Biomedical Engineering Reference
In-Depth Information
1. Denaturation & hybridization
PCR primer sequence
Stuffer sequence (different for
each probe)
5
PCR primer
sequence Y
Hybridization
sequence
5
5
Target
Target
3
5
3
5
2. Ligation
X
X
The two parts of each probe
hybridize to adjacent target
sequences and are ligated by a
thermostable ligase.
5
Y
3
Target
5
5
Y
5
Target
5
3. PCR
All probe ligation products are amplified by PCR using only one primer pair.
Y
X
The amplification product of each
probe has a unique length
(130-480 bo).
Y
X
5
3
5
3
4. Separation of amplification products by electrophoresis
Amplification products are separated by electrophoresis. Relative amounts of probe amplification
products, as compared to a control DNA sample, reflect the relative copy number of target sequences.
Detection of trisomies
SALSA MLPA P001 Trisomy kit
Female
DNA
Female
Tr i 2 1
C
hromosome 21 specific probes
Figure 1.3
Schematic of MLPA. The MLPA reaction is performed using four steps. Genomic DNA
is denatured, whereafter the MLPA probes are added and incubated for 16 h, allowing complete
hybridization adjacent to all target sequences. Probes completely hybridized to sequences either
side of each target region are subsequently ligated to each other, enabling their exponential PCR
amplification and final detection, and quantification by capillary electrophoresis. (See Plate 1.3.)
