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Open Access Research article

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

Colin D Veal1, Peter J Freeman1, Kevin Jacobs23, Owen Lancaster1, Stéphane Jamain456, Marion Leboyer456, Demetrius Albanes2, Reshma R Vaghela1, Ivo Gut7, Stephen J Chanock2 and Anthony J Brookes1*

Author affiliations

1 Department of Genetics, University of Leicester, Leicester, LE1 7RH, UK

2 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, 20892-7335, USA

3 Core Genotyping Facility, National Cancer Institute, SAIC-Frederick Inc., Gaithersburg, MD, USA

4 INSERM U 955, Psychiatrie Génétique, Créteil, 94000, France

5 Université Paris Est, Faculté de Médecine, Créteil,, 94000, France

6 AP-HP, Hôpital H. Mondor – A. Chenevier, Département de Psychiatrie, Créteil,94000, France

7 Centre Nacional d'Analisi Genomica, Barcelona, 08028, Spain

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Citation and License

BMC Genomics 2012, 13:455  doi:10.1186/1471-2164-13-455

Published: 5 September 2012

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.

Keywords:
DNA amplification; DNA denaturation; C + G; Illumina infinium