Bunyan, David J., Bullman, Hilary M.S., Lever, Margaret, Saminathan, Sasi D., Keng, Wee Teik, Araffin, Roziana and Robinson, David O.
Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification
Open Journal of Genetics, 2011/01, (2), . (doi:10.4236/ojgen.2011.12003).
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We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair inser- tion 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 ampli- fied, 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 do- ing so, both the methylated and non-methylated al- leles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methy- lation 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.
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