A method for detecting methylated CpGs involvesthe use of a chemical called bisulfite, which convertscytosine to uracil but leaves methylated cytosine untouched. You want to know whether a particularCpG dinucleotide at one location in the genome ismethylated on one or both strands in a tissue sample.The genomic sequence containing this CpG is:5’...TCCATCGCTGCA…3’. You take genomicDNA from the sample tissue, treat it exhaustivelywith bisulfite, and then use flanking primers toPCR-amplify the region including this CpGdinucleotide. You then want to Sanger sequence(see Fig. 9.7) the amplified PCR product. a. After you treat genomic DNA with bisulfite, the twoDNA strands will melt into single strands. 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 willnot amplify the DNA. What special considerationswould be necessary when you design your PCRprimers for this experiment? Could one pair ofPCR primers amplify both strands of DNA?c. What sequence would you see if you amplified theDNA strand shown and the CpG was methylated?If it was not methylated?d. Using the bisulfite method, can you tell if this CpGdinucleotide in the tissue sample is hemimethylated(methylated on one strand) or methylated on bothstrands? Explain.
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. 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.
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