GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N⁴-methylated analog, N⁴-methyl-2′-deoxycytidine 5′-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N⁴me-dCTP—containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N⁴me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (T_m) clearly below the T_m of the same amplicon synthesized exclusively with the canonical bases.

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Vol. 61, No. 4

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Received 21 March 2016

Accepted 6 July 2016

Published online 16 March 2018

Published in print October 2016

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Author contributions

C.R.F.J. performed the experiments. C.R.F.J. and R.C.P. designed the experiments and wrote the initial manuscript. E.G.J. and A.A. analyzed the data and contributed to the discussion and the final text. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the Secretaría de Investigación y Posgrado of the Instituto Politécnico Nacional (Grant SIP 20131256) and the Consejo Nacional de Ciencia y Tecnología-CONACYT (Grant Ciencia Básica 61322). C.R.F.J. was the recipient of a graduate-student stipend from CONACYT.

Competing interests

The authors declare no competing interests.

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PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2′-deoxycytidine 5′-triphosphate

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References

PCR amplification of GC-rich DNA regions using the nucleotide analog N⁴-methyl-2′-deoxycytidine 5′-triphosphate