A universal primer pair is used to amplify all ligated probes.
The amplification product of each probe has a unique length (130 480 bp).
Separation and quantification by capillary electrophoresis Each peak is the amplification product of a specific probe. Samples are compared to a control sample. A difference in relative peak height or peak area indicates a copy number change of the probe target sequence
Detection of Chr X copy number X Triple X Female Male 283 bp 346 bp
Specificity of MLPA probes is very high. control Homozygous deletion ASPA exon 1-6 1 5 4 3 2 6 1 5 4 3 2 6
Reproducibility of MLPA is sufficient to distinguish homozygotes and heterozygotes. Heterozygous ASPA del. exon 1-6 Control 1 5 4 3 2 6 1 5 4 3 2 6
55 o C hybridisation 60 o C hybridisation 62 o C hybridisation Many variables have only a small influence on MLPA results
10 ng. DNA (less than recommended minimum amount) 50 ng. DNA 750 ng. DNA Amount of DNA used has little influence on MLPA results
Signal will only be present when the mutation is present
Normal Ligation of the two probe oligonucleotides Amplification product Mismatch at the probe ligation site No ligation, no amplification product MLPA discriminates sequences that differ in only a single nucleotide and can be used to detect known mutations. Mismatch Perfect match
MLPA protocol The use of a thermocycler with heated lid is essential. 1. Denature 20-500 ng DNA by heating to 98 o C. 2. Add the MLPA probes and MLPA Buffer. Incubate o/n at 60 o C. 3. Add Ligase-65 and ligase buffer. Ligate at 54 o C for 15 minutes. 4. Inactivate the ligase by heating to 98 o C. Add PCR primers, dNTPs and polymerase and start the PCR reaction. 5. Analyse the products by electrophoresis. 6. Export fragment length’s and peak areas to Excel
Regulation of gene transcription due to methylation of CpG nucleotides located within the promoters
Example: Hypermethylation of the p53 promoter in several tumors
Regions of the genome where the methylation patterns are preserved in the same way as they were inherited from the parents.
Examples: The Chr 15 Prader-Willi/Angelman region and X-linked mental retardation
Detection of aberrant CpG island methylation
MS-MLPA Only undigested (methylated) and ligated probes are exponentially amplified Unmethylated Target M M Methylated Target Denaturation and Multiplex probe hybridization M Ligation and Digestion with methylation sensitive endonucleases M
Control Undigested ME028 PW / Angelman kit Control Hha1 digested Arrows indicate probes for imprinted sequences (1 copy methylated)