PCR using bisulfite-treated DNA as a template, which is followed by next generation sequencing (NGS), has a major potential as a diagnostic tool, by identifying DNA derived from specific tissue based on cell type-specific methylation markers.
To maximize assay sensitivity, multiple loci have to be amplified in parallel.
However, multiplex PCR of bisulfite-converted DNA remains a major challenge.
A new protocol that allows to efficiently co-amplify a large number of loci after bisulfite conversion (multiplex PCR), to generate material that is ready for sequencing without further manipulation.
simultaneously amplifying over 30 different and independent methylation markers
Increased specificity and sensitivity: detects low levels of strongly diluted differentially methylated loci
Compatibility with smaller amount of available bisulfite-converted template
Reduced costs and time of procedure
Most important tool for simultaneous detection of a large number of markers of the same tissue/state, increasing sensitivity and specificity of the test while reducing overall reagent and labor costs
Method validated in multiple studies, including reactions using genomic DNA and second bisulfite-converted cfDNA - the most challenging template
Allows for routine, affordable non-invasive detection of tissue specific cell death in the human body