The SALSA (MS-)MLPA
Probemix P300 Reference-2 is a research use only (RUO)
assay which is developed to be used in combination with ‘home-made’ synthetic (MS-)MLPA probes. This probemix contains reference probes and control fragments specific for unique human DNA sequences. The control fragments and reference probes in this P300 probemix facilitate the data analysis of a synthetic probemix with home-made probes.
MRC-Holland also offers the P200 reference probemix. While the P200 leaves the entire range from 80-170 nt open for the inclusion of synthetic probes, this P300 probemix has reference probes distributed over the whole size range of the probemix. This is especially advantageous for correction of the signal sloping of MLPA amplification products. Signal sloping is the effect that longer probes generate a lower peak height/area on the electropherogram than shorter probes. The exact amount of signal sloping differs between sequencer types and can differ between different samples.
The use of P200 is recommended when a large number of synthetic probes are used, preferably targeting sequences on different chromosomes. Results can be difficult to interpret when P200 is used in combination with a small number of probes that all target the same gene or chromosomal region, or with a small number of probes with lengths much shorter than the reference probes. The use of P300 is recommended when a smaller number of synthetic probes is used.
Synthetic probe design:
Guidelines on how to design synthetic (MS-)MLPA probes and the RaW-Probe software for Tm calculation are available on our website, www.mlpa.com
. Unfortunately, we cannot offer additional support on probe design. Please read our synthetic probe design protocol very carefully before you start designing.
The SALSA MLPA Probemix P300-B1 Reference-2 contains 21 MLPA probes with amplification products between 88 and 274 nt. This includes 14 reference probes, detecting several different autosomal chromosomal locations. In addition, DNA Quantity Fragments (Q-fragments), two DNA Denaturation Fragments (D-fragments), two HhaI-digestion control fragments, one chromosome X and one chromosome Y-specific fragment are included. The Q-fragments give a warning when insufficient amounts of sample DNA are used. The D-fragments give a warning when sample DNA was not completely denatured. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mlpa.com