Comparison and optimization of ancient DNA extraction.
Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.
Rohland
Nadin
N
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. rohland@eva.mpg.de
Hofreiter
Michael
M
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002021:Buffers; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D005580:Fossils; D006863:Hydrogen-Ion Concentration; D010163:Paleontology; D016133:Polymerase Chain Reaction / Q000379:methods; D012492:Salts / Q000494:pharmacology; D017422:Sequence Analysis, DNA / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D001503:Ursidae
D002021:Buffers; D012492:Salts; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D004247:DNA; D004492:Edetic Acid; D019286:Endopeptidase K