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MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS

MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS. Oxford. Somai Man/Kate Gibson. MRC-Holland. Mechanisms causing PWS/AS. Aberrant CpG island methylation-S.Blot, MS-MLPA. MS-MLPA (ME0028). MS-MLPA, detects changes in:

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MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS

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  1. MS-MLPA Methylation Specific Multiplex Ligation Probe Amplification for detection of PWS/AS Oxford Somai Man/Kate Gibson MRC-Holland

  2. Mechanisms causing PWS/AS Aberrant CpG island methylation-S.Blot, MS-MLPA

  3. MS-MLPA (ME0028) • MS-MLPA, detects changes in: • COPY NO. (Standard MLPA) • Differences in methylation status at Chr15q11-13. (HhaI)

  4. Mechanisms causing PWS/AS & effect seen using MS-MLPA kit NB: other methods cannot cannot discern between these

  5. PWS/AS MS-MLPA Probe Mix • Test probes: specific for sequences within & near 15q11-13; 5 contain a HhaI recognition site (SNRPN & NECDIN) • Control probes: for copy number & HhaI digestion

  6. MS-MLPA Procedure Overnight hybridisation Reaction split into 2 tubes Ligation & PCR as with standard MLPA Add HhaI, Ligate & PCR COPY NO. METHYLATION

  7. HhaI • HhaI cuts at unmethylated sites • Only methylated (undigested) & ligated probes are exponentially amplified • Methylated signal • Unmethylated no signal

  8. mUPD IC Deletion (pat) Ch3 Ch3 Ch3 Ch3 Ch3 m m m P m P Doubling of Methylation dose Doubling of Methylation dose One methylation dose; like normal Same (2) Reduced (1) Same (2) Understanding results-PWS Normal Ch3 m P Expression One methylation dose Copy No: 2 NB: UPD and IC indistinguishable. Linked markers

  9. pUPD IC Deletion (mat) m P m P P P No methylation signal No methylation signal No methylation signal Same (2) Reduced (1) Same (2) Understanding results-AS Normal Ch3 m P Expression One methylation dose Copy No: 2 NB: UPD and IC indistinguishable. Linked markers

  10. Normal Results Copy no. Results 100% Control probes Chr15q11-13 probes: in order down csome 15q Control probes Equal heights of probes in PWS/AS critical region = NO 

  11. Normal Results Copy no.Results Methylation Results 100% 0.5 Undigested Controls 1 Differentially Ch3td alleles 0.5 Digested Controls 0 Control probes Chr15q11-13 probes: in order down csome 15q Control probes Equal heights of probes in PWS/AS critical region = NO 

  12. Understanding Results • Deletions: 50% drop in relative peak height. Stats-cfn betwn patient & normal controls • Methyln abnormalities: difference in dose of • methyld allele • Simple pairwise cfn betwn cut & uncut results from same patient • Irrespective of mutation mechanism, methyln ratio for PWS= 1.0, AS= 0 • Bothresults  conclusive diagnosis

  13. Deletion Results Copy no. Results PWS  AS  50% Half heights of probes in PWS/ AS Critical region =  Methylation Results 0.5 Doubling of methylation signal Loss of methylation signal

  14. UPD/ IC Results Copy no. Results PWS UPD/ IC AS UPD/ IC 100% Equal heights of probes in PWS/ AS Critical region = NO  Methylation Results 0.5 Doubling of methylation signal Loss of methylation signal

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