GeneMarker MLPA® Analysis

MLPA® Analysis

Multiplex Ligation-dependent Probe Amplification (Schouten et. al) is a rapid, high-throughput technique for copy number quantification performed with commercially available kits (MRC Holland) or custom chemistries to detect insertions and deletions. Copy number changes and loss of heterozygosity, key factors in the study of human cancers, are readily identifiable using GeneMarker’s MLPA analysis software application, which integrates all of the analysis steps into one software package; providing normalization options and customized clinical research reports. Analysis is rapid and accurate, allowing complete analysis of 96 samples in minutes including MLPA, MS-MLPA (detecting imprinting diseases like Prader Willi) and Luminex®MLPA. GeneMarker is compatible with the output from all major genetic analyzers, including: ABI®PRISM, Beckman-Coulter®, MegaBACE®. Access MLPA panels in GeneMarker’s panel editor, our website, or tech_support@softgenetics.com. GeneMarker’s MLPA application has been validated by research hospitals worldwide, is included in EuroGentest validation report and is an excellent alternative to GeneMapper®, Genotyper™, GeneScan®, CEQ™ Fragment Analysis Software.

Data Normalization

Due to the variations of PCR efficiencies from small to large DNA fragments or from sample to sample, two selectable normalization methods are provided. The first normalization method is the traditional method based upon the control probes as described in reference 4. The second method, unique to GeneMarker, normalizes peak intensities based upon the statically most probable median Figure illustrates the non-linear DNA migration of GeneScan®1200 fragments in a POP7™gel. The migration time is linear for 100-800 bp fragments. The larger and smaller fragments deviate from the linear function. GeneMarker’s new algorithm provides accurate, linear sizing of the data using a DNA derivative migration time correction to large DNA fragments. Current sizing separation technology has its optimum efficiency at 470 bp. GeneMarker’s Large Size algorithm enables accurate sizing from small to large (30-1400) base pair fragments. The peak area in base space accurately determines the copy numbers because the peak area normalization is much intensities. In order to correct for the peak intensity variation over size, an exponential function a*e-bz is used to fit to the square root of peak intensities, where z is size, and a and b are fitting constants. The normalization using the control probes is shown in figure 2. This correction removes the trend of dropping intensities as the DNA fragment size increases, and sets the height ratios of control probes to approximately 1. However the trend of peak intensities vary greatly from one sample to another with the internal control probes. We have found that the use of fewer control probes often results in large errors in the intensity normalization.

Normalization Methods

Traditional Control Probe Normalization.

SoftGenetics’ Population Normalization.

SoftGenetics’ “Population Normalization” addresses the above problems. Median peak intensities are derived from the first five data points, then sliding to data points 2-6, 3-7, etc. to ascertain the local median intensities. Outliers are rejected after applying a median filter. All of the probes (control and test) with the median intensities are then used to fit the exponential function. This methodology creates higher accuracy and lower false positive rates.

Customizable Patient Report available as a .PDF or printed file.

The patient report includes sample ID, analysis parameters, report, graph and electropherogram by sample.

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MLPA® Analysis

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