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

Colin D Veal, Peter J Freeman, Kevin Jacobs, Owen Lancaster, Stéphane Jamain, Marion Leboyer, Demetrius Albanes, Reshma R Vaghela, Ivo Gut, Stephen J Chanock, Anthony J Brookes

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalBMC Genomics
Issue number455
Publication statusPublished - 5 Sept 2012


  • Base Composition/genetics
  • Genome, Human/genetics
  • Humans
  • Nucleic Acid Amplification Techniques/methods
  • DNA amplification
  • DNA denaturation
  • C + G
  • Illumina infinium


Dive into the research topics of 'A mechanistic basis for amplification differences between samples and between genome regions'. Together they form a unique fingerprint.

Cite this