Figure 2. Premature termination of primer extension in the presence of dsGTP. Sequenase was used to synthesize a 25 bp hairpin (HP25) in the presence of diﬀerent combinations of dNTPs. Extension of the radiolabeled primer was monitored by electrophoresis in a 12% sequen- cing gel. dNTPs of the following bases were used: (i) A,T,G,C; (ii) nA,sT,G,C; (iii) A,T,sG,C and (iv) nA,sT,sG,C. The unmodiﬁed hairpin in lane 1 retained a native conformation in 7 M urea and so ran faster than the fully denatured nA/sT-substituted hairpin in lane 2. Table 1. Relative uptake of dGTP and dCTP analogs into SS37 and HP25 primer extension products by f29 N62D DNA polymerase Single-strand Hairpin Single-strand Hairpin dGTP analogs dNTPs of A,T,C G 1.00 1.00 cG 1.07 1.07 NitrocH 0.92 1.17 IcH 1.14 0.35 H 1.03 1.17 cH 1.04 0.89 sG 0.98 0.98 dCTP analogs dNTPs of A,T,cG C 1.07 1.07 NitroC 0.91 0.88 sC 0.69 0.03 MefP 0.87 0.37 P 0.01 0 sCa ND 0.87 dNTPs of nA,sT,C 1.12 1.11 1.16 1.27 1.06 1.01 0.98 dNTPs of nA,sT,cG 0.93 1.03 0.33 0.52 0.01 ND 0.84 0.82 0.66 0.58 0.81 0.69 0.10 0.96 0.62 0.02 0 0 0.78 aTemplate for synthesis contained H in place of G. ND, not determined. a deazaguanine derivative in preparing pseudo- complementary DNA, the reactions with modiﬁed dCTPs contained dcGTP in place of dGTP. This analog of G is readily incorporated into DNA and does not Nucleic Acids Research, 2008, Vol. 36, No. 22 7003 self-associate (19,20,40–42). Yields in each case were determined relative to the amount of each primer exten- sion product obtained when using regular dNTPs. All of the dGTP analogs functioned eﬀectively with A, T and C or nA, sT and C when preparing single-stranded DNAs. However, synthesis of the hairpin was less reliable. In the presence of A, T and C the IcH analog gave reduced yield while in the presence of nA, sT and C all of the analogs were less eﬀective. This was particularly true for sG where synthesis of full-length hairpin was reduced by 90%. Uptake of H and NitrocH was further investigated using Vent (exo-) DNA polymerase to carry out one cycle of asymmetric PCR in the presence of nA and sT. Whereas NitrocH supported synthesis of a 600-mer pro- duct that contained some highly G/C-rich secondary structure elements, H did not. In the presence of the more weakly pairing H analog, primer extension was blocked by these elements. Incorporation of the dCTP analogs into SS37 and HP25 was highly variable. NitroC gave moderate to good yields of all four primer extension products. Conversely, P did not support synthesis of any full-length product and sC only supported synthesis of single-stranded DNAs. MefP improved upon sC by also supporting limited synthesis of hairpin in the absence of nA and sT. In an attempt to improve the utilization of dsCTP, we investigated whether using a template in which H was substituted for G could direct incorporation of sC into the complementary hairpin product. This strategy was supported by an earlier study which had shown that incorporation of rsCTP into RNA was more eﬃcient when templated by H instead of G (43). With steric clash now eliminated by using H as the tem- plating base, sC was incorporated into the hairpin product along with cG or nA, sT and cG. Although use of H in a primer extension reaction is a cause for concern, poly- merases appear to maintain speciﬁcity in the presence of this base (44). Using an analogous approach, the synthesis of hairpin substituted with sG, nA and sT might be made more robust by using a template in which C has been enzymatically replaced by a readily incorporated analog of MefP. Relative pairing strengths of G/C analogs Melting properties of the radiolabeled hairpins prepared using Phi29 DNA polymerase provided a convenient read- out of how each analog decreased hybrid stability either alone or in combination with nA and sT. Apparent melt- ing temperatures were determined using a gel mobility shift assay. Brieﬂy, each hairpin was incubated with a large molar excess of 25-mer probe at temperatures ran- ging from 108C to 808C. The probe, which was comple- mentary to the distal arm of the hairpin stem, was expected to hybridize to the hairpin at a temperature approximating its true Tm. Melting proﬁles were obtained by quenching each hybridization reaction in an ice bath and analyzing for hybrid formation by non-denaturing gel electrophoresis. The midpoint in the melting curve was taken as equal to an apparent Tm. When hybridizing to hairpins that contained A and T, the 25-mer probe was synthesized with a chimeric LNA–DNA backbone to