GeneMarker^® for Multiplex Ligation-dependent Probe Amplification (MLPA*)

GeneMarker now has the capability to analyze data from Multiplex Ligation-dependent Probe Amplification (MLPA). Click here for more information on MLPA tests and test kits.

Current applications include the detection of exon deletions in the human BRCA1, MSH2 and MLH1 genes associated with breast cancer and colon cancer, detection of trisomies as in Down Syndrome (47, XX + 21), categorization of chromosomal abnormalities in cell lines and tumor samples, and mutation detection.

With GeneMarker, MLPA data analysis is an efficient, quick, and easy process, with features such as customized reporting, intensity comparison, data normalization, and printing.

GeneMarker is an excellent tool for nearly all Genotyping applications. The exclusive, high resolution gel image provides a quick overview of the MLPA analysis, allowing rapid discernment of areas of interest. The MLPA analysis accurately identifies exon deletions and duplications through calculating the intensity ratios of sample to control.

MLPA Normalization

GeneMarker can normalize MLPA derived data by an internal control probe method or an inter-lane population method. The user is able to specify the normalization method in the Run Wizard prior to analysis. The normalization is similar to that designed by Graham R. Taylor at St. James’s University Hospital, Leeds. You may find a detailed description of the method titled “Regression-enhanced examination of MLPA (REX-MLPA): towards automated and objective scoring” at http://leedsdna.info/science/dosage/REX-MLPA/REX-MLPA_CMGS04.ppt.

The internal control probe method normalizes the data by comparing the individual peak intensities within one sample lane. The inter-lane population method compares the peak intensities of the amplified probes between sample lanes. Both normalization methods take the square root of the intensity ratio and plot the ratios to model a linear regression, using the control probes as reference points.

MLPA Report	MLPA Intensity Report
GeneMarker is designed to identify copy number changes in genomic sequences. The main interface window after running the data is shown above. The various peaks in the electopherogram represent different amplified probes within the sample and can be labeled accordingly in the Panel Editor. The report displays the presence and absence of the amplified probes within each sample. The green flag is used to represent high quality peaks, whereas a yellow symbol is used to represent peaks with a lower score. This report may be saved as a text file for printing in Excel.	The user may also display the intensity for the peaks representing the amplified probes. Since MLPA is designed to locate exonic duplications and deletions, the peak intensity of the amplified probes is a very important factor in the analysis. The peak intensity is directly correlated to the copy number in the sample trace. This report may also be saved for printing in Excel.
MLPA Regression Plot	MLPA Intensity Ratio
The software forms a regression line utilizing a standard T distribution. The user may select an Increment or Decrement Regression to locate outliers within the data set. The software identifies outliers based upon a userdefined confidence limit. The report shown above displays the peak intensity ratio between sample and control sample for each amplified probe. The sample to control intensity ratio of probe HIRA in sample B5_11881 is 0.415. The user may also display the intensity of each probe, which in this case is 965 for the sample probe and 2323 for the control.	In order to identify copy number changes within the sample, the software uses the intensity ratios of sample to control. The user can define the thresholds dictating the exonic duplications and/or deletions. The report can display the peak intensity ratio, peak intensity, or peak area. Data points which lie outside the user defined threshold for deletion and duplication (indicated by the horizontal green lines) most likely have some significance for copy number change. The report can be saved as a text file and can be imported into Excel for printing.

*MLPA is a registered trademark of MRC-Holland.

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