Background

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.

Our Innovation

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

Opportunity

  • 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