title: A mechanistic basis for amplification differences between samples and between genome regions.
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.
authors.affiliation: Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: Colin D
authors.initials: CD
authors.lastname: Veal
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Freeman
authors.forename: Kevin
authors.initials: K
authors.lastname: Jacobs
authors.forename: Owen
authors.initials: O
authors.lastname: Lancaster
authors.forename: Stéphane
authors.initials: S
authors.lastname: Jamain
authors.forename: Marion
authors.initials: M
authors.lastname: Leboyer
authors.forename: Demetrius
authors.initials: D
authors.lastname: Albanes
authors.forename: Reshma R
authors.initials: RR
authors.lastname: Vaghela
authors.forename: Ivo
authors.initials: I
authors.lastname: Gut
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Chanock
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Brookes
mesh_terms: D001482:Base Composition / Q000235:genetics; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*