Several dna extractions were performed for each specimen as shown in table 1. We describe the effects of dna extraction on the size distribution of sequences obtained from highthroughput sequencing, patterns of dna degradation, library yields, as well as the co extraction of inhibitory substances. A study of postmortem degradation of teeth to advance. Ancient dna as characterized by hummel et al 29 1993 obtained from human remains is, in general, severely damaged chemically or physically. But even in a lab where once outlandish dreams were coming to pass, her project seemed a long shot. This film was produced by external relations, university of. The dramatic degradation of the dna and the presence of pcr inhibitors in the. Briefly, bone powder was digested overnight, and dna was extracted with phenolchloroformisoamyl alcohol, purified and concentrated on microcolumns ultracel ym 100, microcon to 15.
If powder is remaining in the tube, simply transfer the supernatant. Ancient dna extraction from bones and teeth nature protocols. Apr 03, 2018 here, we present an evaluation of three pretreatment methods for the removal of dna contamination from ancient bones and teeth. Sampling, pretreatment of bone powder, and dna extraction. Fernandes1,2, kendra sirak1,3 and olivia cheronet 1 the cortical bone that forms the structure of the cochlea, part of the osseous labyrinth of the inner ear, is now one of the. The dna extraction was performed according to the max plank institute bone or teeth dna extraction,, which is based on the using of silica combined with guanidine thiocyanate gnscn. The tooth will provide ancient dna that will subsequently identify the body as that of king richard iii. Strict protocols were followed to minimize the amount of human dna in the ancient dna laboratory, including the wearing of freshly laundered clothes, a full body suit, shoe covers, boots, facemask, face shield and triple gloving, and no personnel movement from the postpcr environment to the ancient dna laboratory within a single day. Ancient dna, dna extraction, bones, teeth, museumspecimen, silica, column the most abundant faunal remains are partial. And, although this information is very useful, it is more limited in its scope than that which could be obtained from nuclear dna. Ancient dna adna refers to the study of dna extracted from specimens that died decades, hundreds or sometimes thousands of years ago. The hunt for dna indiana jane must have permission to work with each bone.
The dna extraction process varies according to the source material. The extraction and analysis of ancient dna from bone and. In recent studies, pcr has been shown to be an essential tool for the analysis of ancient dna, as it can be used for degraded and chemically modified dna samples. We report here on a mobile laboratory that we setup using commercially available devices, including a compact realtime pcr machine, and describe procedures to perform dna extraction. Feb 04, 20 the tooth will provide ancient dna that will subsequently identify the body as that of king richard iii. Jun 30, 2011 nuclear dna from bones at different states of degradation was isolated using three methods. Comparing ancient dna preservation in petrous bone and. Isolating the human cochlea to generate bone powder for. Alkalisensitive sites in dna can be caused by apurinicapyrimidinic ap sites as well as by modifications of.
A phenolchloroform protocol for extracting dna from. Dna extraction this protocol was adapted from imaizumi et al. Ancient dna extraction from bone was performed following rohland et al. Comparison of the presence of taq inhibitors in the dna extract purified by the previously published isopropanolbased and the new carriermediated methods and determination of the degree of degradation of extracted ancient bone dna. Isolation of nucleic acid from hard tissues bone or teeth 1. Ancient dna extraction laboratory idaho state university.
If polymerase chain reaction pcr amplification starts from a poor extract containing low template quantities, stochastic variation in the amplification of individual alleles may lead to allelic dropout, resulting in a high risk of falsehomozygous typing of a heterozygous. This laboratory protocol or part thereof has been provided. When applied to bones soaked, burned or buried for up to. Most of the current dna extraction protocols for bones and teeth are based on the incubation of powdered material in an ethylene diamine tetraacetic acid edtacontaining extraction buffer. Extraction of dna from human skeletal material springerlink. Issn 586122 pdf published version restricted to repository staff only download 2mb. Nuclear dna study in ancient human bones and teeth. There is a class 100 positive pressure hood and one exhaust hood in the room. Ancient dna research really gained momentum following the invention of pcr, which allowed millions of copies to be made of the few remaining dna molecules preserved in fossils and museum specimens.
Ancient dna extraction from bones and teeth molecular ecology group, max planck institute this method is designed to maximize recovery of pcramplifiable dna from ancient bone and teeth specimens and at the same time to minimize co extraction of substances that inhibit pcr. Largescale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. If necessary, moisten the tissue with hplcgrade water. The use of this protocol entails preparing a set of solutions that will be used throughout the process an extraction or lysis. Twentynine bones and teeth from museum specimens were analyzed. Although founded more than 20 years ago, the field of ancient dna research continues to grow and expand into new areas 3,4. With her age established, scientists then sought to extract her dna from her molars. A study of postmortem degradation of teeth to advance forensic dna analysis as a tool for human identification. The human fossil bones and teeth of 27 individuals from the early iron age, dating from the 10th 7th century b. Ancient dna is of low average molecular size and is often damaged over time by exposure to air.
However, mtdna is susceptible to the same type of environmental degradation as nuclear dna. Samples were decontaminated by wiping the sample with qtips soaked in. Extending the spectrum of dna sequences retrieved from ancient bones and teeth isabelle glocke and matthias meyer department of evolutionary genetics, max planck institute for evolutionary anthropology, 04103 leipzig, germany the number of dna fragments surviving in ancient bones and teeth is known to decrease with fragment length. The extraction and analysis of ancient dna from bone and teeth. Subsequently, the epiphysis was separated from the diaphysis labeled as e and d, respectively.
Our data suggest that the criteria in use for authenticating adna results are not sufficient for work on dna from ancient human tissue. Benefits versus losses erika hagelberg bone dna typing, the molecular analysis of dna from skeletal material, has developed at an extraordinary rate since its beginnings in the late 1980s. The first study of what would come to be called adna was conducted in 1984, when russ higuchi and colleagues at the university of california, berkeley reported that traces of dna from a museum specimen of the quagga not only remained in the specimen over 150 years after the death of the individual, but could be extracted and sequenced. Before that time, the extraction of dna from archaeological bones, or even bones of recently deceased. These exist outside of the nucleus and have their own dna. Poor dna preservation is the most limiting factor in ancient genomic research. Early holocene chicken domestication in northern china pnas. Bone powder was removed from 14 ancient bones and 1 tooth in an ancient dna laboratory using a dentistry drill at the lowest speed. Bbc news sciencenature better dna out of fossil bones. Sep 19, 2018 a box plots of the mitochondrial to nuclear dna ratio logtransformed of human dna in petrous bones p, teeth t, other bones b and controls. Clegg1 1mrc molecular haematology unit, institute of molecular medicine, university of oxford, john radcliffe hospital, oxford ox3 9du, u. In this article, we present a novel strategy to obtain dna from bones based on the use of cetyltrimethylammonium bromide ctab lysis buffer and isoamyl alcoholchloroform extraction with subsequent dna purification using the dna iq tmsystem, or alternatively the qiaquick system.
Sep 09, 2016 teeth are a good source for ancient dna, because their enamel shell helps to preserve its delicate structure and protect it from contamination. Before the dna extraction process, fresh bones were washed with 70% ethanol and old bones were washed with 6% sodium hypochlorite and then placed under uv light for 30 minutes. Groundbreaking scientific research at the museum of london has, for the first time ever, created a detailed picture of the inhabitants of roman london. Most molecular analyses of ancient dna use pcr to amplify the number of copies of the dna. Using sandpaper or a dremel tool, remove the outer layer of tissue. Targeted polymerase chain reaction pcr was used to amplify genes present in. A thesis submitted for the degree of doctor of philosophy at the university of adelaide. Dna was extracted from whole bone powder using the classical phenolchloroform method pce in accordance with the modified protocol described by hochmeister and budowle.
Reducing microbial and human contamination in dna extractions. Even under the best preservation conditions, there is an upper boundary of 0. The results of this work have important implications for future at. Jun 17, 2015 poor dna preservation is the most limiting factor in ancient genomic research. Rinse in sterile water and dry t he bone thoroughly. Jurilovca, satu nou, babadag, niculitel and enisalapalanca. Mitochondrial dna analysis of hair, bone and teeth is particularly successful in part due to the encapsulation of dna by the exterior of the tissue and protection of mtdna within layers of keratin hair and hydroxyapatite bone and teeth. While gene flow between the diqiang and han chinese has been proposed, there is no evidence to support this view. Dna extraction from tissues known to contain preserved biomolecules e. This method is designed to maximize recovery of pcrampli.
Examples include the analysis of dna recovered from archaeological finds, museum specimens, fossil remains and other unusual specimens. High efficiency dna extraction from bone by total demineralization. Ancient dna analysis is a powerful tool to reveal the geographical origins of domesticated species. Simple protocol to extract dna from bone fragmentsa. Here we obtained ancient mtdna sequences from the earliest archaeological chicken bones from northern china as early as 10,000 y ago. Ancient dna reveals ethnicity in roman london museum of london. However, the ancient dnas were highlyalkali sensitive. In the majority of ancient bones and teeth, endogenous dna molecules represent a minor fraction of the whole dna. Pdf the extraction and analysis of ancient dna from bone.
The reduced porosity of teeth compared to bone has led to suggestions that the risk of contamination with modern human or environmental dna is lower in teeth rudbeck et al. Extending the spectrum of dna sequences retrieved from. Areas of extensive mineralization within the bone represent physical barriers to the extraction reagents and therefore prevent the release of dna molecules. Ancient dna protocols were strictly adhered to at all stages cooper and poinar, 2000. This method is designed to maximize recovery of pcramplifiable dna from ancient bone and teeth specimens and at the same. Combined analyses of our ancient sequences with a large dataset of published modern and ancient chicken. Comparison of two different dna extraction methodologies. This film was produced by external relations, university of leicester. Remove the outer surface of the sample with a grinding tool. Ancient dna extraction from bones and teeth which is necessary to dissolve part of the hydroxylapatite matrix specific to bone and teeth samples there are. The bone must be cut before it can be crushed to analyze the bone powder. Ancient diqiang people once resided in the ganqing region of china, adjacent to the central plain area from where han chinese originated. Extraction of dna from bone or teeth using the ez1 dna investigator kit.
Here are described procedures for processing the bone and tooth samples including mechanical and chemical cleaning, cutting and powdering in the presence of liquid nitrogen, complete demineralization of bone and tooth powder, dna extraction, dna purification using magnetic beads, and the precautions and strategies implemented to avoid and. Briefly, bone powder was digested overnight, and dna was extracted with phenolchloroformisoamyl alcohol, purified and concentrated on microcolumns. When she is ready to extract dna, she cuts a small piece of bone or tooth and crushes it into powder. Isolating the human cochlea to generate bone powder for ancient dna analysis ron pinhasi 1, daniel m. Extraction of dna from bone or teeth using the ez1 dna. Methods and protocols expert researchers in the field describe many of the protocols that are now commonly used to study ancient dna. Mobile devices for onfield dna analysis have been used for medical diagnostics at the pointofcare, forensic investigations and environmental surveys, but still have to be validated for ancient dna studies. It is rare to find the fossilised remains of bones from hominid. Due to degradation processes including crosslinking, deamination and fragmentation ancient dna is more degraded in comparison with contemporary genetic material. This method is designed to maximize recovery of pcramplifiable dna from ancient bone and teeth specimens and at the same time to minimize co extraction of substances that inhibit pcr. To separate between authentic adna and modern human contaminant dna we sampled and extracted dna from dogs canis familiaris. If a dna profile is not obtained, extract the bone.
Total demineralisation is the best method for most cases of dna extraction from bones, although it does not provide pure dna. Comparative analysis of extraction methods has shown that this method works reliably for ancient as well as younger, museumpreserved specimens. Richard iii removing a tooth for dna analysis youtube. Using evidence written in their teeth, bones, dna and burial, weve uncovered the extraordinary diversity of these ancient londoners. Extensive human dna contamination in extracts from ancient. The majority of ancient and forensic research using human teeth has focused on different ways to access the internal tooth tissue dentine alakoc and aka, 2009, cobb, 2002, gilbert et. Before at least one hour to start dna extraction, add additionally 150 ul of 20 mgml proteinase k to the tubes and reincubate in a rotary shaker at 56. Since decayed or buried bones may contain large amounts of nonhuman dna, quantitation of total human dna is required. The dna extraction process represents one of the critical stages in the analysis of degraded or ancient dna.
The myriad downstream applications of ancient dna adna analysis all ultimately require that sequence data are generated from extracts of ancient material. This method is designed to maximize recovery of pcramplifiable dna from ancient bone and teeth specimens and at the same time to minimize co. This is because of the physical and chemical barrier that the protein. Dna polymerase, result in the exponential accumula tion of the target dna fragment that can then be sequenced by conventional techniques. Ancient dna research shares a common problem with forensics and other approaches requiring analyses of museum and noninvasively collected specimens. However, during the past few years, ancient nuclear dna studies from human remains of up to 8,000 years have been described with success and. It has been a challenge to extract dna from bones previously soaked in water, burned, or buried for a long time, due to the reduced quality and quantity of dna in the bone samples. Jun 01, 2007 in historical cases, missing persons identification, mass disasters, and ancient dna investigations, bone and teeth samples are often the only, and almost always the best, biological material available for dna typing. We tried a dna extraction on the outside chance some fragments might. Improving access to endogenous dna in ancient bones and. Comparison of three methods of dna extraction from human. Unlike dna extracted from fresh samples, ancient dna is mostly lowquality dna.
The importance of oral and dental health in college students. Ancient dna reveals genetic connections between early di. Dna of ancient humans found in caves even without bones. Ancient dna adna is dna isolated from ancient specimens. Improving access to endogenous dna in ancient bones and teeth. Bones and teeth were prepared in clean room facilities dedicated to ancient dna 5. The majority of ancient and forensic research using human teeth has focused on different ways to access the internal tooth tissue dentine alakoc and aka, 2009, cobb, 2002, gilbert et al. Apr 29, 2017 the dna of our ancient human ancestors can now be found within the sediment rocks in caves even when there are no bones left. The extraction lab is dedicated to ancient dna extraction only. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. The extraction lab has a dedicated positive pressure air handling system to separate the extraction laboratory air supply from sample preparation cleaning and ln grinding and downstream pcr applications. Sterilize the bone with dnase dna away or dip it into ethanol and burn it briefly afterwards. Survival and recovery of dna from ancient teeth and bones. Sep 19, 2005 ancient dna research has so far mainly focused on mitochondria, the tiny powerstations of the cell.
Comparison and optimization of ancient dna extraction. Sinceoligonucleotides 20baseslong canbeprecipitated bytca18, it wasestimated that more than one alkalisensitive site per 20 bp must exist in the ancient dna. The idea was not new, noted viviane slon, a member of. Scientists recover ancient dna from cave dirt clockwise from top left, richard roberts, vladimir ulianov and maxim kozlikin planned the sampling of sediments in the east chamber.