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Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing

Shaw, Richard J.; Akufo-Tetteh, Emily K.; Risk, Janet M.; Field, John K. and Liloglou, Triantafillos (2006) Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing. Nucleic Acids Research, 34 (11). p. 78. ISSN 1362-4962 (online); 0305-1048 (print)

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Official URL: http://nar.oxfordjournals.org/

Cited 29 times in WoS

Abstract

It has been suggested that detection of aberrant DNA methylation in clinical specimens such as sputum or saliva may be a valuable tumour biomarker. Any clinically applicable detection technique must combine high sensitivity with high specificity. In this study we describe methylation enrichment pyrosequencing (MEP), which benefits from the high sensitivity and specificity of methylation specific PCR (MSP) but has a second, confirmatory, pyrosequencing step. The pyrosequencing reaction is rapid, relatively inexpensive and offers significant logistical advantages over previously described validation methods. As proof of principle, we illustrate MEP using assays of p16 and cyclin A1 promoters in a methylated DNA dilution matrix and also in a clinical setting using paired saliva and oral tumour specimens. Our results confirm that mis-priming of MSP, with subsequent false positive results, can occur frequently (perhaps 10%) in assays combining high numbers of PCR cycles and low concentrations of starting DNA. In our clinical example, MEP of saliva-derived DNA was more sensitive than standard non-methylation specific pyrosequencing as illustrated using p16 and cyclin A1 promoter methylation assays.

Item Type:Article
Additional Information:Published Online June 28, 2006.
Uncontrolled Keywords:NECK-CANCER PATIENTS; PROMOTER HYPERMETHYLATION; CPG ISLANDS; TUMORS; HEAD; P16; PCR
Subjects:R Medicine > RK Dentistry
Departments, Research Centres and Related Units:Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Dental Sciences
DOI:10.1093/nar/gkl424
Publisher's Statement:© 2006 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Refereed:Yes
Status:Published
ID Code:92
Deposited On:09 Oct 2007 13:31
Last Modified:20 May 2011 19:43

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