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Improved: SALSA MLPA Probemix ME029 FMR1-AFF2 - Changes to Content and Protocol

Jul 07, 2023

We are glad to inform you that we released an improved version of SALSA® MLPA® Probemix ME029 FMR1-AFF2. ME029 targets the FMR1 and AFF2 genes which contain trinucleotide repeats that are expanded in Fragile X and Fragile XE syndromes. Denaturation of these extremely GC-rich regions requires a product-specific protocol, which has also been updated in the new C1 version. 

Main changes in product content

  • Additional target probes for FMR1 and AFF2 have been added.
  • The digestion control probes and the probes detecting autosomal chromosomal locations have been replaced.
  • Flanking probes have been removed.
  • Multiple reference probes have been replaced.

Main changes to the ME029 protocol

  • Until now, ME029 FMR1-AFF2 was supplied with a vial of Denaturation Buffer (red cap). This denaturation buffer will no longer be supplied. Instead, the user of this product needs to use freshly made NaOH. Instructions for this preparation can be found in the ME029 FMR1-AFF2 product description (page 2).
  • As a consequence of the aforementioned change, ME029 FMR1-AFF2 will now be supplied with SALSA Low pH MLPA Buffer (purple cap). This low pH buffer must be used instead of the normal MLPA Buffer (yellow cap) supplied in MLPA reagent kits.

Left: ME029-B3 supplied with Denaturation Buffer. Right: ME029-C1 supplied with Low pH MLPA Buffer.

Reasons behind this product change

The FMR1 and AFF2 regions targeted in SALSA® MLPA® Probemix ME029-C1 FMR1-AFF2 are very difficult to denature, especially in DNA samples containing long trinucleotide expansions. For this reason, an extra Denaturation Buffer was always supplied with previous versions of the ME029 FMR1-AFF2 probemix. However, it was observed that extremely long trinucleotide repeats would not always fully denature even when using the Denaturation Buffer.

To improve this, the use of 60 mM NaOH solution (freshly prepared by the user) now replaces the use of the ME029 FMR1-AFF2 Denaturation Buffer. More details on the NaOH solution can be found on page 2 of the ME029 FMR1-AFF2 product description. At the start of the ME029 MLPA reaction, sample DNA should be mixed with this 60 mM NaOH solution.

As the addition of NaOH to DNA samples increases pH, potentially reducing ligase activity, MRC Holland will include a low-pH version of the MLPA Buffer (purple cap) with each ME029-C1 order to counterbalance this effect. This Low pH MLPA Buffer should always and exclusively be used with the ME029-C1 probemix, instead of the normal MLPA Buffer (yellow cap) that is included in MLPA reagent kits.

Transition period

We understand that transitioning to a new product version and protocol may require some preparation on your side. Hence, it will be possible to acquire the older ME029-B3 version including the Denaturation Buffer, in reasonable quantities while stocks last, until 29 September 2023. For inquiries regarding ordering the older ME029-B3 version, please contact By default, the new version of the ME029 probemix will be provided if not otherwise specified.

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