long question (1)

Question 1

describe a sequencing method for studying DNA methylation and discuss its uses and limitations

Question 2

what is DNA methylation

Answer 2

the addition of a methyl/hydroxymethyl group to the C5 position of cytosine

Question 3

key factors considered when choosing a sequencing method

Answer 3

• aims of the study
• amount of quality
• sensitivity and specificity
• bioinformatics availability
• equipment & reagent availability

Question 4

sequencing methods for DNA methylation

Answer 4

• bisulfite genomic sequencing (pyrosequencing)
• nanopore sequencing
• single molecule, real-time

Question 5

what is bisulfite genomic sequencing?

Answer 5

• quantitative and efficient approach
• identify 5-methylcytosine at single base-pair resolution
• can detect DNA methylation patterns of dsDNA

Question 6

how does it work?

Answer 6

• cytosines in single-strand DNA will be converted into uracil residues
• recognised as thymine in subsequent PCR amplification
• 5mCs are immune to this and remain as cytosines

Question 7

advantages of this?

Answer 7

• less expensive
• quantitative accuracy
• detection sensitivity
• highly efficiency
• wide spectrum for sample analysis

Question 8

disadvantages of this?

Answer 8

• post next-gen sequencing sequence alignment is difficult
• only works best on short sequences
• bisulfite conversion leads to DNA fragmentation
• single nucleotide polymorphisms where a cytosine is converted to thymidine will be missed
• bisulfite conversion doesn’t distinguish between 5mC and 5hmC