MLPA Fragment Analysis

1. 20 October 2008 MLPA Fragment Analysis

2. Content Visual Troubleshooting on MLPA fragments Fragment analysis steps Size calling separated fragment peaks Software Training Analyzing separated fragment profiles using: Genemapper / Genescan Coffalyser VB CEQ Fragments Analysis Generating Fragment lists 27 Maart 2005 www.mlpa.com

3. Trouble Shooting MLPA data Should always be performed on raw fragment data Troubleshooting prevents wasting time on data analysis of runs that will only produce incomprehensible results Example Electropherograms for troubleshooting and recognition of problems Related to: Sample Quality MLPA Procedure 27 Maart 2005 www.mlpa.com

4. October 5th 2005 www.mlpa.com Each MLPA kit contains concentration control probes Product will be generated at 64, 70, 76 and 82 bp even when no Ligation is omitted or no DNA is present Peaks will be small when DNA concentration is lower than 50 ng Concentration control probes are added in the MLPA mixes at very low amounts and will therefore only be visible if the concentration of the DNA is relatively low to the DNA concentration of the sample DNA concentration check

5. October 5th 2005 www.mlpa.com Low DNA concentration indication

6. October 5th 2005 www.mlpa.com Ligation check Every MLPA kit contains a probe which is ligation dependent and will always form a product if the ligation was succesfull This ligation peak comes from a probes comprised of 2 synthetic probes reflecting the copy number of a 2q14 DNA sequence Methylation kits contain 2 extra control probes which target for Cpg islands. Indicating if the denaturing of the DNA was complete Denaturation control probes give a probe at 88 and 96 bp and should give a signal of about the same height as the 92 bp peak

7. October 5th 2005 www.mlpa.com Incomplete denaturation indication

12. DNA denaturation, Discovered around 1950. Few studies after 1965 Tm is dependent on the % GC. Tm is ~ 16 oC higher if the salt concentration is 10 x higher. Tm is lower in the presence of DMSO / formamide. Effect of other chemicals ? Effect of Salt on DNA denaturation

13. MLPA results when denaturing in PCR buffer

17. Effect of Metal Ions on DNA denaturation Metal ions as Stabilizers or Destabilizers of the Deoxyribonucleic Acid structure. Eichhorn, G.L. Nature (1962) 194, 474-475: � The presence of 0.1 mM MgCl2 in a sample raises the Tm by ~ 12 oC.

19. MLPA experiments: Denaturation of long genomic DNA fragments: AT rich ; DNA in TE: Tm ~ 80 oC. AT rich ; DNA in PCR buffer: Tm ~ 90 oC. GC rich ; DNA in PCR buffer: Tm ~ 98 oC. Long GC islands will not be denatured at 95 oC in PCR buffer. A small amount of degraded DNA will be denatured and can be PCR amplified. The primers and the high % GC will be blamed for the low signal or the large number of cycles required in real time PCR. Long GC islands will be denatured at 95 oC in TE. When DNA is denatured in TE and the PCR buffer + polymerase are added after denaturation, higher signals will be obtained / less PCR cycles are required in ordinary PCR, Real time PCR. Be careful with Real-time PCR for DNA copy number !

20. DNA Denaturation Notes DNA in the vicinity of CpG islands will be denatured but will renature within seconds if the CpG island is not denatured. Completely denatured DNA takes weeks/months to renature. MLPA signals for probes in the vicinity of CpG islands can be reduced if DNA denaturation was incomplete. When DNA denaturation is incomplete, MLPA probe signals will depend on the average length of the chromosomal DNA. For MLPA experiments, DNA is denatured in TE at 98 oC. GC islands will also be denatured at 98oC in TE. The presence of certain chemicals in some DNA samples increases the Tm and prevents complete denaturation of the DNA. Be careful with kits / automatic DNA purification. Any impurities left remaining in the samples can have effects on DNA denaturation and thus on the accessibility of DNA sequences to MLPA probes.

21. Primer Dimer formation The primer peak can be recognized by the largest peak which occurs right after the start around 50 bp, containing the major amount of unused primer. Closely after that a smaller peak may appear, which contain the primer-dimers. Primer dimers may form during the first PCR step unusually when the PCR is not HOT started or when creating the polymerase mix. Large quantities of primer dimers may be formed which negatively affect your PCR reaction by decreasing the amount of available primer. The following guidelines help to minimize primer dimer formation. Always create the polymerase buffer mix on ice. Add the polymerase enzyme as last and mix by pipetting up and down. Pre-heat your ligation mixture with PCR buffer at 60-72 degrees in the therthermocycler and start the PCR reaction a.s.a.p. after adding the polymerase mix. 27 Maart 2005 www.mlpa.com

22. Primer Dimer & Low Signal Intensities 27 Maart 2005 www.mlpa.com

23. Sloping / no Sloping

25. Sloping in Size Marker 27 Maart 2005 www.mlpa.com

26. Effect of sloping on Ratio results (uncorrected/corrected)

27. Irregular Size Marker pattern 27 Maart 2005 www.mlpa.com

28. Fragment Analysis steps Matrix Correction Baseline Correction Peak Recognition Peak quantification Peak to Probe Correlation 27 Maart 2005 www.mlpa.com

29. Baseline Correction

30. Peak recognition A peak should be recognized if several data points (n>10) following after each other have a rising pattern in 45 ?C or more. The relative peak height should be more than 3% of the sum of all the total peak area intensity Probe signals should be at least higher than 100 fluorescence units

31. Determination peak heights / peak areas The peak height should be the intensity of fluorescent units at the top of the peak The peak areas should be comprised of the total area of the peak thus including shoulderpeaks (-1/+1 bp) which are caused by taq errors (slippage) The beginning and end of an peak can be determined by the point where the peak comes back at the 1/100 median line (determined during baseline correction)

32. Recognition of marker values Marker values (ROX, TAMRA or LIZ) should recognized using pattern recognition determined from standard added size marker values (all series 400, 500 and 600 bp) All marker signals should be listed, signals deviating <70% & >130% from the median of all signals should be discarded Signals before and directly after the initial primer dimer peak should also be discarded The remaining signal should be used to correlate the datapoint of the top of peak with the known basepairs of the used marker

33. Size Calling Size calling of MLPA probes by comparison of Size-Marker with MLPA probes All MLPA probes will obtain a specific detected length Size calling will usually estimate the MLPA probe length with a deviation from the cloned length Size calling can be done by Genescan, Genemarker, Genemapper, Ceq software, or the ABI-export tabular (Free provided by Tommy Gerdes in Denmark built in the Coffalyser)

34. Determination of datapoint to basepair formule

35. Size calling of all peaks

36. Software Demonstration Size calling & Data Export using Genemapper Peak Scanner Coffalyser VB 27 Maart 2005 www.mlpa.com