Question

I was told a single peak of T means the Cytosine is methylated, in a CpG context. However, thinking about it, I have trouble explaining how you would get to this conclusion.My though was: Imagine you have:

5' CpG 3'
3' GpC 5'
Now if both are unmethylated you get:
5' TpG 3'
3' GpT 5'
If you now do a PCR, they give rise to two types of DNA molecules (TG/AC) and (CA/GT). If you perform Sanger Sequencing just with the forward primer, this would mean you get 50% T and 50% C at the original position. In other words, the reverse strand (3'-GC-5') should always restore the C at the original position, when I perfom a PCR.

Answer 1

The question deals with the detection of methylation at CpG sites on DNA via PCR and Sanger Sequencing, where methylated cytosines remain as cytosines while unmethylated ones are converted to uracils and then to thymines.

Step-by-step explanation:

The question appears to be related to the methylation of cytosine in a CpG context and its detection through sequencing techniques like PCR and Sanger Sequencing.

Normally, in PCR, both strands of DNA serve as templates for the creation of new strands, where the bases are placed in the correct order due to the base pairing rules—C pairs with G, and A pairs with T. When PCR is applied to a bisulfite-treated DNA, unmethylated cytosines are converted to uracils, which are read as thymines in PCR, while methylated cytosines remain unchanged. Therefore, a sequence with a single peak of T at a CpG site in a sequenced DNA could indicate that the cytosine was methylated, as it did not convert to uracil and then to thymine during the PCR process.

To experimentally determine methylation status, typically, DNA would be treated with bisulfite, which converts unmethylated cytosine to uracil, followed by thymines in the PCR product. Methylation prevents this conversion, so during the sequencing, a cytosine (C) peak means the position was likely to be methylated, and a thymine (T) peak means it was likely to be unmethylated. When performing PCR and then Sanger sequencing, if the original DNA strand had 5' CpG 3', the methylated cytosine will appear as a C peak, and the unmethylated cytosine as a T peak in the sequencing results.