long question (1) Flashcards
1
Q
describe a sequencing method for studying DNA methylation and discuss its uses and limitations
A
2
Q
what is DNA methylation
A
the addition of a methyl/hydroxymethyl group to the C5 position of cytosine
3
Q
key factors considered when choosing a sequencing method
A
- aims of the study
- amount of quality
- sensitivity and specificity
- bioinformatics availability
- equipment & reagent availability
4
Q
sequencing methods for DNA methylation
A
- bisulfite genomic sequencing (pyrosequencing)
- nanopore sequencing
- single molecule, real-time
5
Q
what is bisulfite genomic sequencing?
A
- quantitative and efficient approach
- identify 5-methylcytosine at single base-pair resolution
- can detect DNA methylation patterns of dsDNA
6
Q
how does it work?
A
- 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
7
Q
advantages of this?
A
- less expensive
- quantitative accuracy
- detection sensitivity
- highly efficiency
- wide spectrum for sample analysis
8
Q
disadvantages of this?
A
- 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
