Published online 28 June 2006
© 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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Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing
1 Molecular Genetics and Oncology Group, School of Dental Sciences, University of Liverpool Liverpool L69 3GN, UK 2 University of Liverpool Cancer Research Centre, Roy Castle Lung Cancer Research Programme 200 London Road, Liverpool L3 9TA, UK 3 Regional Maxillofacial Unit, University Hospital Aintree Longmoor Lane, Liverpool L9 7AL, UK
*To whom correspondence should be addressed. Tel: +44 151 706 5265; Fax: +44 151 706 5809; Email: j.m.risk{at}liverpool.ac.uk
Received April 5, 2006. Accepted May 28, 2006.
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.
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