The SALSA (MS-)MLPA Probemix P200 Reference-1 is a research use only (RUO)
assay which contains reference probes and control fragments specific for unique human DNA sequences. The P200 probemix offers a collection of thoroughly-tested MLPA reference probes and control fragments to which ‘home-made’ synthetic (MS-)MLPA probes can be added. Not only do these control fragments and reference probes provide extra assurance about the quality of self-made probes and of the MLPA reaction performed, they also facilitate the data analysis of your synthetic probemix and maximise the number of synthetic probes you can include.
Next to the P200, MRC-Holland also offers the P300 Reference-2 probemix. While the P200 leaves the entire range from 82-173 nt open for the inclusion of synthetic probes, the 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 all targeting the same gene or chromosomal region, and with lengths much smaller than the reference probes. In samples with sloping, it may seem in such cases that all sequences targeted by the synthetic probes are deleted or duplicated. The use of P300 is recommended when a smaller number of synthetic probes is used.
Guidelines on designing synthetic (MS-)MLPA probes and the necessary RAW program are available on our website www.mlpa.com
. Unfortunately, we cannot offer additional support on probe design. Please read our synthetic probe design protocol carefully before you start designing.
The SALSA MLPA Probemix P200-B1 Reference-1 contains 14 MLPA probes with amplification products between 173 and 251 nucleotides (nt). This includes ten reference probes that detect different autosomal chromosomal locations and four quality control fragments. These include: one DNA Denaturation fragment (D-fragment), one digestion control fragment, one chromosome X and one chromosome Y-specific fragment. (see table 1). In addition, it contains 4 DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt. 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