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MLPA slideshow MRC Holland

MLPA slideshow MRC Holland

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  • Transcript

    • 1. MLPA ® Multiplex Ligation Probe Amplification MRC-Holland b.v.
    • 2. Multiplex Ligation-Dependent Probe Amplification (MLPA)
      • “ Multiplex gene dosage analysis made easy”
      • First described by: Schouten JP et al. (2002)
        • Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res . Jun 15;30(12):e57.
      • More than 150 published articles on different subjects using MLPA
      • In routine use in more than 500 laboratories on 6 continents
    • 3. MLPA
      • Detection of aberrant copy number of 45 genomic DNA sequences in one easy to perform, PCR based reaction.
      • Minimum of only 20 ng DNA
      • Partially degraded DNA
        • DNA extracted from paraffin
        • Formalin treated tissues
      • Discriminates sequences that differ in only a single nucleotide.
      • 45 different mRNAs
      • To determine the methylation status of promoters
      • Detection of known mutations and SNPs.
    • 4. Apparatus
      • Thermocycler
      • Sequence type electrophoresis
    • 5. MLPA technique
      • Denaturation
      • Hybridization
      • Ligation
      • Amplification
    • 6. SALSA MLPA probes
    • 7. Hybridysation
      • The MLPA probemix is added to denatured genomic DNA
      • The two parts of each probe hybridise to adjacent target sequences
    • 8. Ligation
      • 3. Probes are ligated by a thermostable ligase
    • 9. Amplification
      • A universal primer pair is used to amplify all ligated probes.
      • The amplification product of each probe has a unique length (130 480 bp).
    • 10. 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
    • 11. Detection of Chr X copy number X Triple X Female Male 283 bp 346 bp
    • 12. 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
    • 13. 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
    • 14. 55 o C hybridisation 60 o C hybridisation 62 o C hybridisation Many variables have only a small influence on MLPA results
    • 15. 10 ng. DNA (less than recommended minimum amount) 50 ng. DNA 750 ng. DNA Amount of DNA used has little influence on MLPA results
    • 16. Detection of point mutations
      • Only perfectly matched probes will be ligated
      • Probes for common mutations or SNP’s can be made
        • Signal will only be present when the mutation is present
    • 17. 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
    • 18. 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
    • 19. Methylation specific MLPA
      • Epigenetics
        • Regulation of gene transcription due to methylation of CpG nucleotides located within the promoters
        • Example: Hypermethylation of the p53 promoter in several tumors
      • Imprinting
        • 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
    • 20. 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
    • 21. Control Undigested ME028 PW / Angelman kit Control Hha1 digested Arrows indicate probes for imprinted sequences (1 copy methylated)
    • 22. Advantages of MLPA
      • Detection of copy number of 45 genomic DNA sequences in a simple to perform, PCR based, reaction.
      • Requires only 20 ng human DNA
      • Discriminates sequences that differ in only a single nucleotide.
      • Only a thermocycler and a sequence type electrophoresis system are required.
      • Identical protocol for many different applications.
      • High throughput ; Results available within 24 hrs.
      • Large lot sizes (> 100.000 reactions) are possible. All reagents have proved to be very stable.
      • For each probe signal, presence of two specific oligonucleotides is required.
      • All reagents are fluid: Simplified quality control.
      • Including electrophoresis, total reagent costs are < EUR 15,- / reaction.
    • 23. Disadvantages of MLPA
      • Results depend on sample quality. For amniotic fluid samples we recommend to use cell lysates rather than purified DNA.
      • Amniotic fluid samples that are contaminated with maternal blood can not be used.
      • MLPA can not detect all triploidies.
      • MLPA can not detect balanced translocations. More than 130 MLPA tests are currently available from MRC-Holland.
      • MRC-Holland is research oriented and is not yet ISO certified.
      • MLPA kits are not yet CE certified.
      • Time consuming and difficult to develop new MLPA based assays.
    • 24. MLPA kits
      • Mixed kits with up to three different probemixes (minimum 20 reactions each)
      • Reagents are the same for MLPA and MS-MLPA kits
      • RNA MLPA kits require additional reagents (included)
      • HhaI enzyme for the ME-MLPA kits not included
      • MLPA kits (including enzymes) very stable
    • 25. MLPA products
      • Detection of aneuploidy of chromosomes 13, 18, 21, X and Y
      • Detection of large chromosomal deletions or duplications: DiGeorge syndrome, Williams syndrome, Spinal Muscular Atrophy (SMA), subtelomeric regions etc
      • Detection of gains and losses of genes in cancer tissues: Her2-neu (ERBB2), TP53, MYC etc.
      • Detection of deletions / duplications of single genes and exons: RHD, BRCA1 and 2, MSH2, MLH1, VHL, SHOX, MECP2, APC and NF2 etc.
      • Copy number of all CFTR exons.
      • Copy number of all 79 DMD exons in 2 MLPA reactions
      • MS-MLPA kits
      • Visit for all our products
    • 26. Future applications MLPA
      • To increase sensitivity of the MLPA assay
      • SC-MLPA: MLPA on single cells / free fetal DNA in maternal plasma
          • Detection of trisomies from fetal single cells obtained in maternal plasma
      • Probemixes for tumor characterisation
      • Probemixes for detection of aberrant CpG island methylation