The Methylamp™ MS-qPCR Fast Kit is a complete set of essential components, which enables the experimenter to perform fast, specific, sensitive and reproducible methylation-specific quantitative PCR. The Methylamp™ MS-qPCR Fast Kit is very suitable for quantitative methylation-specific PCR in a fast format using very minute amounts of DNA. The novel hot start DNA polymerase contained in this kit can specifically reduce the overall time required for MS-qPCR from approximately 2.5 hours to less than 70 minutes. The kit also facilitates sensitivity and specificity of MS-qPCR by significantly increasing primer-bisulfite DNA template annealing, while simultaneously reducing non-specific annealing. This results in considerable time savings and a more efficient MS- qPCR. The kit has the following advantages:

• An extremely fast procedure that can be finished within 70 minutes.
• Abundant yields due to high amplification efficiency.
• Highly accurate and specific in MSP, with reduced false- positive results.
• Convenient master mix format to allow easy reaction setup.
• Simple, reliable, and consistent assay conditions.
• Can be used with any block-based real-time PCR instrument.

Principle & ProcedureThe Methylamp™ MS-qPCR Fast Kit provides a master mix format, which contains a hot start DNA polymerase, dNTPs, an MS-PCR enhancer, an optimized buffer, and an intercalating green dye. This master mix allows for a convenient and easy reaction setup. The unique hot start DNA polymerase is provided in an inactive state at ambient temperature and is reactivated by several minute incubations at 95°C, which can easily be integrated into existing thermal cycling steps. The hot start DNA polymerase in combination with the optimized buffer ensures the MS-qPCR specificity and sensitivity. The green dye allows for DNA detection and analysis without using a sequence-specific probe.

Fig. 1. Schematic procedure for using the Methylamp MS-qPCR Fast Kit.

Fig. 2.MS-PCRamplification of a gene region in euchromatin with internal control primers. Successful GpC methylation and bisulfite conversion were carried out using anEpiQuik Chromatin Accessibility & Methylome Sequencing (CAMe-Seq) Kit (Cat. No. P-1048) and PCR was performed using Methylamp™ MS- qPCR Fast Kit (Cat. No. P-1028).