755a0bb6-4577-4c1d-99a5-73555118dedc
Development and optimization of a silica column-based extraction protocol for ancient DNA
Rapid and cost-effective retrieval of endogenous DNA from ancient specimens remains a limiting factor in palaeogenomic research. Many methods have been developed to increase ancient DNA yield, but modifications to existing protocols are often based on personal experience rather than systematic testing. Here we present a new silica column-based extraction protocol, where optimizations were tested in controlled experiments. Using relatively well-preserved permafrost samples, we tested the efficiency of pretreatment of bone and tooth powder with a bleach wash and a predigestion step. We also tested the recovery efficiency of MinElute and QIAquick columns, as well as Vivaspin columns with two molecular weight cut-off values. Finally, we tested the effect of uracil-treatment with two different USER enzyme concentrations. We find that neither bleach wash combined with a predigestion step, nor predigestion by itself, significantly increased sequencing efficiency. Initial results, however, suggest that MinElute columns are more efficient for ancient DNA extractions than QIAquick columns, whereas different molecular weight cut-off values in centrifugal concentrator columns did not have an effect. Uracil treatments are effective at removing DNA damage even at concentrations of 0.15U/µL (as compared to 0.3U/µL) of ancient DNA extracts.
Rapid and cost-effective retrieval of endogenous DNA from ancient specimens remains a limiting factor in palaeogenomic research. Many methods have been developed to increase ancient DNA yield, but modifications to existing protocols are often based on personal experience rather than systematic testing. Here we present a new silica column-based extraction protocol, where optimizations were tested in controlled experiments. Using relatively well-preserved permafrost samples, we tested the efficiency of pretreatment of bone and tooth powder with a bleach wash and a predigestion step. We also tested the recovery efficiency of MinElute and QIAquick columns, as well as Vivaspin columns with two molecular weight cut-off values. Finally, we tested the effect of uracil-treatment with two different USER enzyme concentrations. We find that neither bleach wash combined with a predigestion step, nor predigestion by itself, significantly increased sequencing efficiency. Initial results, however, suggest that MinElute columns are more efficient for ancient DNA extractions than QIAquick columns, whereas different molecular weight cut-off values in centrifugal concentrator columns did not have an effect. Uracil treatments are effective at removing DNA damage even at concentrations of 0.15U/µL (as compared to 0.3U/µL) of ancient DNA extracts.