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.
See EDTA-RT Sample Collection Requirements