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.
Highlights
Due to the high GC content of differentially-methylated regions (DMRs), the PCR amplification of these sites from untreated genomic DNA often involves the use of special polymerase enzymes or kits which often do not have “hotstart” capabilities
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
After increasing the length of DNA denaturation prior to polymerase enzyme addition, the methylated allele from the MEST DMR was identifiable at the sequencing stage, with the methylated allele starting to appear after a total of 12 minutes of DNA denaturation prior to the PCR cycles
Summary
Due to the high GC content of differentially-methylated regions (DMRs), the PCR amplification of these sites from untreated genomic DNA often involves the use of special polymerase enzymes or kits which often do not have “hotstart” capabilities. We regularly use the FailsafeTM system (Epicentre Biotechnologies, Madison, Wisconsin, USA) which has as its standard protocol an initial DNA denaturation step of 1 - 2 minutes following the addition of the polymerase enzyme. We identified a two-generation family with a 7 b.p. insertion in the MEST gene differentially-methylated promoter region (c.1-99_1-93insdupGGGCTGC). This insertion was shown by previous PCR analysis of bisulphite-treated DNA to be on the methylated chromosome in the unaffected father and on the non-methylated chromosome in the affected child (data not shown). The PCR protocol was modified to include an extra DNA denaturation stage prior to the addition of the enzyme in order to create a PCR product from the methylated chromosome
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