Question

1. How does bisulfite analysis distinguish unmethylated and methylated DNA?
   1. In a bisulfite analysis, antibodies that bind to methylated cytosines enrich methylated DNA sequences.
   2. Bisulfite analysis compares the digestion patterns of two restriction enzymes that cleave only unmethylated, or both methylated and unmethylated DNA to deduce methylation status.
   3. Bisulfite analysis permits direct observation of a cell’s chromosomal number and structure to deduce DNA methylation status.
   4. In a bisulfite analysis, unmethylated cytosines are converted to uracil, which can then be subjected to PCR, sequenced, and compared to a reference genome.

Answer 1

(D) In a bisulfite analysis, unmethylated cytosines are converted to uracil, which can then be subjected to PCR, sequenced and compared to reference genome.

• Bisulphite method/sequencing is used for determination of methylation patterns before DNA sequencing.
• The most common methylation in animals is methlyation of 5' C of cytosines of CpG sequences which are usually responsible for repression of transcription, embryonic development, and chromosomal stability.
• Bisulfite converts unmethylated cytosines to uracil and have no effect on 5-methyl cytosine, therefore leaving only methylated cytosines.
• After dinstinction of methylated cytosines from cytosines PCR is carried out in oredr to determine methylation patterns in chromosome/loci of interest.
• Defects in DNA methylation patterns result in genomic instability and cancer development, therefore bisulfite sequencing provides an efficient and qualitative approach for determination of DNA methylation patternsby identifying 5 methyl cytosine(5mC) at resolution of single base pair.