Cancer Genetics: Troubleshooting in MLPA and FISH Test

CANCER GENETICS
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Cancer genetics
Troubleshooting in MLPA
MLPA (multiplex ligation-dependent probe amplification) is a multiplex PCR technique of identifying
and copying figures of up to 60 diverse genomic DNA arrangements which is capable of distinguishing
orders different by only one nucleotide. Sloping may be perceived as a signal to drop in size where the
longer fragments are lower the shorter ones. This may result in bias in data biasness. When sloping is
severe the signal of longer fragments become very low to fall below the threshold of detection. Hence,
these portions may appear as deletions though it’s not true1.
Causes of sloping
Vaporization when performing hybridization may happen if tubing are not correctly sealed or if
deformations occurs when carrying out hybridization to check for evaporation during hybridization a tube
containing 8 μl water should be added, at least 5 μl water should remain after the hybridization step.
Vaporization can also be checked be use of heated lid, if the lid has not heated there may be no
vaporization although it does not affect the results, they remain the same2. Impurities in the DNA portions
can result in sloping .it more efficient to use small quantities of DNA, dilution of DNA leads to dilution
of the contaminants. For multiplex ligation-dependent probe amplification a DNA input of 50-100 mg is
enough. Alternatively ethanol precipitation can be done to remove all impurities. Sloping in the MLPA
highest design may also improve if capillaries and buffer of the capillary electrophoresis device are
replaced since old capillaries and polymer also cause sloping in the data3. Unfortunately the sloping in the
data is so severe that you can't use the data for interpretation. If the sloping is indeed caused by old
capillaries and/or polymer you can reload the PCR products of this MLPA reaction to see if the data
improves4.
1 S. Schaefer, R. Elise, Pauline Helms, Luc Marcellin, and others Next-generation sequencing (NGS) as a
fast molecular diagnosis tool for left ventricular noncompaction in an infant with compound mutations in
the MYBPC3 gene.( European journal of medical genetics 57 2014).
2 E. Ellard, F. Sian, H. Lango Allen, Z. Elisa De Franco and others, Improved genetic testing for
monogenic diabetes using targeted next-generation sequencing. (Diabetologia 56 2013).
3 M. Patel, K. Ravi , and Mukesh Jain. "NGS QC Toolkit: a toolkit for quality control of next generation
sequencing data. (PloS one7 2012).
4 U. Audo, S. Isabelle, K. Kinga, M. Bujakowska and others. Development and application of a next-
generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal
diseases (2012) Orphanet journal of rare diseases 7, no 1.
2