19.02.04 Methylation detection Flashcards
1
Q
What is DNA methylation?
A
- Epigenetic event that affects cell function by altering gene expression
- Refers to the covalent addition of a methyl group, catalysed by DNA methyltransferase (DNMT), to the 5-carbon of cytosine in a CpG dinucleotide
2
Q
Why do we need to do additional work to assess methylation status?
A
- Epigenetic information is lost during PCR because DNA polymerase does not distinguish between methylated (M) and unmethylated (UM) cytosines
- Therefore the DNA must be modified in a way that allows the methylation information to be preserved
3
Q
What is the common modification process used to assess methylation?
A
Bisulphite modification
4
Q
What are the main steps of bisulphite modification?
A
- UM cytosines in DNA are converted to uracil through treatment of single stranded DNA with sodium bisulphite
- Methylated cytosines have a methyl group attached to the 5-carbon of cytosine in a CpG dinucleotide which protects it from deamination
- Performed under acidic conditions
- Bisulphite- treated DNA is then run on a disease specific PCR
- The sense and antisense strands are no longer complementary after sodium bisulphite treatment (so any primers have to be designed for either strand)
- Uracil residues are replicated as thymine residues and 5-methylcytosine residues are replicated as UM cytosines
5
Q
What are the two main types of methylation PCRs?
A
1) Methylation-independent PCR (MIP)
- primers designed for proportional amplification of M and UM DNA
2) Methylation-specific PCR (MSP)
- primers designed for the amplification of M template only
- tends to give highest analytical sensitivity
N.B. hard to amplify region independent of methylation status - normally get a bias towards amplification of UM DNA
6
Q
Name 10 techniques available for detection of methylation status
A
1) Pyrosequencing
2) Combined Bisulfite Restriction Analysis (COBRA)
3) Methylation-sensitive melt curve analysis
4) Real-Time Quantitative Detection Techniques
5) MS-PCR
6) Array based methylation techniques
7) NGS
8) Southern blotting
9) MS-MLPA
10) Genome wide methylation analysis
7
Q
1) Pyrosequencing
A
- Pro = allows quantification of methylation levels (unlike sanger)
- Peak heights in pyrogram report ratio of cytosine to thymine at each CpG site (represents proportion of M to UM DNA)
- Claims can detect >10% methyation
- Con = requires high number of samples so prone to contamination issues
8
Q
2) Combined Bisulfite Restriction Analysis (COBRA)
A
- The differences in sequence between M and UM DNA after bisulfite modification can lead to the creation of new methylation-dependent restriction sites or the maintenance of restriction sites in a methylation-dependent manner
- COBRA exploits this due to separation of digested PCR products followed by hybridisation of fluorescently labelled probes
- Con = not all sequence changes cause creation/removal of a restriction enzyme site
- Con = prone to error due to the formation of heteroduplexes between the converted M and UM DNA (heteroduplex not cleaved by RE)
- Result shows melting temp of PCR product
9
Q
3) Methylation-sensitive melt curve analysis (MCA)
A
- MCA can us useful as M DNA has a higher CG content
- MIP is performed in the presence of a fluorescent dye that intercalates dsDNA
- The target sequence may be fully M, fully UM, or heterogeneously M
- When a mixture of fully M and fully UM molecules is amplified, 2 distinct melting peaks are observed
- Con = When heterogeneously M molecules are amplified however, the melting pattern can be complex and difficult to interpret
10
Q
4) Real-Time Quantitative Detection Techniques (e.g. MethyLight and HeavyMethyl)
A
- Variety of techniques available that use florescent probes (Taqman)
1) MethyLight - Uses MIP primers and differentiates between M and UM DNA by the use of M and UM specific TaqMan probes labelled with different fluorophores, permitting quantification of the degree of methylation in a sample
- Or uses methylation insensitive TaqMan probes which target regions in between methylation-specific primer sites (2 different primer pairs are used in separate reactions, one specific for M and converted DNA, the other specific for UM and converted DNA).
- Pro = assay permits high throughput and allows determination of the relative prevalence of a particular pattern of CpG methylation
- Con = Can’t provide highly accurate quantitative methylation information about single CpGs within a region of interest as with Pyrosequencing
2) HeavyMethyl - oligonucleotide blockers are used to discriminate between M and UM alleles
- When M - blockers don’t bind, primer site is accessible for MIP primers to bind and amplify, detected with fluorescent oligonucleotide probe
- When U - block occurs and no PCR product is generated
- Pro = blockers significantly increase analytical sensitivity (can detect very low levels of M)
- Pro = extremely low false positive rate (compared to MSP primers)
11
Q
5) MS-PCR
A
- Pro = avoids amplification bias of MIP, as primer paris are methylation-specific
- Pro = highly sensitive
- Con = high false positive rates (due to high number of PCR cycles required)
- Con = get false-priming events (where amplification happens even though primer and template have mismatched)
- Con = Iget incomplete bisulfite convertion of template leading to false-positive results
12
Q
Example of MS-PCR - PWS and AS
A
- BS conversion of DNA
- PCR of exon 1 of SNRPN using common F primer and a maternal specific F primer for the M form, and paternal specific F primer for UN form
- PWS = lack of paternal contribution - so only amplifies mat band (131bp)
- AS = lack of maternal contribution - so only amplifies pat band (164bp)
- Different sized bands separated by Gel
- Controls includes negative, normal, PWS positive, AS positive
13
Q
6) Array based methylation techniques
A
- Provided by Illumina (HumanMethylation27 BeadChip)
- 27,578 CpG loci, covering more than 14,000 genes and >450,000 methylation sites
- Meant to be high throughput and low cost
14
Q
7) NGS
A
- NGS of bisulphite converted DNA is possible for targeted regions or at the genome-wide scale
- Largely within the research setting
- Need to be cautious of depth of sequencing as insufficient reads will yield inaccurate data about methylation levels
15
Q
8) Southern blotting
A
- Non-bisulphite methylation analysis method
- Example of use is FRAX
- Uses methylation specific restriction exnzyme (Eag1) and a methylation insensitive enzyme (EcoR1)
- Produces large 5.2kb M band and smaller 2.8kb UM band
- Gel separation occurs, then transferred to membrane for blotting
- Membrane incubated with labelled probe complimentary to region of interest
- The fragment produced by EcoR1 is further digested to produce a smaller band when the allele is UM and active (remains undigested if the allele is M and inactive)
- Con = Mosaicism hard to detect
- Con = incomplete digestion can be an issue
- Con = time consuming, labour intensive
