Development and Optimization of a Silica Column-Based Extraction Protocol for Ancient DNA
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Development and Optimization of a Silica Column-Based Extraction Protocol for Ancient DNA. / Dehasque, Marianne; Pečnerová, Patrícia; Kempe Lagerholm, Vendela; Ersmark, Erik; Danilov, Gleb K.; Mortensen, Peter; Vartanyan, Sergey; Dalén, Love.
In: Genes, Vol. 13, No. 4, 687, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Development and Optimization of a Silica Column-Based Extraction Protocol for Ancient DNA
AU - Dehasque, Marianne
AU - Pečnerová, Patrícia
AU - Kempe Lagerholm, Vendela
AU - Ersmark, Erik
AU - Danilov, Gleb K.
AU - Mortensen, Peter
AU - Vartanyan, Sergey
AU - Dalén, Love
PY - 2022
Y1 - 2022
N2 - 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 0.15 U/µL (as compared to 0.3 U/µL) of ancient DNA extracts.
AB - 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 0.15 U/µL (as compared to 0.3 U/µL) of ancient DNA extracts.
KW - Bone and Bones
KW - DNA/genetics
KW - DNA, Ancient
KW - Silicon Dioxide
KW - Uracil
U2 - 10.3390/genes13040687
DO - 10.3390/genes13040687
M3 - Journal article
C2 - 35456493
VL - 13
JO - Genes
JF - Genes
SN - 2073-4425
IS - 4
M1 - 687
ER -
ID: 308891795