Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification
Author(s)
David J. Bunyan,
Hilary M. S. Bullman,
Margaret Lever,
Sasi D. Saminathan,
Wee Teik Keng,
Roziana Araffin,
David O. Robinson
ABSTRACT
We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.
Cite this paper
nullBunyan, D. , Bullman, H. , Lever, M. , Saminathan, S. , Keng, W. , Araffin, R. and Robinson, D. (2011) Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification. Open Journal of Genetics, 1, 13-14. doi: 10.4236/ojgen.2011.12003.
nullBunyan, D. , Bullman, H. , Lever, M. , Saminathan, S. , Keng, W. , Araffin, R. and Robinson, D. (2011) Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification. Open Journal of Genetics, 1, 13-14. doi: 10.4236/ojgen.2011.12003.
References
[1] Diede, S.J., Guenthoer, J., Geng, L.N., et al. (2010) DNA methylation of developmental genes in pediatric medu- lloblastomas identified by denaturation analysis of methylation differences. Proceedings of the National Academy of Sciences, 107, 234-239. doi:10.1073/pnas.0907606106
[1] Diede, S.J., Guenthoer, J., Geng, L.N., et al. (2010) DNA methylation of developmental genes in pediatric medu- lloblastomas identified by denaturation analysis of methylation differences. Proceedings of the National Academy of Sciences, 107, 234-239. doi:10.1073/pnas.0907606106