Elimination of band compression in sequencing gels by the use of N4-methyl-2′-deoxycytidine 5′-triphosphate

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Abstract
Taq DNA polymerase, Sequenase, and the large fragment of E.coli polymerase I effectively utilize N4-methyl-2′-deoxycytidine 5′-triphosphate (N4-methyl-dCTP) in the place of dCTP in dideoxynucleotide terminator sequencing reactions on single-stranded templates. When the resulting fragment mixtures are resolved on sequencing gels, they are found to be free of band compressions even in cases where such compressions remain unresolved by the substitution of 7-deaza-dGTP for dGTP. Sequencing reactions using N4-methyl-dCTP instead of dCTP are somewhat more prone to false stops than are sequencing reactions using 7-deaza-dGTP instead of dGTP; this difference is more pronounced when sequencing with Sequenase at 37°C than when sequencing with Taq DNA polymerase at 72°C. For the three polymerases investigated, replacement of dCTP by N4-methyl-dCTP does not fundamentally change the characteristic variations in band intensities seen in the C-lane. N4-methyl-dCTP can also be used for sequencing double-stranded DNA and for DNA amplification by the polymerase chain reaction.