DNA amplification & quantification

The PCR thermocycler amplifies fragments of genomic DNA during the polymerase chain reaction. Diagnostic gene fragments (genetic markers) for taxon identification or characterization of new genetic diversity are amplified in extractions from recent and ancient sedimentary DNA or from organismic material. This step is crucial for the metabarcoding approach.

Library preparation of the entire sedaDNA and quantitative PCR (qPCR) are central to paleometagenomics. qPCR allows for the relative quantification of DNA, which is used to determine the optimal number of amplification cycles for the DNA libraries. This significantly contributes to the reduction of duplicates and improves the comparability of the sedaDNA Next-Generation Sequencing results.

After extraction and/or amplification we can measure the quantity and quality of DNA. The Qubit 4 is a benchtop fluorometer designed to accurately measure concentration of DNA solutions. The 4200 TapeStation System is applied to measure the fragment length distribution of a DNA mixture. The quantity and fragment length (quality) are needed to calculate the input for PCR reactions and the DNA molarity which is crucial for subsequent DNA Next-Generation sequencing.