Journal: BMC Genomics

Article Title: Digital PCR provides sensitive and absolute calibration for high throughput sequencing

doi: 10.1186/1471-2164-10-116

Figure Lengend Snippet: A Schematic of the universal template (UT) PCR assay . The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.

Article Snippet: PCR reaction mix containing the diluted template was loaded onto Fluidigm's 12.765 Digital Array microfluidic chip.

Techniques: Sequencing, Labeling, Activity Assay, Digital PCR, Concentration Assay