CSB349H1 Lecture Notes - Lecture 6: Methylated Dna Immunoprecipitation, Bisulfite Sequencing, Demethylation
14 May 2018
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Lecture 6(b): DNA Methylation
Bisulfite Sequencing/Conversion:
• Bisulfite sequencing is the most common method for detecting DNA methylation
Methods:
• Starting with two CpGs and one of them is methylated
1. Denature both strands to generate ssDNA
2. Add bisulfate to ssDNA to oxidize the nucleotides
o Unmethylated cytosine = Uracil
o Methylated cytosine = Cytosine
3. 1st Round of PCR: generate second-strand synthesis
o Uracil base-pairs with Adenosine
o Cytosine base-pairs with Guanine
4. 2nd Round of PCR:
o Two strands will be denatured to generate
complementary strands
§ All UpA will become ApT
• Complementary strands will undergo sequencing and will detect how
many ApT pairs exist which are representative of unmethylated
cytosines and CpG pairs are representative of methylated cytosines
Methylomics:
• Detecting DNA methylation via genome wide analyses
1. Differential Methylation Hybridization (DMH)
a) Restriction enzymes are used to generate small fragments of DNA
b) Adaptors (linkers) will be ligated to both sides of the DNA
fragments
c) Methylated sensitive restriction enzyme (i.e. SmaI) will be added to
cleave specific regions if cytosine is not methylated
d) PCR will be completed by using primers that recognize the known
adaptors (linkers)
§ If the DNA was methylated, it would not be cleaved,
therefore PCR will generate one product
§ If the DNA was unmethylated, it would be cleaved,
therefore PCR will not work as there are two pieces
• PCR requires two primers for it to work – cleaved
DNA will only have one adaptor sequence (primer)
ð Analyze PCR products via microarray or next-generation sequencing
(NGS)
Document Summary
Lecture 6(b): dna methylation: bisulfite sequencing is the most common method for detecting dna methylation. All upa will become apt: complementary strands will undergo sequencing and will detect how many apt pairs exist which are representative of unmethylated cytosines and cpg pairs are representative of methylated cytosines. If the dna was methylated, it would not be cleaved, therefore pcr will generate one product. If the dna was unmethylated, it would be cleaved, therefore pcr will not work as there are two pieces: pcr requires two primers for it to work cleaved. Dna will only have one adaptor sequence (primer) Analyze pcr products via microarray or next-generation sequencing (ngs: methylated dna immunoprecipitation (medip, restriction enzymes are used to generate small fragments of dna. Sonication can be done as well; to generate fragments: antibodies that are capable of recognizing methylated dna will be added, immunoprecipitate dna fragments. Dna fragments can be analyzed via microarray or next-generation sequencing (ngs)
