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Reduced sloping with the new MLPA buffer

Jan 21, 2019

From March 2019 onwards, all shipments of SALSA MLPA Reagent Kits1 will include a modified SALSA MLPA Buffer. There will also be a small change in the storage buffer of the SALSA Ligase-65 enzyme. These modifications make your MLPA experiments more robust.

Main advantages

The new MLPA buffer strongly reduces sloping that is frequently observed when DNA is extracted by a magnetic bead-based system. Sloping is a characteristic pattern with lower peak heights for larger fragments, which can lead to variation or the potential loss of signals. If you used the S4 Sample Stabiliser for this purpose in the past, you may no longer need to add this reagent2.

Figure: Electropherograms from reactions on the same male DNA extracted with a system based on magnetic beads. Left: Using the old MLPA buffer and Ligase-65. Right: Using the new MLPA buffer and Ligase-65.

The new MLPA buffer will not eliminate other causes of sloping.

What will change?

The new MLPA buffer contains the antioxidant dithiothreitol (DTT). This counters sloping caused by iron ions in the sample DNA, which is a common impurity in DNA samples extracted with magnetic bead-based systems.

The Ligase-65 enzyme remains unchanged, but the β-mercaptoethanol antioxidant in its storage buffer will be replaced by DTT because this is less pungent and less toxic.

The new MLPA buffer and Ligase-65 vials can be recognized by a ‘new’ sticker that will be placed on the caps until the end of 2019. The MLPA protocol has not changed, but old and new reagents cannot be used together in the same experiment.

Why are we making this change?

We noticed that iron-induced sloping has become a more frequent issue due to the popularity of magnetic bead-based extraction systems. In 2015, we introduced the S4 Sample Stabiliser to reduce this type of sloping. Customer feedback confirmed that the S4 Sample Stabiliser greatly reduces, or completely eliminates, iron-induced sloping. With the addition of one of the active ingredients of the S4 Sample Stabiliser (DTT) to the MLPA buffer, this optimization will be freely available to all our customers without the need for an extra pipetting step. The new MLPA buffer can also be used together with probemixes registered for in vitro diagnostic use, while the S4 Sample Stabiliser is for research use only.

Validation

The new reagents have been tested extensively by MRC-Holland and by external laboratories on samples extracted by bead-based methods and non-bead based methods under various conditions and with multiple probemixes. No adverse effects were found. For more details, see this validation summary.

Timeline

  • March – May 31st 2019: All reagent kits (e.g. EK1, EK5) contain both the old and new MLPA buffers and Ligase-65 solutions. Vials of the old MLPA buffer and Ligase-65 are supplied in a separate box (free of charge).
  • June – August 31st 2019: Old reagents are no longer supplied with each reagent kit by default, but are still available (free of charge) with every order of a reagent kit on request. Use reference SMR32 to order the old MLPA buffer, and SMR11 to order the old Ligase-65 solution. Please explicitly mention "old version" to avoid confusion.
  • March – August 31st 2019: Vials of old MLPA buffer and/or Ligase-65 solution can be exchanged for new MLPA buffer and/or Ligase-65 (free of charge). Mention the number of vials of the new reagents that you would like to receive when you order.

Changes to MLPA

We are aware that this change in MLPA reagents may require an additional validation effort for certain laboratories, for which we sincerely apologize. MRC Holland constantly aims to find a balance between making improvements and minimizing inconvenience caused by changes. In this case, we are convinced that the long-term benefits of introducing the new MLPA buffer and Ligase-65 will outweigh the short-term inconvenience. Please do not hesitate to contact us in case we can be of any assistance in successfully making this transition.


1 For information about changes to Ligase-65 kits, see this page.

2 Future of the S4 Sample Stabiliser: With the introduction of the new MLPA Buffer, it is no longer necessary to add the S4 Sample Stabiliser to reactions to counter iron-induced sloping. However, addition of the stabiliser may still be useful to reduce variation in other situations. Therefore, the S4 Sample Stabiliser will remain available as a separate item.

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