To prevent sample depurination it is essential that sample DNAs contain sufficient buffering capacity during the five-minute 98°C denaturation step. The presence of at least 5 mM Tris-HCl pH 8.0–8.5 in the sample DNA is sufficient to prevent depurination. If it is not known what a 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.
|This article contains a summary of the information in this document about depurination.|
During MLPA reactions depurination can occur during the initial five minute 98°C denaturation step if the sample DNA is in a solution with insufficient buffering capacity or a low pH (e.g. water). Sample treatments (e.g. prolonged heating, formalin fixation/paraffin embedding) can also result in depurination of your 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.
The figure below shows a schematic representation of an MLPA 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.