Chapter 17, Problem 27P

A method for detecting methylated CpGs involves the use of a chemical called bisulfite, which converts cytosine to uracil but leaves methylated cytosine untouched. You want to know whether a particular CpG dinucleotide at one location in the genome is methylated on one or both strands in a tissue sample. The genomic sequence containing this CpG is: 5’...TCCATCGCTGCA…3’. You take genomic DNA from the sample tissue, treat it exhaustively with bisulfite, and then use flanking primers to PCR-amplify the region including this CpG dinucleotide. You then want to Sanger sequence (see Fig. 9.7) the amplified PCR product.

a.	After you treat genomic DNA with bisulfite, the two DNA strands will melt into single strands. Page 615 Why?
b.	Your answer to part (a) introduces a potential complication, because if you do not account for this result of bisulfite treatment, the PCR primers will not amplify the DNA. What special considerations would be necessary when you design your PCR primers for this experiment? Could one pair of PCR primers amplify both strands of DNA?
c.	What sequence would you see if you amplified the DNA strand shown and the CpG was methylated? If it was not methylated?
d.	Using the bisulfite method, can you tell if this CpG dinucleotide in the tissue sample is hemimethylated (methylated on one strand) or methylated on both strands? Explain.