Please use this identifier to cite or link to this item:
|Title:||A mechanistic basis for amplification differences between samples and between genome regions|
|Authors:||Veal, Colin D.|
Freeman, Peter J.
|Publisher:||BioMed Central Ltd|
|Citation:||BMC Genomics, 2012, 13:455|
|Abstract:||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.|
|Rights:||Copyright © 2012 Veal et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.|
|Appears in Collections:||Published Articles, Dept. of Genetics|
Files in This Item:
|10.1186_1471-2164-13-455.pdf||Published (publisher PDF)||442.6 kB||Adobe PDF||View/Open|
Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.