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Summary NAD HC10
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NAD HC10 Methylation specific PCRA novel assay for methylation status of CpG islands.
- PNAS, 1996; vol 93, pp 9821-9826
- Recognition of methylation patterns
- In paraffin-embedded coupes
- Pure DNA
- CpG islands in neoplasia
- Imprinted genes
- X-chromosomal genes
Historically
- Methylation occurs in higher order eukaryotes to regulate gene expression
- In lower organisms: protection against cleavage by invading organisms
- Before 1998:
o Restriction analysis, southern blot, input 5 μg DNA
o Restriction analysis, PCR based on non-cut fragments
o Problem: false-positives, insensitive method, PCR picks up also partially digested preps!
Old method
After restriction
- Running on gel
- Southern blotting
- Measure size of fragments
,New method, MSP: PCR based MSP is Methylation Specific PCR
- Requires much less DNA; methylated DNA detected at 0,1% of total DNA
- Amounts as low as 50 pg detected by PCR
- More sensitive than earliers methods
- No radioactivity (at that time an advantage!)
- No sequencing (may be done of course for confirmation of results)
Diagnosis Prader-Willi syndrome and Angelman syndrome
- MSP used not only for cancer diagnosis
- Both syndromes PW and AS diagnosed routinely with MSP
- Brain syndromes, mental retardation, insomnia, high appetite, obesistas
- Incidence 1:15000 births
- Cause of the disease in Methylation pattern
PW and AS Genetic causes
The method
- CpG (CG) often in promoter regions of genes
- Methylation of C
- Methylation leads to inhibition of transcription of the gene
- C is deaminatied to T: via sodium bisulfite The not methylated C becomes a T
- Methylated C is resistant
- PCR on isolated, bisulfite treated DNA
- Primers specific for different strands (with or without C)
, From cytosine to uracil
- Treat with sodium bisulphite at low pH
- Hydrolytic deamination
- Alkalic desulphonation
It starts with good primer design
- Specific software
- Primer3
- Methprimer
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