• A new protocol that allows to efficiently co-amplify a large number of loci in the same PCR reaction (multiplex PCR), to generate material that is ready for sequencing without further manipulations
• A two-step procedure, simultaneously amplifying over 30 different and independent markers, hence substantially increasing the sensitivity and specificity of the information acquired
• Successfully used on bisulfite-treated, small volume plasma samples
Project ID : 6-2018-5690
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