Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification
Bunyan, David J., Bullman, Hilary M.S., Lever, Margaret, Saminathan, Sasi D., Keng, Wee Teik, Araffin, Roziana and Robinson, David O. (2011) Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification. Open Journal of Genetics, 2011/01, (02), 13-14. (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.
|Digital Object Identifier (DOI):||doi:10.4236/ojgen.2011.12003|
|Keywords:||differential methylation, allele-specific PCR, commercial kit|
|Subjects:||Q Science > QH Natural history > QH426 Genetics|
|Divisions:||Faculty of Medicine > Human Development and Health
|Date Deposited:||25 May 2012 09:56|
|Last Modified:||27 Mar 2014 20:22|
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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