Abstract
Detection of low copies of methylated DNA targets in clinical specimens is challenging. The quantitative Methylation-Specific PCR (qMSP) assays were designed to specifically amplify bisulphite-converted methylated DNA target sequences in the presence of an excess of unmethylated counterpart sequences. These qMSP assays are real-time PCR assays utilizing, sequence-specific primers and an intervening, also sequence specific, Taqman probe to cover an amplicon of approximately 100 bp in length. The use of Taqman probes bearing a minor groove binding (MGB) allow for the use of shorter probes and therefore facilitate design and significantly increases the analytical specificity of the reaction. In the context of the biomarker discovery program of the Liverpool Lung Project (LLP), ten gene promoters were selected. qMSP assays were developed, validated and used to screen 655 bronchial washings from patients with lung cancer and age/sex matched controls with non malignant lung disease (Nikolaidis et al., 2012).
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol is adapted from Nikolaidis et al. (2012).
References
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