Logo MRC Holland
  • Products
  • Technology
  • About Us
  • News & Events
  • Support
  • Sign in
    • Products
      • Reagents
      • How to Order
      • Order Form
    • Technology
      • MLPA
      • digitalMLPA
      • Melt Assays
      • Software
    • About Us
      • Our Mission
      • Foundation
      • Careers
      • Contact
    • News & Events
      • Events
      • News Archive
    • Support
      • Help Centre
      • Downloads
      • Contact Us
BSI Assurance Mark - ISO 13485
  • Help Centre
  • Downloads
  • Contact Us
  • Downloads Browse our downloads
  • Contact Us We are here to help
  • Support
  • Help Centre
  • MLPA & Coffalyser.Net
  • Experimental Setup
  • Sample Selection & Requirements
  • What is sample depurination and how do I prevent it?

What is sample depurination and how do I prevent it?

in Sample Selection & Requirements
  • MLPA
  • digitalMLPA

To prevent sample depurination, it is essential that DNA samples contain sufficient buffering capacity during the initial denaturation step of the conventional MLPA and digitalMLPA protocols. The presence of at least 5 mM Tris-HCl pH 8.0–8.5 in a sample is sufficient to prevent depurination.

In a conventional MLPA reaction, if it is not known what the sample DNA is dissolved in, we recommend that 1 µl of 50 mM Tris-HCl pH 8.5 is added to 4 µl of sample DNA.

Background

During (digital)MLPA reactions depurination can occur during the initial denaturation step if the sample DNA is in a solution with insufficient buffering capacity or a low pH (e.g. water). Certain sample treatments (e.g. prolonged heating, formalin fixation/paraffin embedding) can also result in depurination of the sample DNA, even with sufficient buffering capacity. Problems are more likely to arise when these factors are combined.

Depurination of DNA occurs when a purine base (adenine (A) or guanine (G)) is removed from the DNA deoxyribose backbone via hydrolysis. This results in a deoxyribose lacking a purine base, which is known as an abasic or apurinic site.

Depurination at the ligation site of a probe, especially if it has a large number of pyrimidines (cytosine (C), or thymine (T)), will result in inefficient probe hybridization and subsequent limited or failed probe ligation, as these pair with the purines (G, A) that are lost during depurination. As a consequence, the probe signal will be reduced or even absent. Loss of probe hybridization/ligation also leads to variability in the obtained probe signals.

Note
All digitalMLPA probemixes contain specific control probes that are used by Coffalyser digitalMLPA to provide a warning when the DNA is depurinated. These control probes are not present in conventional MLPA probemixes.

The figure below shows a schematic representation of a probe with high pyrimidine content around the ligation site (i.e. purines present in the target DNA). In panel A, the probe hybridises successfully to the sample DNA, which in this case is unaffected by depurination. In panel B, the same probe is hybridised to sample DNA that is heavily affected by depurination: the guanines and adenines of the target on the sample DNA have all been lost. The result is a very poor probe hybridization, leading to an inefficient or failed ligation of the probe oligonucleotides, ultimately causing a reduced or even absent probe signal.

Related Pages

  • Is there a specific DNA extraction method I need to use for (digital)MLPA samples?

Rate the quality of this page

This page was helpful :) :( This page was not helpful
If you have any comments or suggestions on this article, please send us your feedback.

Disclaimer

The information provided in this material is correct for the majority of our products. However, for certain applications, the instructions for use may differ. In the event of conflicting information, the relevant instructions for use take precedence.


Quick Jump
  • « MRCHolland.com
  • Help Centre
  • Downloads
  • Contact Us
Top
Logo MRC-Holland
  • Products
    • Reagents
    • How to Order
    • Order Form
  • Technology
    • MLPA
    • digitalMLPA
    • Melt Assays
    • Software
  • About Us
    • Our Mission
    • Foundation
    • Careers
    • Contact
  • News & Events
    • Events
    • News Archive
  • Support
    • Help Centre
    • Downloads
    • Contact Support
    • BSI Assurance Mark - ISO 13485
  • © MRC Holland. All rights reserved.
  • Privacy Policy.
  • Website designed and developed by Schuttelaar & Partners.