Biomedical Engineering Reference
In-Depth Information
•
Low MLPA peak areas
: The quantity of a probe amplification product is primarily
determined by the target sequence of the probe. MLPA probes located in guanine- and
cytosine-rich areas often have difficulty reaching their target due to incomplete denatura-
tion of the DNA. Better results may be obtained with these MLPA mixes by increasing
the length of the initial denaturation step to 98
◦
C for 10min. The probe signals are
further influenced by the quality of the probe oligonucleotides and the amount of KCl
and polymerase present during the PCR reaction. The polymerase activity may influence
the relative signal strength of some probes; therefore, mixing the master mix well by
pipetting up and down is strongly advised. It is also possible that the DNA input was too
low. This is likely if the average signal of the four DQ (DNA quality) fragments is less
than one-third of the ligation-dependent peak signal.
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The original publication describing MLPA.
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