PMID: identifiant
de la référence Pubmed : |
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 (22950736)  |
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A mechanistic basis for amplification differences between samples and between genome regions. |
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| auteur(s) : |
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Colin Veal1, Peter Freeman1, Kevin Jacobs2, 3, Owen Lancaster1, Stéphane Jamain4, 5, Marion Leboyer4, 5, Demetrius Albanes2, Reshma Vaghela1, Ivo Gut6, Stephen Chanock2, Anthony Brookes ( ) 1 |
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| laboratoire : |
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| résumé : |
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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. |
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| domaine : |
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Sciences du Vivant/Biochimie, Biologie Moléculaire/Génomique, Transcriptomique et Protéomique
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langue du texte
intégral : |
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Anglais |
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| ISSN : |
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1471-2164 |
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| type de publication : |
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Articles dans des revues avec comité de lecture |
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| DOI : |
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10.1186/1471-2164-13-455 |
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| journal : |
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| Audience : |
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internationale |
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| date de publication : |
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05/09/2012 |
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date de publication
électronique : |
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05/09/2012 |
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| volume : |
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13 |
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| numéro : |
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1 |
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| page, identifiant, ... : |
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455 |
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| mots-clés auteur : |
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DNA amplification – DNA denaturation – C + G – Illumina infinium |
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| contrat, financement : |
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This research was supported by Action Medical Research (grants SP4139 and SP4483) and by the European Union's Seventh Framework Programme (FP7/ 2007-2013) project READNA (grant agreement HEALTH-F4-2008-201418). |
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| Projet Européen : |
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| Numéro Cordis |
201418 |
| Acronyme |
READNA |
| Titre |
REvolutionary Approaches and Devices for Nucleic Acid Analysis |
| Financé par |
HEALTH |
| Début |
2008-06-01 |
| Date de fin |
2012-05-31 |
| Identifiant de l'appel |
FP7-HEALTH-2007-A |
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