18.02.04 Methylation detection Flashcards
What are the stages of MS-PCR?
Following bisulphite treatment, PCR is set up using one set of primers specific for the treated methylated DNA sequence, and one set specific for the treated unmethylated DNA sequence.
Even on the methylated parental chromosome, not every C nucleotide is methylated (those not part of CpG are unmethylated).
After bisulphite treatment the unmethylated parental chromosome has all C changed to U, and the methylated parental chromosome has some C changed to U and some unchanged. Therefore both treated parental chromosomes differ from the original sequence. This means that untreated chromosomes will not amplify and skew the result.
The resulting PCR products are run on a gel. As the products from treated methylated and unmethylated are of different sizes they will produce bands at different positions on the gel.
Normal individuals will show 2 bands. Those with UPD will show only 1 band – which band is present will depend whether the maternal or paternal chromosome has been inherited.
What are the advantages and disadvantages of MS-PCR?
Advantages: Does not require parental bloods.
Disadvantages: This method cannot distinguish UPD from a deletion or IC defect – additional testing maybe required to rule out a deletion. Also cannot distinguish segmental UPD.
What are the stages of bisulphite restriction analysis and PCR for methylation detection?
reatment with sodium bisulphite causes changes in CpG sites on the methylated and unmethylated chromosome/region.
Produces differential restriction enzyme sites for the methylated and unmethylated alleles, either creation of new restriction sites, or retention of methylation-dependent restriction sites.
The region of interest is amplified by PCR using primers specific for that region (regardless of methylation status).
The primers used in the PCR reaction do not cover CpG dinucleotides so the amplification step does not discriminate between templates according to their original methylation status [Xiong 1997].
The DNA is then treated with a restriction enzyme that cuts only the unmodified methylated DNA.
Other methods use two different restriction enzyme sites on each allele, so do not rely on the absence of cleavage to detect methylation status [Sadri and Hornsby].
The PCR products are run on a gel. The methylated and unmethylated products are of different sizes therefore they will produce bands at different positions on the gel.
Normal individuals will show 2 bands. Those with UPD will show only 1 band – which band is present will depend whether the mat or pat chromosome has been inherited.
What are the advantages and disadvantages of bisulphite restriction analysis and PCR for methylation detection?
Advantages: Does not require parental bloods.
Disadvantages: This method cannot distinguish UPD from a deletion or IC defect – additional testing maybe required to rule out a deletion. Also cannot distinguish segmental UPD
What are the stages of methylation sensitive melt curve analysis?
Following bisulphite modification, PCR is set up using fluorescently-tagged primers specific for each of the treated methylated and unmethylated DNA sequences.
The PCR products are cooled to 40oC and then heated at 0.05oC per second to 95oC. During this heating the fluorescence in monitored.
Unmethylated PCR products have a lower CG dinucleotide content (as all C’s converted to uracil in bisulphite treatment) and this lowers the melting temperature.
The fluorescence monitoring should therefore show a peak for the unmethylated product and one for the methylated product: Patients with UPD will only show one peak.
What are the advantages and disadvantages of methylation sensitive melt curve analysis?
Advantages: Does not require parental bloods.
Disadvantages: This method cannot distinguish UPD from a deletion or IC defect – additional testing maybe required to rule out a deletion.
How can pyrosequencing be used to look for methylation differences?
Following treatment with bisulphite methylation differences can be detected and quantified by analysing the bisulphite-induced C/T differences at CpG sites.
This method tends to be used to look at several CpG sites within a specific imprinting-related region, so in this sense is more targeted than SNP-array or whole exome/genome sequencing.
