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  • MLPA

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