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Molecular Assays

 

Multiplex Ligation-Dependent Probe Amplification (MLPA)

Multiplex ligation-dependent probe amplification (MLPA) is used to identify large or unknown alpha-thalasssemia or beta-thalassemia deletions.

Each target DNA sequence has a pair of specific probes that contain a unique length stuffer sequence so that each probe pair can be distinguished based on its length. If the target sequence is normal, the probes will hybridize with the target and are then ligated by a ligase.  These ligation products are amplified using only one primer pair and the products separated by capillary electrophoresis.  The amplification product of each target will have a a unique length permitting separation using a sequence analyzer. The relative amounts of probe amplification products correspond to the relative copy number of target sequences. A 50% reduction in PCR product indicates a heterozygous deletion, while complete absence of amplified product indicates a homozygous deletion. MLPA can only determine the extent of the deletion and the approximate location of breakpoints. DNA sequencing is subsequently performed to define the deletion breakpoints.

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Example of MPLA analysis:

MPLA