Methylation Detection Flashcards
What is DNA methylation?
DNA methylation is an epigenetic event that affects cell function by altering gene expression and refers to;
- The covalent addition of a methyl group
- catalysed by DNA methyltransferase (DNMT)
- to the 5-carbon of cytosine in a CpG dinucleotide
Methylation analysis encompasses a large number of techniques.
What types of molecular biology techniques are required to study methylation?
- Most early studies used methylation sensitive restriction enzymes to digest DNA followed by Southern detection or PCR amplification
- Bisulfite reaction based methods have now become standard practice such as methylation specific PCR.
- Global methylation analysis is still a relatively new and evolving technology and is restricted to the research setting
What important consideration must you be aware of when using PCR based mathods to for methylation detection?
Epigenetic information is lost during PCR because DNA polymerase does not distinguish between methylated and unmethylated cytosines
Therefore the DNA must be modified in a way that allows the methylation information to be preserved.
What is ‘Bisulphite modification’?
The ‘deamination’ of unmethylated cytosines in DNA leads to conversion into uracil by treatment of single stranded DNA with sodium bisulphite.
The sequence changes brought about by this process enable us to identify which cytosines are methylated and unmethylated, using a variety of downstream processes.
What is the chemical process of Bisulphite modification?
Bisulfite conversion is performed under acidic conditions and preferentially deaminates cytosine whilst the methyl group on 5-methylcytosine protects the amino group from the deamination.
In diagnostic processes it is advisable to modify control samples of known genotype (e.g. methylation positive and negative) in each assay.
Name one interesting feature which results from bisufite treating dsDNA.
The sense and antisense strands are no longer complementary after sodium bisulfite treatment.
Thus, any probes or primers which are designed for downstream process are designed for either strand.
What are the two broad types of downstream process used to assess methylation of bisulfite modified DNA?
Methylation-independent PCR (MIP) primers are used in most of the available PCR-based methods, which are designed for proportional amplification of methylated and unmethylated DNA.
Methylation-specific PCR (MSP) primers, which are designed to specifically amplify methylated DNA or unmethylated DNA only - gives best sensitivity.
What is the main lmitation to Methylation-independent PCR based assays?
It can be difficult to amplify a target independent of its methylation status.
In most situations, there is a PCR bias toward amplification of unmethylated DNA.
This means that the methylated allele can be ‘missed’ thus reducing sensitivity.
List some of the main methods which utilise Methylation-independent PCR (MIP) primers.
- Pyrosequencing
- Combined Bisulfite Restriction Analysis (COBRA)
- Methylation-sensitive melt curve analysis
- Real-Time Quantitative Detection Techniques
Why would you use pyrosequencing for methylation detection?
Pyrosequencing offers advantages as it permits quantification of methylation levels (as low as 10% methylation).
A disadvantage of Pyrosequencing is that the high cycle numbers involved mean that it can be prone to contamination
What is COBRA anlaysis?
COBRA exploits the creation of new methylation-dependent restriction sites or the maintenance of restriction sites in a methylation-dependent manner after bisulphite treatment.
Analysis then relies on the separation of digested PCR products followed by hybridisation of fluorescently labelled probes.
What are the disadvantages of COBRA?
- Not all sequence changes result in the formation/abolition of a restriction enzyme site
- Any technique that involves restriction digestion post PCR, like COBRA, is prone to error due to the formation of heteroduplexes between the converted methylated and unmethylated DNA. This heteroduplex will not be cleaved by restriction enzyme
Describe the MethyLight RQ-PCR method for detecting methylation.
Provides a quantitative analysis of methylation, two ways to perform assay;
- MIP primers + methylation specific TaqMan probe labelled with different fluorophores for meth/un-meth
- MSP primers + methylation insensitive TaqMan probes
Limitations are that it cannot provide highly accurate quantitative. Not as accurate as pyrosequencing.
Describe the HeavyMethyl RQ-PCR method for detecting methylation.
When the DNA is methylated, the blocker oligonucleotides do not bind, leaving the primer- binding site accessible for the MIP primers to bind and amplify the target.
The amplification is detected with a methylation specific oligonucleotide probe that contains CpG sites, labelled with fluorescent dye (F) and quencher (Q) in a 5′-exonuclease assay, used here as an example for a real-time detection method e.g. TaqMan, Lightcycler.
When the DNA is unmethylated, the blocker oligonucleotides bind, blocking the access of the primers to their binding sites. No PCR product is generated.
What is the advantage of HeavyMethyl over MethyLight?
The use of blocker molecules significantly increases the analytical sensitivity.
The main advantage relative to conventional MSP is that false-positive rates are extremely low.
Due to the blockers providing methylation specificity at every cycle of the PCR, whereas in MSP, a false-priming event needs to happen only once to get the amplification going (i.e. two levels of methylation specificity with blocker and primer).
Describe how the results of MS-PCR assay are interpreted? What controls are run alongside test samples?
- Two reverse primers produce a differently sized products
- In PWS only the maternal band is seen on gel
- In AS only the paternal band is seen on gel
Controls;
- Normal: Has both bands on gel
- No DNA control: No bands
- Unmodified DNA: No bands
Descrive how to interpret the results of a Fragile X Southern Blot.
Name the enzymes and prove used and the sizes/patterns of bands produced on a typical gel image.
- EcoR1 = meth-insensitive enzyme
- Eag1= meth-sensitive enzyme
- StB12.3 probe
- Methylated DNA yields a upper band at 5.2 kb
- Un-methylated DNA yields a upper band at 2.8 kb (as both enzyme cut)
- As the size difference between the alleles increases (due to expansion) then the two alleles can be discriminated in the upper and lower regions of the gel
- Once the expansion becomes so large the allele is always methylated then this allele is only seen in the upper region (methylated DNA) of the gel.
- In females there will just be a single band in the lower region representing the normal allele in cells where that X is not inactivated. In males the lower band is absent as there’s only one allele
Describe how the AmplideX FMR1 PCR assay works
- Long-range PCR to span repeat expansion
- Same primers used in two separate reactions with the only difference being the labelling of the primer (Fam vs Hex)
- The Fam PCR amplifies all alleles irrespective of methylation status
- In the Hex PCR the DNA is exposed to meth-sensitive enzyme.
- If the DNA is un-meth then it is digested and no product is produced and visa-versa
- Thus Hex peaks represent the methylated alleles
- The ratio of Fam to Hex for an allele indicates the level of methylation in the sample
- Normal sized allele should be ~50% in Hex
- Major devations of normal alleles would indicate skewing
- Expanded alleles will be the same height in Fam and Hex because they are always methylated.
What is the advantage and disadvantage of using AmplideX PCR vs Southern?
- Advantages: Cheaper, quicker
- More accurate skewing identification
- PCR is more sensitive for low level mosaicism detection
- Disadvantages: very large expansions can not be spanned by the long-range PCR.
Describe how the MS-MLPA assay works
- MS-MLPA probes for methylation detection are the same as regulat MLPA probes
- Their target sequence contains the restriction site of the methylation-sensitive endonuclease.
- After hybridisation, the reaction is split into two tubes: one tube is processed for copy number, the other is incubated with the enzyme.
- If the sample DNA is methylated the probes are protected from the enzyme and produce a peak after PCR, unmethylated probes are digested away.
HOw are the results of MS-MLPA interpreted?
Analysis of MS-MLPA consists of two parts
- Determining copy number by comparing different undigested samples
- Determining methylation patterns by comparing each undigested sample with the digested sample, this gives a semi-quantified amount of methylation.
Black arrows indicate four SNRPN MS-MLPA probes used for methylation quantification. The white arrow indicates an MLPA probe located within the PWS/AS region used for copy number quantification. The grey arrow indicates an MS-MLPA digestion control probe.
