Ancient DNA extraction from bones and teeth

Abstract

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 1C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days. INTRODUCTION RationaleSince its beginning, research on ancient DNA has suffered from the problem that, in almost all ancient specimens, any DNA that is preserved is present only in small amounts and in various states of degradation. Therefore, it is crucial to make this DNA available as much as possible for further enzymatic manipulation. This usually involves amplification by PCR, but more recent approaches also include ligation into bacterial vectors 1,2 or addition of oligonucleotides for direct sequencing 3-5 . However, this is not a trivial task. First, most DNA extraction methods are designed to deal with fresh tissue containing intact cells and high molecular weight DNA. In ancient specimens, usually no cell structures are preserved (but see ref. 6 for rare exceptions) and, owing to as yet uncharacterized chemical modifications, it may even be difficult to get the DNA into aqueous solution 7 . Second, ancient DNA is damaged in various ways [8][9][10][11] , so extraction methods also have to avoid overly aggressive treatments, such as high temperatures or use of strong detergents 12 . Although these treatments might increase DNA release, they would decrease overall DNA yield by inflicting further damage to the ancient DNA molecules. Third, ancient bones and teeth often contain large amounts of PCR inhibitors 13-15 that interfere with DNA amplification and are co-purified with ancient DNA. Thus, ancient DNA extraction methods have to deal with a number of problems that are sometimes difficult to reconcile. The method described here is the result of testing, on a number of Pleistocene samples, a wide range of conditions and ingredients from one published method 16 , followed by a final comparison of the optimized procedure to other published ancient DNA extraction methods 12 . It provides a trade-off between DNA release, DNA degradation during extraction and separation of DNA and inhibitors, thereby maximizing the DNA available for further applications. We have applied it suc...

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy)

“…The two laboratories involved (one in Florence and one in Barcelona; SOM) adhered to standard criteria and precautions for the analysis of ancient samples (Handt et al, 1994;Cooper and Poinar, 2000). For DNA extractions, three powder aliquots were globally used, as described in Rohland and Hofreiter (2007). Multiple PCR amplifications with multiple primer pairs that cover the entire hypervariable region I (HVR-I) of mitochondrial DNA (mtDNA; Caramelli et al, 2006) were performed as described in the SOM.…”

The origins of the enigmatic Falkland Islands wolf

“…DNA was extracted using a modified silica-based method 29 designed to maximize recovery of PCR-amplifiable DNA from ancient bone and tooth specimens while minimizing coextraction of PCR inhibitors.…”

Complete mitochondrial DNA genome sequences from the first New Zealanders

“…Because the material was part of a museum collection and likely to have been extensively handled, the teeth were submerged in 5% bleach (wt/vol) for a minimum of 15 min (32) and rinsed in ultrapure water to reduce surface contamination before grinding for DNA extraction. Teeth were ground by using a sterile mortar and pestle, and 160-550 mg of dentin was used for DNA extraction following the silica based extraction protocol (33). Extractions were conducted in batches of five samples and one extraction blank (repeat extractions account for the total number of samples not being a multiple of five).…”