. 9); (b) examined on denaturing gels, the band found after second strand synthesis at twice the mRNA length is converted back to mRNA length upon cleavage by single-strand specific Sl nuclease; (c) on native gels, the full length band is unchanged upon digestion with increasing nuclease concentrations (Figs. 11 and 12). Some second strand synthesis probably does not begin from the hairpin primer, however, since not all duplexes snap back (Fig. 9). Oligo(dT) or messenger fragments (which might be generated by the RNase H activity of reverse transcriptase by contaminating nucleases (9), or by thermal scission during the boiling step) could serve as primers for non-hairpin syntheses. Any hybrids formed during the DNA polymerase reaction are likely to be poorly matched. No hybrids stable to Sl nuclease digestion at 37" are formed (Fig. 1); imperfectly base-paired structures may form which, although unstable at 37", are suitable primers for DNA polymerase during the 15" incuba-tion. Rougeon and Mach (2) have reported that even purified first strands are sometimes converted to non-snap back duplexes by E. coli DNA polymerase I, but some oligo(dT) fragments may persist in their first strand fraction. Ovalbumin-directed second strand-labeled products often, but not always, display more fast moving, low molecular weight radioactivity (less than 900 bases) than do first strand labelings done in parallel (compare Fig. 1OA (Truck 1) to Fig. 1OB  (Track 11, Fig. 7A (Truck 3) to Fig. 7B (Track 2)). Such a result is predicted if priming sometimes occurs not at the hairpin but from an internal priming site not covalently attached to the first strand i.e. from oligo(dT) or messenger fragment primers as discussed earlier.
Several reaction conditions were critical in determining the length of second strands made by DNA polymerase I. Salt concentration, monovalent cation, temperature, time, and the amount of polymerase added each had marked effects. Some of the observations warrant further comment. 1) The time course data, comparing Figs. 1 and 3, suggests that some incorporation into second strands occurs even after the Sl resistance of first strands has reached a maximum plateau. When we investigated the rate of the reaction between 2 and 24 h, exactly as described in Fig. 3 (using either labeled dGTP or dCTP), we were surprised to find that second strand synthesis continued at a linear rate at approximately 10% of that found over the first 2 h (data not shown); net incorporation into second strands thus doubled between 2 and 24 h. But when 2-, 12-, and 24-h second strand-labeled products were examined on denaturing gels, no significant differences were seen. No change in first strand size distribution (Fig. 2B) or total first strand radioactivity was found over this period. We have not yet identified the material synthesized by DNA polymerase I after 2 h. 2) The DNA polymerase dependence data with ovalbumin mRNA (Fig. 5) suggests that a polymerase molecule does not simply bind at the 3'-hydroxyl end of the first strand primer and elongate without dissociation until completion (processive polymerization).
Were that the case, one would expect subsaturating enzyme levels to generate small amounts of "normal" full length products. Instead the data is compatible with the enzyme dissociating from the template during second strand synthesis. Both nonprocessive (17) and processive (18) mechanisms for E. coli DNA polymerase I have been suggested. 3) At 42" DNA polymerase I synthesizes large products regardless of which template is used. Since each reverse transcriptase reaction is primed by oligo(dT), a relatively unstable dA:dT tract should be present at the end of a completed second strand, whether copied from a full length or partial first strand. Use of low salt (Fig. 6) or elevated temperature (Fig. 8), conditions which should further destabilize this region, result in the synthesis of slowly migrating, high molecular weight products. One possible mechanism for the formation of extraordinarily long molecules by DNA polymerase I is that the dA tract being synthesized opposite the first strand oligo(dT) primer is so unstable in low salt or high temperature that the enzyme effectively "slips" and synthesizes a long stretch of poly(dA). If this were the mechanism responsible, then second strands made at 42" should have a much higher adenosine content than those made at 15". This was demonstrated not to be the case in an experiment in which we determined the ratio of ovalbumin directed second strand incorporation at the two temperatures using either 13H]dCTP or 13H]dATP. The 42":15" ratio is the same with both isotopes (25.9 with dCTP, 26.1 with dATP).