Hi all,
We have recently published a paper that provides insight into why different samples (or even different preps of the same sample) can have varying success for methods that involved DNA amplification such as WGA and PCR. The details are below and hopefully it will be useful to some of you out there.
all the best
Colin
http://www.biomedcentral.com/1471-2164/13/455
A mechanistic basis for amplification differences between samples and between genome regions
Background
For many analytical methods the efficiency of DNA amplification varies across the genome and between samples. The most affected genome regions tend to correlate with high C + G content, however this relationship is complex and does not explain why the direction and magnitude of effects varies considerably between samples.
Results
Here, we provide evidence that sequence elements that are particularly high in C + G content can remain annealed even when aggressive melting conditions are applied. In turn, this behavior creates broader ‘Thermodynamically Ultra-Fastened’ (TUF) regions characterized by incomplete denaturation of the two DNA strands, so reducing amplification efficiency throughout these domains.
Conclusions
This model provides a mechanistic explanation for why some genome regions are particularly difficult to amplify and assay in many procedures, and importantly it also explains inter-sample variability of this behavior. That is, DNA samples of varying quality will carry more or fewer nicks and breaks, and hence their intact TUF regions will have different lengths and so be differentially affected by this amplification suppression mechanism – with ‘higher’ quality DNAs being the most vulnerable. A major practical consequence of this is that inter-region and inter-sample variability can be largely overcome by employing routine fragmentation methods (e.g. sonication or restriction enzyme digestion) prior to sample amplification.
-colinveal-
does this mean you want us to cite you?
-Curtis-
Well I find it interesting. But most problems with template variability (though intra in ths case) I've seen in CHIP samples, which are all sonicated to the same degree, immunoprecipitated and purified and yet I often can get very different results between replicates. Of yourse one of the reasons is high Ct, but the variability is higher even than it should be in that case. Also insuficient purification can play role, for sure, but I'm personaly very curious about the reasons and it would be interesting if someone found out.
-Trof-
