A mechanistic basis for amplification differences between samples and between genome regions

BMC Genomics

Research Article

Ivo Gut¹  Reshma R Vaghela²  Owen Lancaster²  Colin D Veal²  Peter J Freeman²  Anthony J Brookes²  Demetrius Albanes³  Stephen J Chanock³  Kevin Jacobs⁴  Stéphane Jamain⁵  Marion Leboyer⁵ 

[1] Centre Nacional d'Analisi Genomica, 08028, Barcelona, Spain;Department of Genetics, University of Leicester, LE1 7RH, Leicester, UK;Division of Cancer Epidemiology and Genetics, National Cancer Institute, 20892-7335, Bethesda, USA;Division of Cancer Epidemiology and Genetics, National Cancer Institute, 20892-7335, Bethesda, USA;Core Genotyping Facility, National Cancer Institute, SAIC-Frederick Inc., Gaithersburg, MD, USA;INSERM U 955, Psychiatrie Génétique, 94000, Créteil, France;Université Paris Est, Faculté de Médecine, 94000, Créteil, France;AP-HP, Hôpital H. Mondor – A. Chenevier, Département de Psychiatrie, 94000, Créteil, France;

关键词: DNA amplification;    DNA denaturation;    C + G;    Illumina infinium;   

DOI  :  10.1186/1471-2164-13-455

 received in 2012-05-01, accepted in 2012-08-28,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundFor 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.ResultsHere, 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.ConclusionsThis 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.

【 授权许可】

Unknown   
© Veal et al.; licensee BioMed Central Ltd. 2012. This article is published under license to 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.