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The Beothuk Indians were an extinct group of Amerinds who were among the earliest founders of Newfoundland. Mitochondrial DNA (mtDNA) analysis was performed on a Beothuk chief.
The Beothuk Indians were an extinct group of Amerinds who were among the earliest founders of Newfoundland. Mitochondrial DNA (mtDNA) analysis was performed on a Beothuk chief.
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The Beothuk Indians were an extinct group of Amerinds who were among the earliest founders of Newfoundland. Mitochondrial DNA (mtDNA) analysis was performed on a Beothuk chief.
Droits d'auteur :
Attribution Non-Commercial (BY-NC)
Formats disponibles
Téléchargez comme PDF, TXT ou lisez en ligne sur Scribd
A THESIS SUBMITTED TO THE GRADUATE SCHOOL IN PARTIALFULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENCE IN BIOLOGY BY APRIL MAY REED Date "7 t/ 1 3),/ Date Date Departmental Approval: C2P--, 1-/5-01 Dr. Carl Warnes, Departmental Chairperson Date Graduate Office Check: 7 / / ~ /D/ . g ~ 21. ~ 4 ,j;1t4 Dean of the Graduate School Date BALL STATE UNIVERSITY MUNCIE, INDIANA JULY 2001 11 ,/MITOCHONDRIAL DNA ANALYSIS OF NONOSABASUT, A BEOTHUK INDIAN CmEF/ A THESIS SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE MASTER OF SCIENCE IN BIOLOGY BY , APRIL MAY REED -, CHAIRPERSON DR. CAROLYN N. V ANN BALL STATE UNIVERSITY MUNCIE, INDIANA JULY 2001 ABSTRACT ....... f', ;i <:I:;" .R. 1.(1 THESIS: Mitochondrial DNA Analysis ofNonosabasut, a Beothuk Indian Chief STUDENT: April May Reed DEGREE: Master of Science COLLEGE: College of Science and Humanities DEPARTMENT: Biology DATE: July 2001 PAGES: 104 The Beothuk Indians were an extinct group of Amerinds who were among the earliest founders of Newfoundland. In literature, the Beothuk were described as perhaps being phenotypically more similar to Europeans than Asians (Gatschet 1890, Lloyd 1875, 1876a, Marshall 1996). In this research, mitochondrial DNA (mtDNA) analysis was performed on a Beothuk individual in order to determine his haplotype and, perhaps, shed light on the origins of the Beothuk. For this analysis, a tooth ofNonosabasut, a Beothuk chief who died in 1819 was loaned from the Royal Museum of Scotland. Ancient DNA was extracted from 172 mg of dentin from the tooth. The DNA was cut with two blunt-end restriction enzymes, RsaI and HaeIII. Double-stranded DNA adapters were ligated to the blunt ends. A single adapter was used to amplify the resulting fragments using PCR. In this manner, two libraries ofthe DNA were created that could be readily reamplified using a small amount 111 ABSTRACT ....... f', ;i <:I:;" .R. 1.(1 THESIS: Mitochondrial DNA Analysis ofNonosabasut, a Beothuk Indian Chief STUDENT: April May Reed DEGREE: Master of Science COLLEGE: College of Science and Humanities DEPARTMENT: Biology DATE: July 2001 PAGES: 104 The Beothuk Indians were an extinct group of Amerinds who were among the earliest founders of Newfoundland. In literature, the Beothuk were described as perhaps being phenotypically more similar to Europeans than Asians (Gatschet 1890, Lloyd 1875, 1876a, Marshall 1996). In this research, mitochondrial DNA (mtDNA) analysis was performed on a Beothuk individual in order to determine his haplotype and, perhaps, shed light on the origins of the Beothuk. For this analysis, a tooth ofNonosabasut, a Beothuk chief who died in 1819 was loaned from the Royal Museum of Scotland. Ancient DNA was extracted from 172 mg of dentin from the tooth. The DNA was cut with two blunt-end restriction enzymes, RsaI and HaeIII. Double-stranded DNA adapters were ligated to the blunt ends. A single adapter was used to amplify the resulting fragments using PCR. In this manner, two libraries ofthe DNA were created that could be readily reamplified using a small amount 111 of the PCR product. mtDNA type was determined by amplifying specific regions and performing Restriction Fragment Length Polymorphism analysis and sequencing. It was determined that the Beothuk individual had a 9-bp deletion at nucleotide position (np) 8272, an AZul restriction site at np 5176, and heteroplasmy for a HincH restriction site at np 13,259, indicating that the Beothuk individual falls into the Native American Haplogroup B. Haplogroup B is not present in modem Siberian populations, whereas the remaining Native American mtDNA haplogroups are. It has been hypothesized that Haplogroup B arrived in the Americas at a different time than haplogroups A, C, D, and X, about 16,000-13,000 YBP (Years Before Present) (Starikovskaya et aZ. 1998). Haplogroup B can be found in some modem Taiwanese, Japanese, Korean, Evenk, and other Asian populations. Sequencing ofthe D-Loop region revealed a G to A transition at np 16303. To our knowledge, this transition was never previously reported in a Native American. This transition has been reported in Tibetans, Koreans, Hans, and Japanese, all considered to be southeast Asian Causacoids (Torroni et al. 1993b, 1994b). This transition, also frequently described in the Caucasian Haplogroup H, is especially prevalent in Spain and among the Basque. It is described as a root haplotype of Hap log roup H whose expansion was estimated to be between 12,300-13,200 YBP (Torroni et aZ. 1998). This time estimate coincides with the expansion ofHaplogroup B. One possible explanation for this transition may be some admixture of the Beothuk with a Caucasian population. IV ACKNOWLEDGEMENTS Without the encouragement, support, and advise of my family and friends, completing this project would have been very difficult. I would especially like to thank my husband, Kris, for his constant support throughout my time at Ball State. Dr. Carolyn Vann has been my great motivator. Her faith in my abilities has meant the world to me. I would like to thank her for all that she has taught me. Most of all, I would like to thank her for her friendship. I would also like to thank the rest of my thesis committee. Dr. Clare Chatot has given me advice and encouragement often throughout this project. Dr. James Mitchell did not hesitate to jump in and help when I needed it. Without Dr. James Pyle's encouragement, I never would have pursued research with Dr. Vann. Dr. Carl Warnes has always supported and encouraged me. Fresia Steiner has been a constant wealth of information. She has also been a good friend. I would like to thank the Royal Museum of Scotland for loaning us the Beothuk tooth. I hope they will be pleased that we were able to amplify the DNA. I also hope they will find the results interesting. I would like to thank Dr. Neal Lambert for drilling the tooth for me. He was very generous with his time. His quick response allowed me to get this project finished in time for graduation. I would also like to thank Tara Angel Keels for starting this project. She obtained the tooth from the Royal Museum of Scotland. In addition, she sent me an entire box of background material on the Beothuk Indians. Lastly, I would like to thank my parents and grandparents. Grandpa, maybe I can fly the plane now. v X. RESULTS AND DISCUSSION 63 Powdering of Dentin 63 Gel Analysis of Library Amplification 63 Gel Analysis of Marker-Specific Amplification 65 RFLP Analysis 76 Sequencing 81 European Marker 85 Analysis of the RsaI Restriction Site Loss at np 16303 85 Apendices A & B- Compilation of Published mtDNA Analyses 95 XI. CONCLUSIONS 96 XII. REFERENCES 100 Vll - - - - - - - - ~ - - - . - - - LIST OF FIGURES AND TABLES Figure 1. Map of Newfoundland and the Areas the Beothuk Were Known to Inhabit 2. Map of Newfoundland Showing L'Anse aux Meadows, the Viking Settlement 3. Rendering of Demasduit 4. Replica of a Canoe Taken from Nonosabasut's Burial Hut 5. Shanawdithit's Drawing of the Killing ofNonosabasut 6. Library Creation Flowchart 7. D Loop Replication System Mechanism Model 8. Diagram of Native American mtDNA Polymorphism Locations 9. Distribution of mtDNA Haplogroups Among Native Americans 10. Gel analysis of the Initial Amplification of the RsaI and Haem Libraries 11. Gel Analysis of Libraries After Ethanol Precipitation 12. Example of Failed Attempts at Marker-Specific Amplification 13. Example of Spurious Bands Observed During Optimization Attempts 14. Amplification ofAluI-5176, Hinell-13259, and Haem-663 Markers 15. Amplification of 9-bp Deletion Marker 16. Amplification ofD-Loop Region 17. Gel Analysis Showing Contamination in the Negative Controls 18. RFLP Analysis of HaeIll-663, Hinell-13259, and AluI-5176 PCR Products 19. Re-Digestion of Hinell-13259 PCR Product with Hinell and AluI 20. Re-Amplification of Desired PCR Product from Agarose Extraction 21. Tibetan Phylogenetic Tree 22. Phylogenetic Tree of Caucasian mtDNAs 23. Diffusion of European Haplogroups Table 1. Polymorphisms Which Characterize Each Native American mtDNA Haplogroup 2. Primers and Primer Sequences Used to Analyze the mtDNA Markers in the Beothuk Chief 3. The Library Used for Amplification of each Native American mtDNA Marker 4. Final Thermocycling Conditions for the D Loop and Native American Marker Amplification Vlll Page 6 8 13 17 23 29 36 39 40 64 66 67 68 70 71 72 74 76 78 80 87 90 92 Page 41 52 53 55 - ~ . - - ----- 5. Digestion Reaction Conditions for RFLP Analysis of Native American Markers 58 6. Final Thermocycling Conditions for the AluI-5176 mtDNA Marker Amplification 60 7. Expected RFLP Products and Results 8. Beothuk mtDNA Sequences 9. Sequencing of Investigator's D-Loop Region Appendix A. D-Loop Polymorphisms B. Previously Published mtDNA Polymorphisms IX 77 83 85 Page attached as a disk attached as a disk INTRODUCTION Contemporary Amerindian mitochondrial DNA (mtDNA) types group into 5 mtDNA lineages designated as Haplogroups A, B, C, D, and X based on the presence or absence of single base pair alterations (Stone and Stoneking 1993). The existence of these lineages in modem Asian populations is used as evidence for hypotheses that the founding Native Americans were of Asian descent. Further analysis of Native Americans can be performed through sequencing ofthe non-coding Displacement Loop (D-Loop) region of the mtDNA, the most heterogeneic portion of the mtDNA (Anderson et al. 1981). The objective of this study was to determine ifNonosabasut (No-nos-baw-soot), a Beothuk (Bee-oth-ick) Indian chief, belongs to a Native American (Asian) or a European mtDNA haplogroup using DNA extracted from his tooth. The Royal Museum of Scotland loaned a tooth from the skull ofNonosabasut to Ball State University. The hope was that mitochondrial analysis of this individual would provide some evidence as to the origins of the enigmatic group of people, the Beothuk Indians, candidate founders of Newfoundland. The origins of the now extinct Beothuk have been argued since the early 1800's. Based on archaeological evidence, it is believed by some that the Beothuk were a relic group of Maritime Archaic peoples that separated from a mainland Algonquin population 2 at an early date (Lloyd 1876a). However, others believed, due to the apparent European reatures ofthe Beothuk, that the Beothuk were descendants of one of the earliest European outgroups (Marshall 1996). Still others believe that the Beothuk were not descendants ofthe Maritime Archaic or an ancient European outgroup, but a people entirely unique (Blake 1888). The last known Beothuk individual, Shanawdithit (Shaa-naan-dith-it), died of tuberculosis in 1829. It has been reported that a few surviving Beothuk went back to the mainland, Labrador, to join other Indian groups. However, such reports have never been substantiated. Thus, the origin and history ofthe Beothuk remains a mystery. However, the Beothuk Indians could still possibly communicate their origin to us through their remains via molecular techniques allowing mtDNA analysis. The results of this study could have important implications for the current controversy surrounding the initial colonization of the Americas. The analysis will reveal if the Beothuk individual belongs to one of 5 Native American haplogroups. This will, perhaps, answer some questions about the Beothuk's role in American pre-history and about their relationship to other populations and migration times. Previous genetic studies have strongly implied that all modem Native Americans derived from Asian ancestors. Because mtDNA is primarily maternally inherited, has a high mutation rate, is found in high copy within the cell, and does not undergo recombination, it is a very useful genetic system for studies into human origins and migrations. It is particulary useful for studies involving ancient DNA. Ancient DNA is highly fragmented and degraded. Due to the high copy ofmtDNA versus nDNA in cells, mtDNA amplification is often more successful than nDNA in ancient DNA. 3 The accumulation of mitochondrial mutations has created continent -specific po1ymorphisms. The linked occurrence of these polymorphisms within a group of people bids to the formation of specific mtDNA haplotypes (a collection of all polymorphisms held by an individual's mtDNA). Groups of related haplotypes are termed a "haplogroup." Haplogroups are defined by one or more unique mtDNA mutations that not only relate the individual haplotypes within the haplogroup to each other, but also set the haplogroup apart from other mtDNA haplogroups. All mtDNA haplotypes found to date in contemporary and ancient Native American populations group into 5 categories (Brown et al. 1998). Haplogroup A is defined by a HaeIlI site at nucleotide position (np) 663, Haplogroup B by a 9-bp deletion in the intergenic region between COIl (cytochrome oxidase subunit II) and tRNA Lys (lysine tRNA) at np 8272, Haplogroup C by a combined HindI site loss at np 13259 and an A luI site gain at np 5176, and Haplogroup X by a HaeIlI site at nps 16223 and 16278 in the D-Ioop region and a loss ofa DdeI restriction site at np 1715. Two libraries of the individual's DNA will be created to be used as templates for mtDNA marker-specific amplification as described in Weiss et al. 1994. Library creation is necessary due to the limited nature of the irreplaceable DNA. The creation of two hbraries is necessary because the four-base cutters being used, RsaI and HaeIII, are found frequently in the genome. Thus, if the portion ofthe mtDNA containing the marker of interest contains, for example, an RsaI site, the HaeIII library can be used as a template instead. The libraries will be created by :first digesting the Beothuk DNA with RsaI and HaeIII. The resulting blunt-ended fragments will be ligated to a common, double- 4 sbanded, blunt-ended adapter. The fragments will then be amplified to form the two Iiharies using a shorter, single-stranded oligonucleotide within the adapters as a PCR primer. As needed, the library can be reamplified using the same oligonucleotide as a , . PCR primer. In this manner, a potentially inexhaustible supply of the irreplaceable material will be created. Mitochondrial marker-specific amplification will then be carried out using PCR primers (modified from Stone and Stoneking 1993) to amplify specific segments of DNA, less than 250 base pairs (bp) long, which contain previously described markers. The resulting PCR products will be used in Restriction Fragment Length Polymorphism (RFLP) analysis and/or sequencing. The Beothuk individual will then placed into a Native American haplogroup, if possible, based on the mtDNA analysis. In addition, PCR primers will be designed and used to amplify a 340-bp fragment of the D-Loop region. This PCR product will then be sequenced and compared to the Cambridge reference mtDNA sequence (Anderson et al. 1981) to determine any polymorphisms. Any polymorphisms found in this sequence will be compared to previously published D-Loop polymorphisms in Asian and European populations. This information will be used in conjunction with the marker-specific analysis to aid in demystifying the origins of the Beothuk. LITERATURE REVIEW Beothuk Indian Background The Beothuk Indians were thought to be the aboriginal inhabitants of Newfoundland (which is about the size of New York state) (Gatschet 1885). There is evidence that the Beothuk inhabited Labrador and Newfoundland at the same time (Gatschet 1890). The Labrador Eskimo tradition is that the Beothuk were inhabitants at one time of Battle Harbour, Labrador (Lloyd 1875). The Beothuk were nomadic people who were hunters, fishers, and gatherers. They lived along the Exploits River in the late fall to hunt caribou (see Fig 1). In the early winter they were together at the eastern end of Red Indian Lake. They were scattered along the east coast of Newfoundland for the duration of the year. The Beothuk were thought to be secluded from other Indian people (Lloyd 1875). However, they may have had peripheral involvement with the Indians on the mainland. For example, it was reported that the Eskimos crossed Straight of Belle Isle every day in the 1690's (Gatschet 1886). The Beothuk lived in a state of isolation far apart from the inhabitants of the mainland (Lloyd 1876b). The Micmac said that the Beothuk had "magic powers" that would allow them to know about the approach of strangers. They would flee on their snowshoes before anyone saw them (Gatschet 1890). Perhaps this ability to avoid contact is why so little is known about the Beothuk. .-.. - - - - - - - - - - - - = - - ~ - - - - ~ - - - . ~ - - - - - - ~ ~ - - - - 6 LABRADOR .... ....... ... ... aui:8E:C Figure 1: Map of Newfoundland and the Areas the Beothuk Were Known to Live (Marshall 1996). The blue asterisk denotes the Exploits River that the Beothuk inhabited in the Fall. The yellow asterisk denotes eastern Red Indian Lake, which the Beothuk inhabited in the winter. The Beothuk were scattered along the east coast for the duration of the year. The orange arrow points the Straight of Belle Isle the Eskimos were known to cross. 7 Some described the Beothuk as "cruel and austere" (Marshall 1996). However, others held the same opinion as Captain Hayes that the Beothuk were "altogether harmless" (Blake 1888). The Beothuk differed from other North American tribes in that they had a "lightness of complexion" (Lloyd 1876a). The Beothuk were further described as being different because they had European features (Lloyd 1875). In addition, the Beothuk Indians are distinguishable in historical literature due to their extensive use of red ochre. The Beothuk would smear their bodies and, perhaps inadvertently, their tools and clothes with a mixture of the red ochre and oil. Due to this ritual, the Europeans called the Beothuk the "Red Indians." The Beothuk's first encounter with the Europeans was probably with the Vikings, circa 1000 AD. The Viking Lief Erikson, "Lief the Lucky," built a settlement in what is now agreed to be L'ans aux Meadows in northwestern Newfoundland (Fig 2). The Vikings (Nordic) inhabited Newfoundland for about 8 years. During that time they encountered the aboriginal natives (which were probably Recent Indians, ancestors of the Beothuk). The Vikings called the red people "Skraelings" (Marshall 1996). John and Sebastian Cabot recorded an encounter with the Beothuk Indians when they arrived in Newfoundland in 1497 (Lloyd 1875). In addition, the Beothuk were described by Gaspar de Corte-Real in 1500 and Jacques Cartier in 1534 (Marshall 1996). Basque, Breton, Spainish, Portuguese, French, and English fishermen all described the presence of the native Beothuks (Blake 1888). Captain Richard Whitboume reported that in 1615 that the Beothuk helped the French kill, cut, and boil whale in exchange for bread (Lloyd 1875). However, this encounter was most likely with the Micmac rather than the Beothuk due to the Beothuk's unwillingness to interact with the European settlers. - ~ - - - - - - ~ - - - - - - - - - - - - - - - - - Port au:-:: Ch 0 ix: Gros Morne National Park I I :' Fenies to Nova Scotia .r ". J ................. ,. 150 km Figure 1: Map of Newfoundland Showing L'Anse aux Meadows, the Viking Settlement (htip:!/www.nfld.net/whitefeather/map2.html). The purple arrow indicates the position of the Viking settlement, circa 1000 a.d. The Vikings wrote of interactions with the aboriginals they called "Skraelings" which were probably the Beothuk. 8 9 THE ORIGIN OF THE BEOTHUK INDIANS About 5000 years ago, the ice increased around Greenland (Blake 1888). Later, as the glaciers retreated, Ice Age hunters followed them into Labrador. It is believed that some ofthese individuals migrated across the Straight of Belle Isle from Labrador to the Northern Peninsula of Newfoundland (see Fig 1) due to the arrival ofthe Palaeo-Eskimos in Labrador. Those who remained in Labrador and Newfoundland became the Maritime Archaic Culture. However, most of the Maritime Archaic Indians died or left Newfoundland before 1050 B.C. (Marshall 1996). Most scholars believe that a second Indian population (thought to be direct descendents of the Maritime Archaic) called the Recent Indians migrated from Labrador into Newfoundland in around 50 B.C. (Marshall 1996). Conversely, a small population of Maritime Archaic Indians may have remained on Newfoundland when the remainder of the population left. Then they may have mixed with other surrounding populations (pastore 1991). The Recent Indians gave rise to Beaches Complex around 1000 YBP. The Beaches Complex then gave rise to the Little Passage Complex circa A.D. 1100. Upon arrival ofthe Europeans to Newfoundland in the 1500's, Little Passage Complex Indians historically became known as Beothuk. The Beothuk are thought to have derived from the Little Passage Complex due to the similarities in tools they used (Pastore 1991, Marshall 1996). There is much discussion concerning the origins of the Beothuk in historical literature. William Strong thought that they might be a remnant of an "old stone" archaic culture (Strong 1930). Frank Speck thought that the Beothuk might have been an archaic Algonquian remnant. However, he also indicated that they may also be related to the Eskimos, Labrador Indians, or the Red Paint people (Speck 1922). Others felt that the appearance, customs & manner of the Beothuk indicated that they were a race separate from Algonquians & Eskimos (Gatschet 1886). Charles Willoughby also believed that the Beothuk might have been a surviving branch of the Red Paint people of Maine (Willoughby 1915, Dixon 1913, and Birket- Smith 1918). Warren Moorehead, the foremost expert on the Red Paint people disagreed. He felt that there was little cultural similarity between the Beothuk and the Red Paint people of Maine (Moorehead 1917) The conjecture concerning the origins of the Beothuk seemed to have no end. 10 Due to the occasional appearance of blue eyes and the pale skin ofthe Beothuk, some people even believed they originated in Scandinavia while others thought it was more probable that they were driven out of Canada by the Iroquois and Hurons (Lloyd 1876a). It was once stated that "it would not be impossible that ... some ofthe old Viking blood ran in the veins of the Beothuk tribe" (Blake 1888). The Beothuk have even been affiliated with Polynesians (Campbell 1892) while Santu, a woman who claimed to be halfBeothuk and half Micmac said that they were mixed Eskimo (Speck 1912). Physical Characteristics of the Beothuk Almost without exception, the Europeans who encountered the Beothuk Indians took notice of their European-like features. Captain Buchan stated that the skin of the Beothuk was much fairer than most Indians and closer in fairness to most Europeans 11 (Marshall 1996, Lloyd 1886). Numerous times the appearance of the Beothuk was compared to that of the Basque or the Spanish. Lloyd stated that the shape of their head was the same as Europeans, but they had black eyes and hair like Esquimos and skin like Spaniards (Lloyd 1875). John Peyton stated that the Beothuk were darker than Spaniards, sometimes having blue eyes. He likened their overall appearance to that of Spanish gypsies (Busk 1876). The Micmac of Nova Scotia spoke ofthe 'other tribe' (the Beothuk) that had white faces (Gatschet 1890). In addition, Beothuk skulls purloined from a grave by McConnack in 1826 were descn"bed as being more European in their frontal elevation that the skulls of surrounding Indians (Busk 1876). Shanawdithit, a Beothuk woman who was held captive by the English for 5 years, was described as having a striking similarity to Napoleon. She had an olive complexion, black hair and perfect teeth. When she made a sketch of her father, she depicted a Romanesque nose (Fraser 1886). Elsewhere, the Beothuk were again described as having aquiline noses (Lloyd 1876a). Another Beothuk woman was captured in 1803 while collecting bird eggs from a canoe. She was described as being docile, having copper color skin, black eyes, and the hair of a European (Blake 1888). The story of two Beothuk men who were captured and taken to the King's court (Palace of Westminster) is often told as an example of the European resemblance of the Beothuk. After the men were dressed in fine clothing, someone who had not seen them in two years had to be told who they were because he did not know they were not Englishmen (Lloyd 1875). ~ - - - ~ ~ ~ - - - Beothuk Captives Most of the known information about the Beothuk derived from the captives Demasduit (Dee-mas-doo-weet), whom the English called Mary March, and Shanawdithit, whom the English called Nancy April. 12 Demasduit (Fig 3) was captured on March 5, 1819 (Gatschet 1885). A group of Beothuk was crossing the Exploits River when a group led by John Buchan began to pursue them. Demasduit, who was weak from being ill and just delivering a child fell behind the rest ofthe Beothuk when her snowshoe broke. Buchan captured her on the ice. Her husband, Nonosabasut, was shot trying to save her. Her infant child died two days later (Blake 1888, Marshall 1996). Demasduit herself died in less than a year of tuberculosis (Pastore 1991). Demasduit spent the year of capture living with the family of Rever and John Leigh in Twillingate. Leigh developed a written record of about 180 Beothuk terms for English words taught to him by Demasduit (Marshall 1996). Shanawdithit (called Nancy April by the English), her sister, and her mother gave themselves up to William Cull in the winter of 1823 because they were starving (Pastore 1991). Cull attempted to return them to the Beothuk. However, Shanawdithit, her mother, and her sister returned because they were afraid they would be killed due to their contact with the Europeans and the Micmac in the settlement. Shanawdithit's sister and mother were both sick and died soon after their capture. Cull sent Shanawdithit to live with John Peyton. She was Peyton's servant for five years. Shanawdithit was more forthcoming with information on the Beothuk than Demasduit had been. She communicated with W.E. Cormack ofBeothuk life mainly through sketches she made. Cormack would then copy the sketches or make notes on the 13 Figure 3: Rendering of Demasduit (Marshall 1996). Miniature portrait of "A female Red Indian ofNfld. 'Mary March' painted by W. Gosse at St. Joh's Nfld. July 1941 from an original by Lady Hamilton May 1821." This rendering portrays Demasduit. Her huband, Nonosabasut, who was killed during her capture, is the subject of this study. - ~ - ~ - - - - - - - - - - - - - 14 sketches about Shanawdithit's explanations. Cormack, like John Leigh, made a dictionary ofthe Beothuk words Shanawdithit spoke and their meanings {Marshall 1996). Shanawdithit died of consumption on June 6, 1829 in St. John's (Fraser 1886, Pastore 1991, Marshall 1996). Beliefs and Customs of the Beothuk The legend among the Beothuk was that they sprang from an arrow{ s) stuck in the ground {Marshall 1996). Whitbourne said they believed that the Great Spirit struck an arrow in the ground and they arose from the arrow. These beliefs of the Beothuk were not unlike the beliefs of other North American Indians. The Choctaws believed they were from the earth, the Oneidas believed they sprang from a large boulder, and the Hurons believed they arose from a hole in the mountainside (Blake 1888). Similarities were noted between the sleeping habits and burial rituals ofthe Beothuks and other American Indians. A tribe at the foot of the Rocky Mountains, the "Atnahs," dug holes in ground and would inlay them with grasses or branches. This strongly resembled the sleeping places (trenches) built by the Beothuk (Blake 1888). 1.P. Howley found a "mummy boy" in 1886 that resembled an Alaskan mummy preserved in the National museum in Washington, D.C. (Gatschet 1890). It was a nine- or ten-year old boy wrapped in birchbark, doubled together and laid on his side under a heap of stones. In addition, the Beothuk disposed of their dead like the Western Indians of sources of Mississippi by wrapping them with birch bark. They also often put their dead on scaffolding made up of four posts like the Mississippi Indians (Lloyd 1875). 15 Linguistics of the Beothuk Many parallels were drawn between the Beothuk language and the languages of other American Indians. However, few linguists agreed on the basis of the comparisons. Some stated that the Beothuk language was not unlike Inuit (Eskimo) language. Some parallels were also found with the Tinne in the Rocky Mountains. No parallels with the Iroquois, who once lived in St. Lawrence (close to Newfoundland), were found (Gatschet 1886). Many linguists believe that the Beothuk language is in the Algonquian family of languages (Pastore 1991, Latham 1862, Hewson 1968, 1971). Lloyd thought that the Innu (Naskapi or Montagnais) of Quebec-Labrador's language most closely resembled Beothuk language (Lloyd 1886). Dr. R.G. Latham stated that from the vocabulary ofthe Beothuk, they were of Algonkin relation rather than Eskimo. However, he thought they were not a branch of the Micmac, etc, but a division of their own (Lloyd 1876a). Gatschet was the foremost proponent that the Beothuk were of a separate linguistic family. Gatschet made many statements in support ofhis belief He stated that the Beothuk and Algonquin phonetic systems differed largely. The Beothuk language had an objective case while the Algonquin did not. The words for numbers in the Beothuk and Algonquin vocabulary differed greatly. The terms for human, body, celestial bodies, nature, colors, animals, and plants (which would be expected to be similar if they shared ancestry) differed completely. Additionally, Gatschet stated that the Beothuk language was a totally separate language from Inuit, Tinne, and the Iroquois, (the other mainland Indians) (Gatschet 1886). 16 Connack thought that the Beothuk language resembled European language more than other Indian languages (Gatschet 1885). In support ofGatschet's hypothesis that the Beothuk language was unrelated to surrounding tribes, the language of the Beothuks was unknown among Canadian Indians (Lloyd 1875). Archaeological Evidence Archaeological excavations, written descriptions, and drawings of the Beothuk tools, weapons, and canoes were also used to infer the origins of the Beothuk. Some parallels have been drawn between the Beothuk and the Eskimos. Their harpoons resembled harpoons used by early Eskimo peoples and the Eskimo of the northeast coast of Labrador (Lloyd 1876a, Pastore 1991). In addition, stone tools found were similar to Eskimo stone tools (Lloyd 1876b). The canoes the Beothuk used were unlike Micmac and Nakaspi canoes. The Micmac used skin for their canoes, not birch bark like the Beothuk (Lloyd 1875). The Beothuk canoe had a large, rounded keel (Fig 4). In addition, the Beothuk were the only Indians to use a sail. However, they may have started using sails after contact with the Europeans. The Beothuk would often steal sails from European settlements. With these peculiar canoes, the Beothuk were able to sail as far as Funk Island (about 60 km north of Newfoundland) to hunt Great Auks and collect sea bird eggs. The Beothuk craftsmanship of the canoes was so clever that they were able to develop a method to make a canoe that could fold up for transport and still be waterproof when unfolded (Lloyd 1876b). 17 Figure 4: Replica of a Canoe Taken From Nonosabasut's Burial Hut (MarsbaIl1996). A birchbark canoe replica collected in 1827 by W.E. Cormack from Nonosabasut's burial hut (Copyright The Trustees of the National Museums of Scotland, 1996, neg. 0418). The distinctiveness of the Beothuk canoe from the canoes of the surrounding Indians was used as support for the hypothesis that the Beothuk were not related to the other Native American populations. 18 The Beothuk made deer fences that were up to 30 miles long. They would do this by strategically falling trees and tying branches to the trees to create impenetrable fences alongside rivers. They would then leave small passageways open that the deer would pass through where the Beothuk would be lying in wait. It was said that the upkeep that would be required on the miles offences was an indication of the sheer number of the Beothuk at one time. The Columbians frightened and drove the deer in a method similar to the Beothuk by placing bright colored rags in the bushes that would wave in the wind (representing men) and drive the deer (Lloyd 1876a). The Beothuk differed from other American Indians in that they did not make pottery (Blake 1888, Lloyd 1886). Scholars pointed to this fact as suggestion that the Beothuk were a remnant of an ancient group. The Beothuk carved their pots out of soapstone. However, some believe that many archaeological finds, such as soapstone dishes, have been attributed to the Beothuk that are, in fact, assemblages of materials from many aboriginal inhabitants such as the Maritime Archaic and the Paleo-Eskimos (Holly 1999). Beothuk Interactions With Other Native Americans The Beothuk Indians were described in literature as being isolated. However, the Beothuk interacted with many of the "mainland" Indians. The Beothuk were on good terms with the Labradorian Algonkins of the coast and interior. The Naskapi and the Montagnais (whom the Beothuk called "Shoudamunk" meaning "good spirit") traded with them (Gatschet 1885, Lloyd 1875). Shanawdithit talked oftrade with the Labrador Indians (Jukes 1842). They were friendly and traded with the Montagnais from the - ~ - - - - - - - - - - - - - - - - - - - - North. In fact, there may have been intermarriage among Beothuk: and Indians residing between Newfoundland and the northern Quebec-Labrador peninsula (Pastore 1991). It is believed that the last surviving Beothuk: may have joined the Montagnais in Labrador (Marshall 1996). 19 It was Micmac tradition that the relations with Beothuk: were good in earlier times (Gatschet 1890). At one time, the Beothuk: may even have intermarried with the Micmac who were from Nova Scotia (Marshall 1996). However, the relations between the Micmac and the Beothuk: had soured before the arrival of the European settlers. For example, Shanawdithit dreaded the Micmac, especially a Micmac named "Mudty Noel"- Wicked Noel. She said that he shot at her for no reason across Exploits River when she was cleaning venison. She then showed shot wounds to W.E. Cormack (Gatschet 1890). Shanawdithit said that the Beothuk: name for Micmac was "Shonack" which meant "bad Indians" (Lloyd 1875). It is believed that once the Micmacs were given firearms by the French and moved to southern Newfoundland, relations soured irrepairably (Marshall 1996). The Eskimos were afraid of the Beothuk: because they were unlike Mountaineers, the Indians of eastern Canada. The Eskimos thought the Beothuk: were more fierce. There was an encounter between the Beothuk: and the Eskimos in 1831 in the Bay of Seven Islands in Labrador (Lloyd 1875). Shanawdithit said that the Beothuk: "hated" the Eskimo because they were dirty and always harassed the Beothuk: (Blake 1888). In addition to interacting with the surrounding natives, the Beothuk: were being forced to interact with the Europeans settling in Newfoundland. Newfoundland became a - - - ---- sanctuary for deserters from the navy, refugees from Ireland, ''reckless and unruly characters of all kinds who dare not return to their own country, sought an asylum in Newfoundland (Blake 1888)." 20 Many seasonal fishing settlements were springing up in Newfoundland in the 1800's. The Basque, Bretons, Spanish, Portuguese, French, and English were all settling in Newfoundland (Marshall 1996). It is believed that some ofthe Beothuk may have been raped, captured, and! or killed by the Europeans. There may also have been some blame on the part of the Beothuk. John Buchan claimed to have seen a captive European mother with children in a Beothuk camp in 1811 (Howley 1915). Shanawdithit said that their custom was that the first white men whom they encountered were called "good spirit" (probably the Vikings), the second white men were called ''bad spirit," and the Micmac were called ''bad spirit" (Fraser 1886). Some have even gone so far as to state that it is even possible the Beothuk may have encountered Turkish pirates in late 1500's (Marshall 1996). BEOTHUK DEMISE The largest estimate for the number ofBeothuk on Newfoundland at the time of the first European contact does not exceed 2000 individuals (Pastore 1991). After the Micmac began to settle Newfoundland and Europeans began to settle, the Beothuk withdrew into the interior because the coast was no longer safe. However, there were few available food sources on the interior. The ecological system the Beothuk depended upon made them vulnerable. Sea resources may have been unavailable along the coast for a season due to inclement weather changes in fish migratory patterns (Pastore 1991). In addition, eventually they were completely unable to fish, gather oysters, eggs, or catch water fowl because they were forced to remain in the interior (Fraser 1886). 21 The Beothuk were unable to keep up with their deer fences due to their diminishing population (Fraser 1886). Shanawdithit said that in the Beothuk settlement in Exploits River in 1811, 20 men, 22 women, and 30 children were left. There were 20- 30 Beothuk in Newfoundland by 1822 (Pastore1991). By 1823, only 14 of the 72 from 1811 survived (Howley 1915). It is reported that the French may have offered a reward for Beothuk heads in the late 17 th century (Gatschet 1885). However, others said that there was no truth to stories that the Micmacs were brought to Newfoundland by French to kill Beothuk (Pastore 1991). French and English furriers may also have shot Beothuk for furs they were wearing (Blake 1888). In the end the Beothuk were confined to the Southeast interior (Marshall 1996). Shanawdithit was believed to be the last remaining Beothuk Indian. She died in June 1829 of tuberculosis. Some believe that some surviving Beothuk could have been taken into the fold ofthe Montagnais of Labrador. NONOSABASUT Nonosabasut was described as being a strong and powerful chief Unlike most Beothuk, he had a beard. He stood well over six feet tall. Shanawdithit said he was the most powerful hunter and chief in the tribe (Marshall 1996). Others said that Nonosabasut was "noble" (Fraser 1836). 22 John Peyton, Jr., an important furrier & fisherman in Notre Dame, killed him while capturing Nonosabasut's wife, Demasduit in March 1819 (Pastore 1991). Nononsabasut tried to negotiate with the captors, but when they refused, he became enraged (Marshall 1996). Members of Peyton's group shot Nonosabasut in the head and killed him. Shanawdithit made a sketch ofthe capture ofDemasduit and the killing of her husband (Fig 5). W.E. Cormack found the burial hut which contained the remains ofNonosabasut, his wife (Demasduit), and their infant child along with others on the northwestern shore of Red Indian Lake during his last attempt to find any living Beothuk in Newfoundland in 1826. There was a boy wrapped in birchbark. Nonansabut was on a scaffold in the burial hut. There were two skulls. One skull (Nonosabasut's) appeared to be the skull ofa warrior. It had several wounds. The lower jaw had been cleaved and reconnected in healing. Another wound was apparent that had been caused by a shot. Both the female and the male skull had perfect teeth (Fraser 1836, Marshall 1996, Blake 1888). The two skulls were presented to the late Professor Jameson by Cormack and placed in the Edinburgh Museum (Lloyd 1876, Marshall 1996). They eventually made their way to the Royal Museum of Scotland. Analysis of Ancient DNA DNA molecules which are preserved in ancient biological material are considered to be "ancient DNA" (Brown and Brown 1992). DNA has been extracted from bones as old as 11,000 years old (Brown and Brown 1992). Short sequences of mtDNA from 50,000 to 100,000 year old mammoths preserved in the permafrost have -,': _ .... ' / .. ', --- . " f. "' ... ' "r.. ' / ; /" ;- --", .. ...... :. - ....;: .- .;.- .' Figure 5: Shanawdithit's Drawing of the Killing of Nonosabasut (Marshall 1996). The blue arrow indicates the dead Nonosabasut lying on the ice. The yellow arrow indicates the captive Demasduit. The orange asterisk indicates the paths the remaining Beothuk took as they were fleeing Buchan's group. 23 24 been retrieved (Lindah11997). The most favorable conditions for DNA preservation are those that are cold and dry (Stone and Stoneking 1999). The standard technique for extraction of DNA from bone is to powder the core of the bone. Then, powdered bone is soaked in a buffered salt solution in order to draw out the charged biomolecules. Protein is extracted with phenol. The DNA is then precipitated out using ethanol. More often, molecular biologists trying to isolate ancient DNA are using teeth. This is because the DNA in the dentin is protected by the enamel surrounding the tooth. Due to this, sometimes less degradation is observed in DNA extracted from teeth than, for example, DNA extracted from bones such as a rib. One of the largest problems in dealing with ancient DNA is fungal, algal, and bacterial contamination that can occur both before and after excavation (Brown and Brown 1992). In addition, ancient DNA undergoes fragmentation over time. This fragmentation varies depending upon the conditions of preservation such as the amount of heat and humidity and the extent of microbial attack. Most ancient DNA molecules are about 100 to 200 bp in length, although some have been found to be as large as 500 bp (Brown and Brown 1992). However, this fragmentation appears to occur very soon after death (within 4 years) (Paabo et al. 1989). Therefore, although more fragmentation may occur over time, the age of the DNA may be less of a factor than previously expected. Overlapping fragments of the fragmented DNA can be sequenced and combined in order to reconstruct longer sequences. 25 Contamination Concerns with Ancient DNA Contamination can occur with the DNA of those who handle the remains of a body before it is buried. In addition, contamination from modem DNA may occur during handling ofthe remains by archaeologists, museum keepers or laboratory personnel. Ancient DNA can be further contaminated by the sloughing off of modem dead skin cells, dandruff, sweat, saliva, or blood (Brown and Brown 1992). Other examples of contamination that can occur are bacteriological, fungal, or algal contamination. The presence of previous PCR products on tools used in genetic analysis are one of the most common contaminants of ancient DNA. Many laboratories that work with ancient DNA have gone so far as to create separate laboratories for this sensitive work. These laboratories often contain separate ventilation systems. In addition, lab workers are often required to wear face and hair coverings to prevent modem DNA contamination (Paabo 1993). Further methods of preventing contamination of ancient DNA have been described in the literature. These methods include the use of blocked pipette tips to prevent aerosol contamination, irradiation ofthe outer surface of bone with UV light, ''negative'' extractions (no sample, but all other components), sequencing ofPCR products being performed on both the light and heavy strands, and wearing gloves and surgical masks throughout all procedures (Ribeiro-Dos-Santos et aZ. 1996). PCR as a Method to Study Ancient DNA The first time PCR was used to study ancient DNA was the amplification of DNA from a 7000-year-old brain found in Little Salt Spring in Florida (Paabo et aZ. 1988). It 26 has been stated that PCR products amplified from ancient DNA more than a few hundred base pairs in length should be considered as suspect (Kurosaki et al. 1993). Due to its degradation, nested PCR is often required in order to get amplification of ancient DNA. In nested PCR, an initial amplification is performed using external primers. A subsequent amplification is then performed using more stringent annealing temperatures or primers that are more internal to the PCR product. Whenever PCR is performed on D N ~ there is always a chance that the polymerase being used will introduce, substitute or skip bases during polymerization. There are several steps that can be taken in order to minimize this possibility. The first step is to use higher annealing temperatures to increase stringency. It is also often helpful to include a G or C residue at the 3' end of a PCR primer. G's and C's have a triple hydrogen bond versus the double hydrogen bond ofthe A's and T's. This helps to prevent mis-priming, when the primer matches with sequences of partial homology during PCR. Additionally, several types of polymerase can be employed to ensure more stringent base-pairing during amplification. High fidelity polymerases such as Red Taq polymerase (Sigma-Aldrich, St. Louis, MO), Biolase polymerase (Midwest Scientific, St. Louis, MO), and Tjl polymerase can be used. Also, polymerases with proofreading ability, such as Pfu polymerase (Stratagene, La Jolla, CA), are available for use in PCR. Finally, PCR results can be verified through repetition ofPCR from the beginning template to verify the reliability of previous results. 27 Various molecular methods have been used to study ancient DNA. In one instance, cloning was performed on 23 mummies dating from 2370-2160 BC by Svante Paabo (1985). DNA was extracted from dried tissues and blunt-ended with Klenow fragment in the presence of trace amounts of radioactively-labeled nucleotides. The reaction mixture was then run through a gel. Larger fragments were extracted and cloned into the bacterial plasmid, pUC8. The clones were then transferred to nitrocellulose fibers and probed with a radioactively labeled probe containing a member of the Alu family of repetitive human sequences. The cloned inserts were then sequenced. However, this cloning method was found to introduce errors into the ancient DNA. In addition, the specificity of hybridization is reduced by the presence of chemical modifications within the target DNA (Paabo 1985). A more recently used method to study ancient DNA is the study of Y- chromosomes. Y -chromosome analysis allows for information about population relationships along with information about the paternal genetic contribution. Y- chromosome analysis was used to study the origins of the Japanese and the origins of Neolithic European farmers. In Y-chromosome analysis, male-specific DNA fragments are amplified using PCR primers. The PCR products are sequenced and the individuals are placed into Y -chromosomal haplogroups based on sequence analysis. This information is then compared to information provided by maternal lineages. Through Y- chromosome analysis, a paternal Asian haplotype was found providing evidence of a paternal Asian contribution to northern European populations (Zerjal et al. 1997). This method provides a comparison for samples on which mtDNA analysis has already been performed and will probably become more prevalent in future studies. 28 Another method of studying ancient DNA is micro satellite analysis. Microsatellites are nucleotide repeats, usually around 10 bp in length, that are highly polymorphic throughout the genome. In one method of micro satellite analysis, ancient DNA is amplified using peR primers to areas where the specific micro satellites being used are more prevalent (micro satellites can be selected from previously published papers or genome databases). The peR products are then run on gels, transferred to blots, and probed with radioactively-labeled micro satellite probes. Polymorphisms are evident between individuals when the probes hybridize to peR products of different sizes. However, this method has not been found to be easily repeatable between laboratories. An additional use for peR when working with a limited supply of irreplaceable DNA is to create a library as described by Weiss et al. (1994) (Fig 6). In order to create this library, ancient DNA is digested with a four-base specific restriction enzyme (which is a frequent cutter, such as HaeIII) that produces a blunt end. Adaptors are then ligated to the blunt ends of the digested fragments. Primers specific to the adaptors can then be used in order amplify the fragments which were pooled to create a library. Lastly, a second set of amplification can then be performed using primers specific to areas of the mtDNA of interest for haplotyping. Mitochondrial DNA Analysis Properties of Mitochondrial DNA The mammalian mitochondrial genome is 16,569 bp in length and encodes 13 proteins (Kiechle et al. 1999, Anderson et al. 1981). The mtDNA is double-stranded, containing a light and a heavy chain. The light chain is the template for replication while 29 Figure 6: Library Construction Flowchart (adapted from Weiss et ale 1994). Steps in the library creation. (1) Ancient DNA template is digested with the 4-base cutter restriction enzyme to create blunt ends. (2) A purchased, double-stranded DNA adapter oligionucleotide is ligated to the blunt ends of the digested DNA. (3) The ligated fragments are amplified using an oligonucleotide that contains the sequence of the smaller adapter strand as a PCR primer. (4) The library is ready for marker-specific PCR amplification. 30 s ..... ~ . ........................ MDNA ~ lone adapter --"-"--,-,--"., . ,."'_111,. adapter annealing '1,,,1,., __ ~ . denaturation ==_ Gene-specific primers annealing -IIIII1III. extension 31 the heavy chain is the chain from which protein is transcribed. Most of these proteins are involved in the electron transport system. Mitochondria are unusual in that all of the rRNAs and tRNAs needed for mitochondrial translation are encoded for in the mtDNA (Anderson et al. 1981). Four ofthe genes the mtDNA encodes are the genes for cytochrome c oxidase subunit I (COl), COIl, ATPase, and cytochrome h. The other proteins needed for mitochondrial replication are encoded in the nDNA, synthesized in the cytosol, and imported into the mitochondria (Kiechle et aZ. 1999). The origin of replication in the mtDNA occurs in the displacement loop (D loop). The D loop, also referred to as the Control Region (CR) or First Hypervariable Region (HVI), is the only region of the mtDNA where noncoding bases are prevalent (Anderson et al. 1981). Because of this, most sequence divergence between individuals in the mtDNA exists in this region. Through advances in molecular techniques, it is now sometimes possible to recover DNA from ancient skeletal remains. Although this DNA is most often highly fragmented, it is usually possible to amplify revealing regions ofthe DNA using PCR. The most successful, therefore most widely used, analysis of ancient DNA is mitochondrial DNA (mtDNA) analysis. Although mtDNA represents only a small percentage of the DNA in an individual, it is present in high copy number (Brown and Brown 1992). Therefore, even though nDNA may be highly degraded, there is often enough mtDNA still present in ancient DNA to be amplifiable. Thus, the use of mtDNA as a template for polymerase chain reaction (PCR) increases the likelihood of successful amplification. 32 Mitochondrial DNA has a high mutation rate. This ensures differentiation between populations. The high mutation rate exhibited by mtDNA facilitates more informative analysis than nuclear DNA (nDNA). This is due to the greater number of nucleotide differences observed in the mtDNA when a nDNA and mtDNA sequence strand ofthe same size are compared. In addition, the mtDNA is inherited maternally (there is very little, ifany, recombination with the paternal mtDNA) permitting the tracing of a maternal lineage. This means that mutations occur sequentially with time on the mtDNA, in essence forming a biological recordbook. Previously, it was thought that all children inherit maternal mtDNA, but only daughters will pass that mtDNA on (Kiechle et al. 1999). However, one study has accredited from one one-thousandth to one one-hundredth of mtDNA in an individual to paternal mtDNA (Gyllensten et al. 1985). This paternal mtDNA was found to rarely recombine with the maternal mtDNA. Both normal and mutated forms of mtDNA may be inherited maternally (Kiechle et al. 1999). Heterogeneity Different forms ofmtDNA can exist within the same mitochondrion (Kiechle et al. 1999). Heteroplasmy, the existence of both normal and mutated mitochondrial DNA in a cell, was previously described in literature (Liang and Wong 1998). The opposite of heteroplasmy, homoplasmy, means that a tissue only contains one form ofthe mtDNA, normal or mutated. It has been found in recent studies that, contrary to what was previously believed, homoplasmy is very unusual within an individual (Kiechle et al. 1999). Genetic heterogeneity does occur in mitochondrial DNA (Liang and Wong 1998). Cell fusion studies have shown that at least two mitochondrial genotypes can coexist in the same cell in vitro (Oliver and Wallace 1982). One study of bovine mitochondrial D loops concluded that somatic and genn line mammalian cells are likely significantly heteroplasmic (Hauswirth et al. 1984). 33 Mutations of the mtDNA usually occur more often and accumulate in eukaryotic cells as aging commences. This heteroplasmy has been shown to occur at different rates within an individual in different tissues. For example, one study showed that a set of deletions occurred at a more rapid rate in skeletal muscle than in cardiac muscle (Kopsidas et al. 2000). However, no study was found in which bone or dentin mtDNA mutation rates were compared to other tissues. The presence ofheterplasmic mtDNA in an individual's tissue can complicate genetic analysis. When using PCR to ampli:fY heteroplasmic mtDNA, one fonn of the mtDNA can be preferentially amplified over the other, especially if one sequence of DNA is much more common than another. However, ifboth forms are amplified, assigning an individual to a specific haplotype by restriction analysis of an amplified region may be difficult. A study was perfonned that showed that single bands of mtDNA PCR products could show sequence heterogeneity when they underwent restriction analysis (Baumer et al. 1994). Methods Used to Detect Heterogeneic mtDNA In order to detect heteroplasmic mutations in mtDNA, multiplex PCR has been perfonned to ampli:fY the area of mtDNA of interest. Then, probes to the mutant and the normal fonn of the mtDNA were used in a dot blot. Binding of both the normal and 34 mutated probe indicated heterogeneity in the individual (Liang and Wong 1998). In order to perform this procedure, however, the possible points of mutation would have to be known to create the appropriate oligonucleotide probes. Long PCR (XL-PCR) is another method that can be used in order to detect heterogeneic mtDNA. The entire mtDNA chromosome can be amplified using XL-PCR. Changes in the sequence length when XL-PCR is performed are indicative ofmtDNA deletion or insertion mutations (Kopsidas et al. 2000). However, it is possible that the the DNA may be altered in such a way, such as a point mutation, that size differences indicative of heterogeneity would not be observed. This is a weakness of this PCR technique. XL-PCR utilizes a Taq polymerase along with a proofreading polymerase (such as Plu). It has been reported that XL-PCR may preferentially amplifY shorter, deleted mtDNA sequences over full-length mtDNA (Nagley and Wei 1998). There is also a concern that XL-PCR may generate PCR artifacts that could be interpreted as deletions (Lightowlers et al. 1999). However, a study addressing the previous concerns was carried out. It indicated that the use ofXL-PCR to study mtDNA heterogeneity is reliable if the PCR conditions are optimized, a proofreading enzyme is used, appropriate controls are used, and good primer design is implemented (Kopsidas et al. 2000). Mechanism for D Loop Replication Heterogeneity is often found in a 5' homopolymer-rich region of the D loop. A mechanism for generation of the heterogeneity has been suggested. In this model, a primer or template "slips" in relation to its complement because of the presence of a 35 repeating nucleotide to which the primer can align (Fig 7). A DNA polymerase is then able to generate insertions or deletions due to the slippage. Usually, between 2-6 extra C's were inserted within a stretch of6 C's found at np 568-573 (Torroni et al. 1994a). This resulting stretch ofC's is near to two 7-bp repeats at np 302-308 (designated box 1) and np 567-573 (designated box 2). Box 2 was found to be responsible for the 270 bp portion between box 1 and box 2 sometimes being replicated twice. This is thought to occur when a newly synthesized box 2 (which after homoplasmic insertion ofC's sometimes yields as many as 12 of 13 bp homologous with box 1) aligns with box 1 in the template strand. In this manner, heteroplasmy in the D-Ioop region is and has been created (Hauswirth et al. 1984, Torroni et al. 1994a). Pseudogenes Another source of difficulty in genetic analysis arises because there are pseudogenes in the nuclear DNA (nDNA) that may be amplified using primers specific to the mtDNA (Kiechle et al. 1999). nDNA sequences which are homologous to portions of mtDNA have been found for almost the entire mtDNA genome. These homologous regions are evidence of past and continuing evolutionary lateral movement of DNA within cells. IfPCR is used to amplifY mtDNA and the nucleotide sequence is unusual or if more than one band or more bands than expected are seen, it is a possibility that a pseudogene and surrounding nDNA sequences have also been amplified (Hu and Tilly 1995). Other studies have proposed that the previously described heteroplasmic mtDNAs were, in fact, derivatives of ancient mtDNA variants preserved as nuclear pseudo genes (Wallace et al. 1997). 36 additional C's w Box 1 A Dllit , Daughter light strand i
polymerization Box 2 0" B I 1 , 1""1 -r- ON t , Mispairing of the newly C r= I synthesized box 2 with .. 10 box 1 and extension OR t , , D J I .... 2 1":-
../ \..J Next mtDNA replication / Loop excision
0" 2 t , 2 i I liIIop'"l!UNlO'I r:J __ .-L ___ __ __ ... ;z
l 2 Breakpoint joI'( ? II Polymerization and Ligation Figure 7: D Loop Replication System Mechanism Model (Torroni et al. 1994a). Hypothesized model of the origin ofthe 270-bp tandem duplication in mtDNAs with the insertion of2-6 C's in box 2. During the polymerization of the daughter light strand (thinner line), by use of the parent heavy strand (thicker line) as a template (A), the newly synthesized box 2 with the additional C's could pair with box 1 on the parental heavy strand (B and C). The region between boxes 1 and 2 would then be copied twice (D). A wild-type mtDNA molecule will be generated if the loop is excised. Otherwise, at the next DNA replication, an mtDNA molecule with two copies of the 270-bp sequence that are separated by an additional box 2 would be produced. ON , 37 Mitochondrial D Loop The mtDNA displacement loop (D loop) is a 1,122 bp noncoding region of the mtDNA in which the initiation of DNA replication occurs (Kiechle 1999). The D loop is sometimes referred to as the first hypervariable region (HV1) (Stone and Stoneking 1999) and is the most heterogeneic portion of the mtDNA (Kurosaki et al. 1993). Since no functioning genes are present, the D-Ioop region is the most variable region of the mtDNA in both its sequence and its length (Greenberg et al. 1983). Mutations within the D loop are used to help define haplogroups (individuals grouped by mtDNA due to its variability). Therefore, sequencing of the D loop is highly informative. It often provides critical information on the origins of an individual. NATIVE AMERICAN mtDNA HAPLOGROUPS Development of American mtDNA Haplogroups Previous genetic studies ofmtDNA have strongly implied that all modem Native Americans derived from Asian ancestors. That some mtDNA polymorphisms are specific to a continent has turned out to be very useful for the purposes of identifying human origins. For example, the first clear evidence that mtDNA variation correlated with the geographical origin of an individual came from studies on African mtDNAs. They showed that roughly 70% of all sub-Saharan African mtDNAs contained a single C to T base change at mtDNA np 3594. This base change defines the most common African mtDNA haplogroup, which is designated Haplogroup L. In other words, all individuals that contain the np 3594 C to T mutation in their mtDNA belong to 38 Haplogroup L. The np 3594 polymorphism (and thus Haplogroup L) is essentially unique to Africa and thus allow for a rapid assignment of an individual to Africa based on mtDNA analysis (Batzer et al. 1994) Most ofthe planet's major population groups (Africans, Asians, Caucasians, Native Americans) are comprised of only a small number of mtDNA haplogroups which are typically unique to that group. There is very little overlap between the groups. Analysis of over 800 Native American mtDNAs from North, Central, and South America has identified only 5 founding mtDNA haplogroups. These haplogroups are characterized by the presence or absence of polymorphisms within mtDNA genes (Fig 8). They have been designated as haplogroups A, B, C, D, and X. Together, these five haplogroups account for roughly 98% of all Native American mtDNA. However, mtDNA haplogroups are not found homogeneously among specific Native American groups (Fig 9). Rather, one haplogroup is usually predominant within a group while other haplogroups are also present. Haplogroups A, B, C, and D have also been identified among modem Asians (Lorenz and Smith 1996). Haplogroup X is rarely found in Asians but is often found in Europeans. There are 10 mtDNA haplogroups assigned to Europeans: H, I, J, K, M, T, U, V, W, and X. They account for nearly 98% of European-derived mtDNAs. A summary ofthe polymorphisms which characterize each Native American mtDNA Haplogroups is shown in Table 1. Haplogroup A is characterized by the presence of a HaeIII restriction site at np 663 (Lorenz and Smith 1996, Schurr et al. 1990, Torroni et al. 1993a). Haplogroup A is the most common haplogroup found in Native Americans. In addition, it is the only
DNA 9-bp deletion 39 Figure 8: Diagram of Native American mtDNA Polymorphism Locations (Stone and Stoneking 1993) Human mtDNA genome, showing the approximate location of four of the mtDNA markers that delineate contemporary Native American mtDNA lineages. Gene abbreviations: 12S rRNA, 12S ribosomal RNA gene; N2 and N5, subunits 2 and 5 ofNADH-dehydrogenase, respectively; COIl, subunit II of cytochrome oxidase; and lys tRNA, lysine transfer RNA gene. Arctic/ Subarctic UtD .5 f ABC 0 Northwest
,$ _____ J o A Be Q ' - ,'-.,... -- . ,.on ':1l .J A seD "':.' Figure 9: Distribution of mtDNA Haplogroups Among Native Americans (Lorenz and Smith 1996). Frequency distributions of 4 ofthe Native American mtDNA haplogroups (Haplogroups A, B, C, and D) by geographic regions. 40 Table 1: Polymorphisms which Characterize Each Native American mtDNA Haplogrou 41 MtDNA Lineage (Ha logroup) 9-bp Deletion Haell-663 AluI-5176 Hincll-13259 A + + + B + + + c + D + x + + haplogroup found in the Arctic/Subarctic region (Lorenz and Smith 1996). This haplogroup is also commonly found in the Han Taiwanese (Torroni et af. 1994b). Haplogroup B is characterized by the presence of a 9-bp deletion at np 8272 (Lorenz and Smith 1996, Schurr et al. 1990, Torroni et af. 1993a). It has been reported that the Southwest Native American groups have the greatest frequency of Hap log roup B (Lorenz and Smith 1996). Haplogroup B is almost entirely absent in the northern Native American groups. This haplogroup is also found in modern Asian, Melanesian, and Polynesian groups (Torroni et af. 1994c). Haplogroup C is characterized by the presence of an AfuI site at np 13,262 and the loss ofa HincH site at np 13,259 (Lorenz and Smith 1996, Schurr et al. 1990, Torroni et af. 1993a). Haplogroup C occurs at a frequency of about 17% in North American Native Americans (Lorenz and Smith 1996). Haplogroup C is also found at high frequencies in the Aboriginal Siberian populations (Torroni et at. 1993b). Haplogroup D is characterized by the absence of an AfuI site at np 5176 (Lorenz and Smith 1996, Schurr et al. 1990, Torroni et af. 1993a). In addition to Native Americans, haplogroup D is found in numerous Asians and Siberians. 42 Haplogroup X is characterized by the creation of HaelII restriction sites at np 16223 and 16278 in the D-Ioop region along with the loss of a Ddel restriction site at np 1715 (Stone and Stoneking 1999, Brown et aZI998). Haplogroup X has been found primarily in northern Amerindian groups of the North American plains. Unlike the more common Native Amerian haplogroups A, B, C, and D, Haplogroup X appears to be absent in northeastern and eastern Asia (where the origin for the remainder of the Native American haplogroups has been assigned). Haplogroup X is also found in low frequencies (about 4%) in modem European populations (Brown et aZ. 1998). Thus, this mtDNA lineage is found in Europe and the Americas, but not in Asia. This implies the possibility that some Native American founders were of Eurasian ancestry (Stone and Stoneking 1999). European Haplogroups Haplogroup H does not have the Ddel site at np 10394 while Haplogroups I-K have this site at this position (Torroni et aZ. 1994a). Haplogroup H, which also lacks an AZul site at np 7025, represents 40% ofall Caucasians (Torroni et aZ. 1994a). The markers which define Haplogroup H have rarely been found in Africans or Asians. About 7 Ya % of all Caucasians belong to Haplogroup I. Haplogroup I is found to a greater extent in Northern Europeans. This haplogroup has the np 10394 Ddel restriction site. In addition, it is characterized by G to A transition at np 1629, 16223, and 16391 in the D loop, a HaeIII site loss at np 4529, a Ddel site loss at np 1715, anAZul site gain at np 10028, and an AvaIl site gain at np 8249 (Torroni et al. 1994a). "\ ~ - - ~ --. -... ~ -. - - ; - ~ ~ - : .. - - ~ -. :.. .. ~ --- --- About 9% of all Caucasians fall into Haplogroup J. Haplogroup J has the DdeI restriction site at np 10394. In addition, it is characterized by a BstNI site loss at np 13704 and a HinjI site loss at np 16065 (Torroni et al. 1994a). 43 About 7 ~ % of all Caucasians belong to Haplogroup K. Along with the presence of the DdeI site at np 10394, Haplogroup K is characterized by a HaeIII site loss at np 9052 (Torroni et al. 1994a). - - - - - - . ; ~ - - . - - ~ . - - ~ - - . -- - -.- y y- - MATERIALS AND METHODS SAMPLE A tooth belonging to the Beothuk chief, Nonosabasut, was obtained from the Royal Museum of Scotland. W.E. Cormack obtained the skull containing the tooth in 1826. It was presented to Dr. Jameson in 1921 and eventually placed in the Royal Museum of Scotland. In order to rid the tooth of surface contaminants, the following procedure was performed. The tooth was washed in a 10% bleach solution for 2 min. It was then rinsed in RNase- and DNase-free water for 3 min. The outside of the tooth was irradiated with ultraviolet light (260 run) for 7 min (Ribeiro-Dos-Santos et al. 1996). Dr. Neal Lambert, DDS powdered the remaining dentin of the tooth (a previous drilling had been performed on the tooth). Dr. Lambert washed the burr used for the drilling in alcohol and autoclaved it. Dr. Lambert wore powder-free gloves, a long- sleeved lab coat, and a face mask while drilling the tooth. The integrity of the crown remaining was maintained. Dr. Lambert stated that the enamel of the tooth was worn completely off in some spots. Precautions Taken When Working With the Ancient DNA Several precautions were taken when working with the Beothuk DNA in order to prevent contamination. From the time the tooth was drilled, only one individual handled the tooth or any of the reagents involved in the research. That individual's D loop was sequenced for comparison with the Beothuk sequence to confirm that there was no contamination. 45 Negative control extracts were carried throughout the entire process. If contamination was seen in the negative control, all results were considered suspect. Reagents such as molecular grade water and the PCR lOX buffer were irradiated with UV light (260 nm) for 15 min along with pipettemen, pipette tips, and all tubes used. Gloves were worn at all times when working with the ancient DNA. In addition, hair was pulled back and a long-sleeved lab coat was worn at all times. Separate pipettemen were used for negative controls. Pipette tips had aerosol barriers and were set aside for ancient DNA research. DNA Extraction The powdered dentin was first preincubated overnight in a soaking solution of proteinase K (Promega, Madison, WI). The proteinase K was reconstituted in 50 mM Tris-HCI (PH 8.0) and 10 mM CaCh (calcium chloride). The overnight soaking solution composed of 5 mL of 0.5 M EDTA (PH 8.0), 200 f-lL of 10% SDS, and 200 f-lL of20 mg/mL proteinase K. The powdered dentin was rotated at 37C overnight in the overnight soaking solution. It was then placed in a 65C water bath for 15 min to inactivate the proteinase K. DNA was extracted from the dentin using the GENECLEAN Kit for Ancient DNA (Bio 101, Vista, CA). In parallel to the extraction, a negative control was performed in which the same procedures were carried out without the powdered dentin. 46 One rnL of the Dehybridization A solution from the GENECLEAN Kit for Ancient DNA was added to the tooth/proteinase K solution. It was rotated for 2 ~ hr at 60C. The sample was then centrifuged at 14,000 x g for 5 min in order to pellet the particulate material. The supernatant was transferred to a new micro centrifuge tube and 1.2 mL glassmilk: and 3 mL Dehybridization A solution were added. The sample was then rotated at 37C for 2 hr. The samples were centrifuged for 1 min at 5,000 x g in order to pellet the Ancient DNA Glassmilk containing the DNA. The supernatant was discarded and 0.5 mL of Salton Wash #1 was used to resuspend the pellet. The suspension was then transferred to a Spin Filter and centrifuged at 14,000 x g in order to clean the Glassmilkl DNA complex. A second wash was performed in the same manner using 0.5 rnL of Salton Wash #2. In order to ensure a clean product, a 1: 1 acetone:ethanol wash was performed that was not called for in the GENECLEAN Kit for Ancient DNA protocol (Sambrook and Russell 2001). Then, 0.5 rnL of Ancient DNA Alcohol Wash was added and the solution was centrifuged. The Glassmilk Spin Filter was dried by additional centrifugation. The dry filter was placed into a new microcentrifuge tube and 50 ~ L of DNA-free Elution Solution was added to the filter. The pellet was resuspended by a 2 s vortex. The tube was centrifuged for 1 min to transfer the eluate to a catch tube. An additional 50 ~ L of the Elution Solution was heated and added to the filter. Centrifugation ensured complete elution ofthe DNA. The two 50 ~ L eluates were combined to create eluate #1. A third elution was then carried out using 1 00 ~ L heated Elution Solution to create eluate #2. Eluates # 1 and #2 were stored at -20C. -- -- _ __ ~ . r - ~ _ ~ ~ . _ - ~ . .._. _ ._ 47 Library Creation The library was created as descnbed in Weiss et al. 1994 as follows: Digestion The DNA extracted from the sample was digested in order to create blunt -ended DNA for ligation with the blunt-ended adaptors. A parallel procedure was performed on the negative control from the DNA extraction. The reaction mixture consisted of 100 flL of the DNA from eluate #1, 11.3 fLL of the lOX buffer provided with RsaI, and 1.5 fLL of 10 U/fLL (15 U total) RsaI (New England Biolabs, Beverly, MA). The total reaction volumes of 113 fLL were incubated overnight in a 37C water bath to insure complete digestion. The reactions were then inactivated at 65C for 20 min. This was done to prevent the RsaI from re-digesting the DNA and, thus, preventing the adaptors from ligating. A second library was needed in order to amplifY areas of the mtDNA (containing the markers of interest) that, it was determined from the Cambridge reference sequence (Anderson et al. 1981), had RsaI restriction sites. A parallel procedure was performed with the negative control from the DNA extraction. Therefore, eluate #2 was digested with HaeIII (New England Biolabs). The reaction mixture consisted of 100 fLL of eluate #2, 11.3 fLL of the lOX buffer provided with the HaeIII, and 1.5 fLL of 10 UI fLL HaeIII (15 Units total). The DNA was digested overnight at 37C. The HaeIII was inactivated at 80C for 20 min. The eluate were not quantitated because most of the material would have been lost during quantitation. - -- ~ - ~ - . . . - - ~ - - ~ - 48 Ligation Two single-stranded, complementary oligonucleotides, LLSa12A and LLSa12B (5'-pTCGAGTCGACTATATGTACC-3' and 5'-pGGTACATATAGTCGACT-3', respectively) described by Weiss et al. 1994 were purchased (Integrated DNA Technologies, Inc. Coralville, IA) that, when annealed, became double-stranded blunt- end adaptors with a three nucleotide overhang on one side. The oligonucleotides were purchased phosphorylated on the 5' end in order to block the ligation of the adaptors to themselves. In addition, the phosphorylation would aid in the ligation of the blunt-ended adapters to the blunt-ended fragments. The single-strand oligonucleotides were added in equal portions (10 f-LL each) to a microcentrifuge tube. They were placed in an 80C water bath for 5 min. The water bath was removed from the heat and allowed to slowly cool to room temperature. This allowed the single-stranded oligonucleotides to anneal to each other to form double-stranded adaptors with a protruding, phosphorylated 5' end and a blunt end for ligation with the blunt-ended (digested) DNA The blunt-ended DNA and the separate negative control was ligated to the blunt- ended adaptors using T4 DNA Ligase (New England Biolabs). Fifty f-LL of the RsaI or HaeIII-digested DNA (or the corresponding negative controls) were placed in a micro centrifuge tube with 2 f-LL of adaptors (final concentration of each of the primers was 0.5 mglmL), 6 f-LL 10X buffer with ATP provided with the T4 DNA Ligase and 2 f-LL ofthe 400 VI f-LL T4 DNA Ligase (800 Vnits total). Ligation was performed at room temperature for 15 min, at 15C for 2 Y2 hr, and then at 4C overnight in an effort to maximize ligation of adaptors. Library Amplification In order to amplifY the entire hbraries prior to haplotyping, DNA fragments with the adaptors ligated to them were amplified using the shorter single-stranded oligonucleotide, LLSa12B. Again, a parallel procedure was performed on the negative controls. The 100 pL PCR reaction mixture contained 20 J1L of the ligated material, 10 J1L of the 10 X buffer provided by the manufacturer, 5 J1L of 50 mM MgCh, 2 uM:final concentration ofLLSal2B, 2 J1L of 12.5 uM dNTP master mix, 1.0 mL of 100 X BSA, 4 Units ofBiolase Red Taq polymerase (Midwest Scientific, St. Louis, MO), and autoclaved molecular grade water to volume. Master mixes (excluding the polymerase and nucleotides) were made in order to ensure consistency between samples. A second master mix containing the heat-sensitive polymerase and nucleotides was added after an initial hotstart of 5 min at 94C in a Perkin Elmer 2400 Thermal Cycler. PCR conditions were as follows: 30 cycles of 94C for 30 s, 53C for 1 min, 72C for 2 min and, finally, 72C for 7 min. Twenty J1L of each PCR product was electrophoresed on a 3.5% agarose gel in 1 X TBE prestained with 2.5 J1L of 1 mg!mL ethidium bromide (EtBr) in order to determine if the library had amplified. The remaining 80 J1L of each sample was stored at -20C until needed for haplotyping. Ethanol Precipitation An ethanol precipitation was performed on the ligated material due to what appeared to be excess salts observed in the gel analysis of the PCR products. Two 49 50 volumes of cold 95% ethanol and 1110 volume of3 M sodium acetate (pH 5.4) was added to each ligation mixture. The mixture was placed in a -70C freezer for 20 min. The mixture was spun at 14,000 x g until the DNA pelleted. The supernatant was poured off and the pellet was allowed to dry completely (about 10 min). The pellet was resuspended in 50 f-LL of molecular grade water. Ten f-LL of the ligated, ethanol- precipitated DNA was used as template to repeat the PCR to amplifY the hbrary. The remainder ofthe ethanol-precipitated DNA was stored at -20C. Additional Library Purification Two additional methods of purification were used on the amplified library to remove excess buffer (salts), nucleotides, and primers that would have interfered with subsequent amplifications. A kit already available in our laboratory, Wizard PCR Preps DNA Purification System (Promega, Madison, WI), and a newly purchased product, Microcon PCR (Millipore, Bedford, MA) were both used. The library amplification products were originally cleaned with the Wizard PCR Prep. The entire PCR product (50-100 f-LL) was added to 100 f-LL of Direct Purification Buffer in a 1.5 f-LL micro centrifuge tube. One mL of the resin was added and the mixture was vortexed 3 times over 1 min. A 30 cc syringe barrel was attached to a Wizard minicolumn. The resin and PCR product mixture were pipetted into the syringe barrel and slowly pushed through the minicolumn. The syringe was detached and the plunger removed. The barrel was then reattached to the minicolumn. Eighty percent isopropanol was added to the barrel and slowly pushed through the minicolumn using the syringe plunger to rinse the DNA adhered to the resin. The minicolumn was removed and 51 transferred to a clean 1.5 mL microcentrifuge tube. Then 50 pL of molecular grade water was added to the minicolumn. After waiting 1 min, the minicolumn was centrifuged at 10,000 x g for 20 s to elute the bound DNA fragments. The purified amplified horary was used in a subsequent gene-specific amplification or stored at -20C. Microcon PCR was used in order to purifY and concentrate PCR products rather than the Wizard PCR Purification Prep after contamination was observed in subsequent PCR negative controls and poor sequencing results were obtained. The Microcon PCR column was placed in a 1.5 mL micro centrifuge tube. The PCR product and 400 ILL of molecular grade water were placed in the sample reservoir. Additional water was added to bring the total volume up to 500 ILL. The column and tube with the sample were spun in a centrifuge at 1000 x g for 15 min. The column was removed from the tube, inverted, and placed in a clean 1.5 mL microcentrifuge tube. Twenty ILL of molecular grade water was added to the reservoir without touching the membrane surface. The column and tube were centrifuged at 1000 x g for 2 min. One ILL of the cleaned, concentrated PCR product was then used for subsequent gene-specific PCR amplification or for sequencing. The remainder ofthe PCR product was stored at -20C. ANALYSIS OF NATIVE AMERICAN MARKERS Marker-Specific Amplification A hierarchical strategy was employed in order to analyze the mtDNA of the Beothuk chief The most definitive sites for Native Americans were looked at first. These sites included the HindI site at np 13259, the AZul site at np 5176, the HaeIII site 52 at np 663, and the 9-bp deletion in the gene for subunit II of the cytochrome oxidase. In addition, the D-Ioop region, where most heterogeneity is seen was amplified. When testing for Caucasian- European mtDNA haplogroups, the 7025 AZul restriction site was examined first. The absence of the site in large part defines Caucasian Haplogroup H (the most common mtDNA haplogroup in European-derived populations). Haplogroup H is found in about 40% of all modem Europeans and North American CaucaSian. In addition, it is the most common mtDNA haplotype found in ancient Basque samples. All of the Native American primer sequences are shown below in Table 2. The D-Ioop and the European Alul-7025 primer sequences were designed using the primer Table 2: Primers and primer sequences used to analyze the mtDNA markers in the Beothuk chief Marker Primers 5'->3' Sequence 9bp-deletion 9bpLI ACAGTTTCATGCCCATCGTC 9bpH I ATGCTAAGTTAGCTTTACAG HincII-13259 HincIIL l CCCTTACACAAAATGACATCAA HincIIH 2 TCCTATTTTTCGAATATCTTGTTC HaeIII-663 HaeIIIL l ACCTCCTCAAAGCAATACACTG HaeIIIH2 GTGCTTGATGCTTGTTCCTTTTG AluI-5176 AlulL l TAACTACTACCGCATTCCTA AluIH 2 GTGCTTGATGCTTGTTCCTTTTG D Loop* L15997 CACCATTAGCACCCAAAGCT H16401 TGATTTCACGGAGGATGGTG AluI-7025* 7025L ACATCGTACTACACGACACGT 7025H GGCGTAGGATTGGACTAGGGT IL denotes a primer to the light chain of the mtDNA, while H denotes a primer to the heavy chain. 2Primer sequences taken from Stone and Stoneking 1993 Those primers with an * were designed in our laboratory using the Integrated DNA Technologies website (www.idtdna.com) 53 design program on the Integrated DNA Technologies website (www.idtdna.com). The Cambridge mtDNA sequence was used as reference sequence (Anderson et al. 1981). When possible, primers were designed that were between 18-25 bp long, had about 50% GC content, had annealing temperatures between 45 and 60C, did not form primer dimers, did not form hairpin loops, had more G's and C's at the 5' end and more T's and A's at the 3' end. In addition, the primer pair had annealing temperatures within 5 of the each other. Lastly, the area of the mtDNA being amplified had to be free of either HaeIII or RsaI restriction sites. Four PCR reactions were set up in order to amplify regions of the Beothuk mtDNA containing the Native American markers that were less than 250 bp long. In addition, a PCR reaction was set up to amplify a 440 bp portion of the D loop. The initial reactions were performed as described in Stone and Stoneking 1993. The 100 J-LL PCR reactions contained 5 J-LL of the appropriate cleaned library (Table 3), 10 J-LL ofl0 X buffer supplied with the polymerase, 2.5 mM:final concentration of the dNTPs, 40 pmoles each primer, 1 J-LL 100 X BSA (New England Biolabs), 5.0 J-LL 50 mM MgCh, 2.0 Units of Red Taq polymerase (Sigma-Aldrich, St. Louis, MO), and molecular grade water to volume. TabJe 3: The library used for amp1i1ication of each Native American mtDNA marker mtDNA Marker Library HaeIII-663 RsaI library AluI-5176 RsaI library 9-bp deletion RsaI library HincII-13259 HaeIII library D-Ioop region HaeIII library 54 A master mix ofthe lOX buffer, MgCh, BSA, and water was made in order to ensure consistency. The negative controls for each library were carried throughout the extraction and hbrary creation process. The samples were placed in the Perkin-Elmer 2400 Thermocycler for 30 cycles of denaturation at 94C for 1 min, annealing at 50C for 1 min, and elongation at 72C for 1 min. A:final elongation at 72C for 7 min was then performed. Each PCR reaction required optimization in order to initially obtain PCR products and then to increase specificity. A series of reactions were performed in which the following PCR reaction conditions were optimized: MgCh concentration, primer concentration, amount of polymerase, type of polymerase, amount of template, and thermo cycling conditions. The following conditions were varied in order to optimize the thermocycling conditions: presence/absence and length of time ofthe hot start, lowering/raising annealing temperature, and single annealing temperature versus touchdown, a PCR reaction in which annealing temperature drops from highly stringent to less stringent during the PCR reaction. The final, optimized thermo cycling conditions for the D loop and Native American mtDNA markers are listed in Table 4. The library templates were reamplified and cleaned as needed. The final, optimized 100 J-LL PCR reaction for the amplification of the mtDNA markers was 5 J-LL library template, 10 J-LL lOX buffer supplied with the polymerase, 5' J-LL 50 mM MgCh, 2 J-LL 50 X dNTP Master Mix (250 J-LM final concentration), 1 J-LL 10 mglmL BSA, 0.4 J-LM each primer, 4 Units Biolase Red Taq polymerase, and autoclaved, molecular grade water to volume. 55 Table 3: Final Thennocycling Conditions for the D loop and Native American Marker Amplification Marker Hotstart Touchdown Cycling Final Extension HincII- 94C for None 94C for 30 seconds 72C for 7 13259 5 minutes 48C for 1 minute minutes 72C for 2 minutes for 40 cycles AluI-5176 94C for 94C for 30 seconds 94C for 30 seconds 72C for 7 5 minutes 66->47C (-1 oC) 48C for 1 minute minutes for 1 minute 72C for 2 minutes 72C for 2 minutes for 30 cycles for lO cycles HaeIII-663 94C for None 94C for 30 seconds 72C for 7 5 minutes 54C for 1 minute minutes 72C for 2 minutes for 40 cycles 9-bp 94C for 94C for 30 seconds 94C for 30 seconds 72C for 7 deletion 5 minutes 57->48C (-loC) 47C for 1 minute minutes for 1 minute 72C for 2 minutes 72C for 2 minutes for 30 cycles for lO cycles Dloop 94C for None 94C for 30 seconds 72C for 7 5 minutes 54C for 1 minute minutes 72C for 2 minutes for 40 cycles An initial hotstart of 94C for 5 minutes was used. A second master mix ofthe polymerase and dNTPs was added to each reaction after the hotstart. The tubes were cooled to 4C, removed from the thennocycler, and spun in a centrifuge briefly before the second master mix was added. This was done in order to avoid cross-contamination of the samples. In addition, aerosol-barrier tips and a separate pipetteman for the negative controls only were used. 56 There was a problem with contamination in the negative controls at one point of time in the process of optimization. However, this contamination was traced through the process of elimination (new reagents, new molecular grade water, and new primers) back to the Wizard PCR Purification Prep Kit. After the contamination was traced, a new method, previously described, employing the use of Micro con PCR devices was used to clean and purify PCR product. Due to the contamination, the remaining ligated material was amplified to create new hbraries. The contaminated libraries were disposed of The new libraries were then successfully used as templates for the marker-specific amplification. Gel Analysis of peR Products The PCR products were electrophoresed on a 3.5% NuSieve 3: 1 agarose gel (FMC Bioproducts, Rockland, ME) in 1 X TBE. NuSieve 3: 1 is a high-resolution gel suggested for analysis oflow molecular weight products. The manufacturer suggested a 3.5% gel for products in the 100 bp range. Fifteen fLL of each PCR product was mixed with 3 fLL 6 X sample buffer and loaded in the gel. A 100 bp ladder (0.5 fLg) (Invitrogen, Carlsbad, CA) was used as a molecular weight and concentration reference. The gel was electrophoresed in 1 X TBE [40 mM tris-borate, 1mM EDTA] at 50V until the blue tracking dye was three-quarters of the way down the gel. The gel was post-stained in 60 mL of 1 X TBE and 6.0 fLL of 10,000 X GelStar (FMC Bioproducts, Rockland, ME) for 20 min. The gel was rinsed in tap water. A photograph of each gel was taken using a transilluminator and UV light at 260 nm. 57 RFLP Analysis After the mtDNA of interest was amplified, restriction digests were performed on the amplified products containing the markers HaeIII-663, HincII-13259, and AluI-5176. This was performed in order to determine if the markers contained the definitive restriction sites. Initially, the peR products were digested without purifying as described in Stone and Stoneking 1994. Later, the peR products were purified using the Microcon peR device in order to remove the excess salts, dNTPs, and primers from the mixture. This was done in order to prevent these contaminants from interfering with the complete digestion of the peR products. Several steps were taken in order to ensure complete digestion. All of the digestions were performed overnight. An excess of restriction enzyme was used in each reaction. Each digestion was repeated at least 3 times. Along with each digestion, a control was performed in which all of the components of the restriction digest were present except for the restriction enzyme. This was to ensure that the cutting was not occurring due to contamination of a buffer or water. Although the peR products were purified using the Microcon peR device, BSA was added to the digestion to prevent the interference of any contaminant in the cutting action of the enzyme. The digestion conditions for all of the RFLP analyses are listed in Table 5. The HaeIII -663 product was digested with HaeIII. The HincII -13259 product was digested with HincH in one reaction and AluI in another reaction. The AluI digestion was performed because the mutation responsible for the HincII site loss (an A to G substitution at np 13263) creates an AluI-13262 site gain (Stone and Stoneking 1993). The AluI-5176 product was digested with A luI. Table 5: Digestion Reaction Conditions for RFLP Analysis of Native American Markers 15.0 fLL cleaned PCR product 2.0 fLL 10 X buffer supplied by manufacturer 0.2 fLL 10 mglmL BSA 2.0 fLL of appropriate 10 UI fLL restriction enzyme 0.8 fLL autoclaved molecular grade water 20.0 fLL total volume * All digestions were performed overnight at 37C Sequencing 58 The PCR products (except AluI-5176) were sent to Davis Sequencing (Davis, CA) to be sequenced in order to verify the validity ofthe RFLP results, or, in the case ofthe D-Loop region PCR products, to obtain sequence results. The initial PCR products were cleaned and concentrated using the Microcon PCR device before sequencing. Re-Amplification of peR Products from Agarose Gel In some cases, sequencing ofPCR products was not possible due to multiple signals. Therefore, an attempt was made to extract a small portion ofthe band containing the marker of interest from the gel and reamplify it for sequencing in order to obtain a 59 clearer signal. The PCR products were run on a 1 % Low Melting Point (LMP) agarose gel (FMC Bioproducts, Rockland, ME) in 1 X TAE [40 mM tris-acetate, 1mM EDTA]. The gel was run in a cold room at 50 V in order to keep it from melting. The end of a 1000 ILL pipette tip was cut off. The tip was then pushed into the band of interest in the gel. A plug of agarose containing the product of interest was drawn up into the tip and released into a microcentrifuge tube. Fifty ILL of 1 X TE was added to the plug. The tube was then heated to 65C for 5-10 minutes (until melted). Twenty ILL of the melted solution was used as template for a duplicate PCR reaction. The new PCR products were analyzed by gel electrophoresis. GenElute Minus EtBr Spin Columns When the previous approachs yielded poor results, all subsequent PCR products that were to be sequenced were prepared in a different manner. Forty ILL ofthe PCR product was electrophoresed on a 1 % Low EEO Agarose Gel (Midwest Scientific) in 1 X T AE. The band of interest was cut from the agarose using a sterile scalpel. A GenElute Minus EtBr Spin Column (Sigma-Aldrich, St. Louis, MO) was prepared by adding 100 ILL of 1 X TE to the column, placing the column in a 1.5 mL micro centrifuge, and performing a 5 s spin at 14,000 x g in a centrifuge. The column was then removed and placed into a new 1.5 mL micro centrifuge tube. The extracted agarose containing the band of interest was placed in the prepared column. The device was centrifuged at 14,000 x g for 10 min to elute the DNA and the column was thrown away. DNA in the eluate was subsequently sequenced. 60 After analysis of the Native American markers was completed, the European marker AluI-7025 was amplified using the primers listed in Table 1. The HaeIII lIbrary was used as a template. Amplification was carried out using the optimized PCR reaction for the other markers. The thermocycling conditions used are listed in Table 6. The PCR product was then extracted from the gel using the GenElute Minus EtBr Spin Columns previously described. The cleaned PCR product was sent to Davis Sequencing to be sequenced. Table 6: Final thermo cycling Conditions for the AluI-5176 mtDNA Marker Amplification Marker Hotstart Cycling Final Extension AluI-5176 94C for 94C for 30 seconds 72C for 7 minutes 5 minutes 54C for 1 minute 72C for 2 minutes for 40 cycles AMPLIFICATION AND SEQUENCING OF INVESTIGATOR'S D- LOOP REGION After all ofthe PCR work was finished with the Beothuk DNA, a sample of the investigator's cheek cells was taken. A sterile toothpick was used to scrape the inside of the cheek. The cheek cells were then place on the triangle of an IsoCode Stix (Schleicher & Schuell) where the triangle was allowed to dry for 10-15 min. The triangle was then placed in a 1.5 mL microcentrifuge tube with 500 fLL of distilled water using the cap of the tube to break off the triangle. The tube was vortexed 3 times over 6 s in order to remove any PCR inhibitors. The triangle was then removed from the micro centrifuge tube (any excess water was wrung from the triangle by twisting the triangle around the tweezers and pushing against the inside of the micro centrifuge tube) and placed into a PCR tube containing the first master mix previously used for amplification of the Beothuk D loop. This was performed using isopropanol and flame-sterilized forceps. 61 The triangle and master mix were heated to 95C in a thermo cycler for 30 minutes in order to elute off the DNA. The PCR reaction was then cooled to 4C, removed from the thermo cycler, and centrifuged. The second master mix containing the nucleotides and the polymerase was pipetted into the mixture. The PCR reaction was then replaced in the thermo cycler and the cycling was performed as with the Beothuk D loop. The PCR product was analyzed by gel electrophoresis. A one percent Low EEO Agarose gel in 1 X T AE stained with 0.5 ILL of 10 mg/rnL EtBr was run at 90 V for 25 minutes. The resulting PCR product band (about 420 bp) was cut from the gel using a sterile scalpel. The PCR product was eluted from the agarose using the Gene1ute EtBr Minus Spin Column described previously. The cleaned PCR product was then sent to be sequenced. CONSTRUCTION OF mtDNA HAPLOTYPE CHARTS Throughout the process of this study, two charts were created using EXCEL (Microsoft Office 2000), which compiled mtDNA data from previously published research since the data in the published research was not reported in a consistent format (the papers the data was compiled from are marked with a superscript letter in the Reference section that refers to the letter representative in the chart reference column). This made it difficult to glean information needed from the papers. These charts were created in order that the results ofthe Beothuk mtDNA analysis could be readily compared to previous findings when the analysis was finished. The charts were also created for eventual use by other groups in the laboratory who are performing mtDNA analysis on additional Native American groups. 62 All of the markers reported in the published reports were converted to a format in which a "1" denotes the presence of a restriction site (or presence of the 9-bp deletion), and a "0" denotes the absence of a restriction site (or absence of the 9-bp deletion). D loop sequences were reported in this same form (as having or not having restriction sites at certain nucleotide positions) in this chart (Appendix A). In a second chart, some D loop sequences were entered as reported differences from the Cambridge (Anderson et a.1 1981) sequence (Appendix B). In all cases, a reference to the paper and the haplotype and/or haplogroup assigned were entered also. In addition, after the mtDNA analysis was completed, the Beothuk sequence was entered. All markers in the chart in common with the Beothuk were highlighted. Ifa marker was not looked at for an entry, a "-" or a blank space was entered (Appendices A & B, attached as a disk). RESULTS AND DISCUSSION POWDERING OF THE DENTIN We were able to obtain 172 mg of powdered dentin from the previously drilled Beothuk tooth. We had hoped to obtain 116 to 113 of the beginning tooth material. We began with 1479 mg of material. Therefore, with the second drilling, we were able to obtain only about 118 to 119 of the beginning material. The tooth had split at a fracture during the previous drilling. However, we were able to maintain the integrity of the crown remaining during the second drilling. GEL ANALYSIS OF LIBRARY AMPLIFICATION After the libraries were amplified, the peR products were run on a 3.5% agarose gel in 1 X TBE with 2.5 fJ.L of 1mg/mL ethidium bromide (EtBr) in order to determine if the library had amplified (Fig 10). Although we expected to see a broad smear of different sizes of amplified fragments, this was not clearly seen. This suggested that sufficient amplification had not occurred. In addition, spreading of the bands near the bottom of the lanes with samples of amplified material (lanes 2, 3,5, & 6) was apparent but not seen in the molecular weight marker lane (lane I), suggesting excessive salt was present. Therefore, an ethanol precipitation was performed. The library was reamplified M RsaI library NC HaeIII NC library M 64 Figure 10: Gel Analysis of the Initial Amplification of the RsaI and HaeIII Libraries. Lanes 1 & 7 contain 0.5 f-Lg of a 100 bp molecular weight marker (Midwest Scientific, St. Louis, MO). Lanes 2,3,5, and 6 contain 15 f-LL ofa 100f-LL PCR reaction. The lanes labeled "M" (Lanes 1 & 7) contain the molecular weight marker. The lanes labeled ''NC'' contain the no DNA negative controls (Lanes 3 & 6). The arrow at the left indicates the spreading occurring in the sample lanes not present in the molecular weight marker lanes. 65 and analyzed by gel electrophoresis (Fig 11). A broad smear was observed in the lanes containing the libraries (lanes 2 and 6). No such smear was seen in the negative control for the RsaI library. A smear was also observed in the HaeIII library negative control (Lanes 5 & 6), perhaps as a result of lane spillage or contamination in this control. The low molecular weight smear at the bottom of the RsaI hbrary negative control (Lane 3), also observed in the RsaI Library (Lane 2), was due to primer-dimers and excess adapters. Although it appeared some library amplification had occurred, a reference was found later that stated the library may not be visible until the second, gene-specific amplification (Weiss et aI1994). GEL ANALYSIS OF MARKER-SPECIFIC AMPLIFICATION Initially, many failed attempts were made to perform marker-specific amplification of the libraries. In the first few amplifications, only very small bands near the bottom of the gel were seen which were believed to result from primer-primer interaction (Fig 12). Once peR products were obtained, they often were not specific enough conditions, and spurious bands were sometimes observed (Fig 13). The 150 bp band in Lane 1 is the desired peR product. The 95 bp band in Lane 1 is a spurious band. A peR product of 400 bp was desired in Lane 2. However, peR products of 460 and 260 bp were observed. Reaction conditions were optimized and annealing temperatures were lowered until specific amplifications occurred. M RsaI NC M library HaeIII NC library M 66 Figure 11: Gel Analysis of Libraries After Ethanol Precipitation. Lanes 1, 4, & 7 contain 0.5 j.Lg of 100 bp DNA Molecular Weight Marker (M). Lanes 2,3,5, & 6 contain 15j.LL ofa 100 j.LL PCR reaction amplifying the RsaI & HaeIII hbraries and their negative controls (NC). The arrow to the left indicates the area of primer- dimer and adapter amplification. 67 M 9-bp NC HincII NC HaeIII NC M Figure 12: Example of Failed Attempts at Marker- Specific Amplification. Lanes I and 8 contain 0.5 fLg of 100 bp molecular weight marker (M). Lanes 2,4, & 6 contain IS fLL of 100 fLL marker-specific PCR reactions. Lanes 3, 5, & 7 contain 15 fLL of a 100 fLL PCR reaction of the respective negative controls (NC). The arrow at the bottom indicates primer-dimers. 68 AluI D Loop M It--- -460 bp * 11--- -260 bp * -150 bp ---. -95 bp*---' Figure 13: Example of Spurious Bands Observed During Optimization Attempts. Lane 3 contains 0.5 jLg of a 100 bp molecular weight marker (M). Lanes 1 & 2 contain 15 JLL of 100 JLL peR reactions. The negative controls are not shown. The asterisks indicate the spurious bands. ----:;;=--- - ~ - - - - - - - - - - ~ 69 First Successful Amplification The first successful amplification of a specific product that occurred was with the AluI-5176, HincII-13259 and HaeIII-663 PCR products (Fig 14). The amplified bands were the sizes expected and no spurious bands were observed. The AluI-5176 PCR product (Lane 2) was 150 bp as expected, the HincII-13259 PCR product (Lane 3) was 210 bp as expected, and the HaeIII-663 PCR product (Lane 4) was 180 bp as expected. Smaller bands (primer-dimers, less than 100 bp indicated by the blue arrows) were present in both these lanes and the negative controls (lanes not shown). The 9-bp deletion would not amplify even at very low annealing temperatures (42C). The primers for the 9-bp deletion region were re-examined. It was determined that the primer sequences were incorrect on the initial order form. The correct primers were ordered and the PCR reactions were quickly optimized (Fig 15). The 9-bp deletion PCR product (Lane 2) was ~ 1 2 0 bp, as expected. The blue arrow indicates prime-dimers present in both the sample and the negative control. When the D-Ioop region was amplified, 2 bands were observed, one at 440 bp as expected, but another at 650 bp (Fig 16). Repeated amplifications were performed at various annealing temperatures. Both bands amplified even when the annealing temperature was increased. Whenever the annealing temperature was changed by a few degrees, additional bands ranging in size from 100 bp to 650 bp were present. It was determined that those conditions which were optimal for amplification of the desired 440 bp product also yielded the ~ 6 5 0 bp product. Three attempts were made to extract the 650 bp band from the gel and sequence it. Informative sequence information was not obtained. -210 bp -150 bp ~ 70 M AluI HincH HaeIH -180 bp Figure 14: Amplification of AluI-5176, HincH13259, and HaeHI-663 Markers. Lane 1 contains 0.5 fLg of a 100 bp DNA molecular weight marker (M). Lanes 2,3, & 4 contain 15 fLL of 100 fLL PCR reactions. The black arrows indicate PCR products of desired size. The blue arrows indicate bands also observed in the negative control lanes (not shown) due to primer- dimers. 71 M 9bp -120 bp Figure 15: Amplification of 9-bp Deletion Marker. Lane 1 contains 0.5 f-ig of 100 bp DNA molecular weight marker (M). Lane 2 contains 15 f-iL of a 100 f-iL PCR reaction. The desired PCR product in Lane 2 is marked with a black arrow. The blue arrows indicate low molecular weight bands also present in the negative control lane (not shown) due to primer dimers. 72 M DLoop NC -650bp --440 bp Figure 16: Amplification of D-Loop Region. Lane 1 contains 0.5 fLg ofl00 bp DNA molecular weight marker (M). Lanes 2 & 3 contain 15 fLL of 100 fLL PCR reactions. The black arrows indicate the desired 440 bp PCR product and the additional 650 bp band that was amplified. The blue arrows indicate low molecular weight bands also present in the negative control (NC) due to primer-dimers. 73 There are several possible explanations for this additional 650 bp band. One explanation is that slippage mechanism occurred as described earlier whereby a homoplasmic insertion of C's creates an opportunity for misalignment of primers. In the published research, this led to a 270 bp insertion (Torroni et af. 1994a). However, it is possible that the Beothuk D Loop differs enough from the Cambridge sequence (we only analyzed about 200 bp of the Beothuk D Loop) that an insertion of about 110 bp could be observed. An additional explanation may be that a pseudo gene in the nDNA was also amplified by the primers to the D Loop. One last possible explanation is that there may have been some sort of contamination in the PCR reaction. However, this is unlikely due to the absence of the 650 bp band in the negative (no DNA) control. Contamination in the Negative Controls At one point in the research, strong PCR products were observed in all of the negative controls (Fig 17). For example, a 150 bp band was observed in the AluI-5176 negative control (Lane 3). This indicated that there was marker-specific amplification without the presence of the library template. In addition, bands ranging in size from 100 to 200 bp were observed in the negative controls and not observed in the marker-specific amplification of the library (Lanes 5 & 7). These bands were also attributed to contamination of the negative controls. It was determined by the process of elimination (as described before) that this resulted from contamination of the Wizard PCR Purification Prep by a previous researcher. The contaminated libraries were discarded. The remaining ligated material (which had never been cleaned with the Wizard PCR 74 M AluI NC HincH NC HaeIH NC M i i i Contamination in the Negative Controls Figure 17: Gel Analysis Showing Contamination in the Negative Controls. Lanes 1 & 8 contain 0.5 f.-Lg of 100 bp DNA molecular weight marker. Lanes 2-7 contain 15 f.-LL of 100 f.-LL peR reactions. These reactions were performed directly after cleaning the initial libraries with a contaminated Wizard peR Prep Kit. Notice the amplification of the marker-specific band 150 bp band (red arrow) in the negative control (Ne, Lane 3) and additional bands not observed in the marker-specific amplification of the library in the negative control lanes (Lanes 5 & 7) indicated by green arrows. Purification Kit) was amplified to create new libraries. The new libraries were used as template to reamplify using gene-specific primers and the optimized peR conditions. The resulting peR products were purified for sequencing using new Microcon peR devices. RFLP ANALYSIS Initial Results 75 The HaeIII-663, HincII-16259, and AZul-5176 peR products were digested overnight as previously described. The undigested controls and digested products were run on an agarose gel (Fig 18). Lane 2 contains the uncut, 175 bp HaeIII-663 peR product. After digestion with HaeIII, the peR product remained uncut at 175 bp (Lane 3). This indicates that the Beothuk individual did not have the HaelII-663 polymorphism. Lane 4 of Fig 18 contains the uncut, 210 bp HincII -13259 peR product. After digestion with HincH, a digested band of about 180 bp was observed (Lane 5). In addition, a band was remaining at 210 bp in Lane 5 (the yellow asterisk indicates the 210 bp peR product remaining after digestion of HincII-13259 peR product with HincII). It was initially thought that this was due to incomplete digestion. It was decided that this peR product should be digested again to verify the results (discussed later). The HincII-13259 peR product was also digested with AZul. This was performed because the during the transition in which the HincII site is lost, an AZul site is created. Therefore, if the HincH does not digest the peR product, then the AZul should (and vice .
M uncut HaeIII uncut HincII AluI uncut AluI cut cut cut cut 76 Figure 18: RFLP Analysis of HaeIII-663, HincII- 13259, and AluI-5176 peR Products. Lane 1 contains 0.5 ILg of 100 bp DNA molecular weight marker (M). Lanes 2, 4, & 7 contain 15 ILL of20 ILl digestion controls in which the restriction enzyme was not added and, therefore, cutting did not occur. Lanes 3, 5, 6, & 8 contain 15 ILL of20 ILL digestions of cleaned peR products. Two bands of21O bp and 180 bp are apparent in Lane 5. The yellow asterisk indicates a partial digestion. 77 versa). After digestion of the HincH-13259 peR product with AZul, a remaining, uncut 210 bp product was observed (Lane 6 of Fig 18). This indicated that the HincH-13259 peR product did not contain an AZul restriction site. This was thought to be further verification that the HincH site was present and the partial cutting with the HindI was a problem with the digestion. After digestion of the AZul-5176 peR product with AZul, the 150 bp band observed. in the undigested control (Lane 7) was no longer present (Lane 8). The digested bands of 77 and 72 bp migrated along with the primer-dimers and adapters. This indicated that the AZul-5176 peR product contained an AZul restriction site. This indicated that the Beothuk individual had the AIuI-5176 polymorphism. The RFLP results are summarized in Table 7. Table 7: Expected RFLP Products and Results (+) (-) Beothuk 9-bp deletion 112 bp 121 bp (+) (deletion present) (deletion absent) AluI-5176 77 & 72 bp 149bp (+) HincII-13259 158 & 53 bp 211 bp Partial digestion (+1-) (AIuI should cut) AIul (-) heteropZasmic HaeIII-663 101 & 75 bp 176 bp (-) Redigestion of HincII-13259 and AluI-5176 Because of evidence of a partial digestion of the HincH -13259 peR product, it was redigested overnight (Fig 19). At first, it was assumed that the partial digestion (indicated by the presence of both 210 bp product and 180 bp product) was due to a problem with the digestion reaction and that the HincH restriction site was present. In addition to an extended period of digestion, a different stock of excess enzyme was used M HincH AluI M digested 78 210bp ____ ~ 180bp ~ Figure 19: Re-Digestion of HincII-13259 PCR Product with HincII and the AluI-5176 PCR Products with AluI. Lanes 1 & 4 contain 0.5 J-Lg of 100 bp DNA molecular weight marker (M). Lane 2 contains 15 iLL of the HincII-13259 PCR product digested with an excess of the new HincII restriction enzyme overnight. Lane 3 contains 15 J-LL ofthe Alul -517 6 PCR product digested with and excess of AZul overnight. 79 in order to rule out the possibility of a faulty enzyme. The uncut 210 bp band was still present along with the 180 bp digested band. This indicated that the partial cutting of the HincII-13259 PCR product was not a problem with the digestion, but indicative of heteroplasmy. The AZul -517 6 PCR product was again completely digested with AZul verifying the presence of an AZul restriction site at np 5176 (Lane 3). Re-Amplification of peR Products from Agarose Gel An attempt was made to re-amplifY PCR products from Low Melting Point (LMP) agarose gels (Fig 20). This was performed in order to isolate bands of interest for sequencing. For example, in order to determine if the HincII-13259 site was heteroplasmic, the 210 bp undigested band was extracted from the agarose and reamplified for sequencing. This was performed in order to verify that the HincII site was not present. In addition, sequences for the other PCR products were difficult to obtain due to multiple signals probably from background. Therefore, the PCR product of interest was extracted from the gel and reamplified for sequencing. However, this was largely unsuccessful. In the gel analysis of the reamplification, more background was observed rather than less. Lane 2 contains the reamplifiedAZul-5176 PCR product, Lane 3 contains the undigested 210 bp HincII-13259 PCR product reamplified, Lane 4 contains the HaeIII -663 PCR product reamplified, Lane 5 contains the 9 bp product reamplified, and Lane 6 contains the 440 bp D-Loop region PCR product reamplified. Background smears (green brackets) and a spurious band (yellow asterisk) can be observed. The use of GenElute Minus EtBr Spin Columns previously described, however, was successful at isolating the PCR products of interest without the background for sequencing. 80 M AluI Haem DLoop M Figure 20: Re-Amplification of Desired peR Product from Agarose Extraction. Lanes 1 & 7 contain 0.5 f-Lg of 100 bp DNA molecular weight marker (M). Lanes 2-6 contain 15 f-LL of 100 f-LL peR reactions. The green brackets indicate examples of non-specific background. The yellow asterisk indicates a non-specific band. 81 SEQUENCING Sequencing was performed in order to verify RFLP results. The HaelH -663 PCR product was sequenced to verify that the HaeHI site was not present. The absence of the HaeHI site was verified through a hand search of the sequence obtained. The entire HincH-13259 PCR product was sequenced in both directions (Table 7). When the heavy chain, the chain of mtDNA that is transcribed, was sequenced, a HincH restriction site was found at np 13259. When the light chain, the template chain for replication, was sequenced, there was noise (the sequence was ambiguous) around np 13259. In order to ascertain the cause of the partial digestion of the HincH-13259 PCR product with HincH, sequencing was obtained on the 210 bp band remaining after digestion (extracted from the agarose and purified). The sequencing of the 210 bp band was performed only once, yielding a partial sequence (Table 8). The undigested band did not have the HincH site at np 13259. The sequencing obtained from the 210 bp band indicated that there was an A to G transition at np 13263 that created an AZul site gain and a HincH site loss. The RFLP results correlate in that the HincH-13259 210 bp PCR product (Lane 6, Fig 18) when digested with AZul appears to be less intense than the uncut 210 bp product in Lane 4. If the AZul digested the HincH-13259 PCR product at np 13263, fragments the size of 154 and 57 bp would have been expected. The 57 bp fragment would not be visible because it would migrate along with the primer-dimers. The 154 bp band, however, should be visible at about half the intensity as the remaining 210 bp band in Lane 5. Although the Cambridge reference sequence (Anderson, et aZ. 1981) does not contain an A lui site within the mtDNA sequence the HincH-13259 82 primers amplified, the Beothuk individual may have had an additional AZul site within the first 154 bp. A simple one base transition at np 13245, 13280, 13311, or 13347 would have created this site. In this case, the HincH -13259 PCR product would be cut two times and the resulting fragments would all migrate along with the adapters and primer- dimers. The sequencing ofthe remaining HincH-13259 210 bp band in Lane 5 was not complete enough to indicate if this had occurred. The 9-bp deletio-n PCR product was sequenced (Table 8). Through a hand search of the sequence, it was determined that the Beothuk individual did not have the repeat, CCCCCTCTA, at np 8272 to np 8280. In previous studies, when this repeat was not present, the individual was characterized as having a 9-bp deletion. Therefore, the Beothuk individual has a 9-bp deletion. This was unexpected because the size of the PCR product as about 130 bp was extrapolated from an EXCEL (Microsoft Office 2000) chart plotting the logarithm ofthe molecular weights versus the distance migrated of the standard. This indicated that the 9- bp deletion was probably absent. However, the measuring of the distance migrated across the gels is objective making misinterpretations of up to 25 bp common. The 9-bp deletion is the definitive marker for Native American Haplogroup B. Haplogroup B is further characterized by the absence ofthe HaeIH-663 polymorphism, presence ofthe HincII-13259 polymorphism, and presence of the AZuI-5176 polymorphism. After complete characterization of the Native American mtDNA markers essential for haplogrouping, it was concluded that the Beothuk individual belonged to Native American Haplogroup B. Haplogroup B is prevalent in Southwestern and Central American Native Americans (Fig 9), indicating that the Beothuk may share ancestors 83 Table 8: Beothuk mtDNA Sequences PCRProduct Starting np* Sequence HaeIII-663 590 5' -CCTCCTCAAAGCAATACACTGAAAAT GTTTAGACGGGCTCACATCACCCCATAA ACAAATAGGTTTGGT* CCTA GCCTTT*CTAT TAGCTCTTAGTAAGATTACACATGCAAG CATCCCCGTTCCAGTGAGTTCACCCTCTA AATCACCACGATCAAAAGGAACAAGCAT CAAGCAC-3' * The HaeIII site is not here. HincII-13259 13211 5' -GCCCTTACACAAAATGACATCAAAAAA ATCGTAGCCTTCTCCACTTCA*GTCAAC*TAG GACTCATAATAGTTACAATCGGCATCAAC CAACCACACCTAGCATTCCTGCACATCTG TACCCACGCCTTCTTCAAAGCCATACTATT TATGTGCTCCGGGTCCATCATCCACAACCT TAACAATGAACAAGATATTCGAAAAATAGG-3' * The HincII site is here},2 9-bp Deletion 8204 5' -ATGCCCATCGTCCTAGAATTAATTCCCC TAAAAATCTTTGAAATAGGGCCCGTATTTA CCCTATAGCA*CCCCCTCTAGAGCCCACTG TAAAGCTAACTTAGCAT -3' * The 9-bp deletion is here D-Loop 16005 5' -T AATTT AAACTATTCTCTGTTCTTTCATG Region GGGAAGCAGATTTGGGTACCACCCAAGTA TTGACTCACCCATCAACAACCGCTATGTAT TTCGTACATTACTGCCAGCCACCATGAATA TTGTACGGTACCATAAATACTTGACCACCT GTACGGTACCATAAATACTTGACCACCTGT AGTACATAAAAACCCAATCCACATCAAAA CCCCCTCCCCATGCTTACAAGCAAGTACAG AATCAACCCTCAACTATCACACATCAACT GCAACTCCAAAGCCACCCCTCACCCACTAG GATACCAACAAACCTACCCACCCTTAACA*A* TACATAGTACATAAAGCCATTTACCGTACA TAGCACATTACAGTCAAATCCCTTCTCGTCC CCATGGATGACCCCCCTCAGATAGGGGTCC CTTGACCACCATCCT -3' * At 16303 there is a G-> A transition which deletes an RsaI restriction site. " The bght chain sequence obtaIned was amblguous around the HincH slte .... GNNAAC ... (np 13259-13264) 2The 210 bp portion sequence at np 13259-13264: ... GTCAGC ... An A to G transition at np 13263. 84 with these groups. Also, no Innuit in several studies, to our knowledge, has ever been reported as belonging to Haplogroup B. This indicates that, despite what Santu said, the Beothuk probably were not part Eskimo. In order to perform some further characterization, sequencing on the D-Loop region 440 bp PCR product was obtained (Table 8). The D-Loop 440 bp PCR product was extracted from the gel and purified using spin columns. It was then sent for sequencing. A hand search of the obtained D-Loop sequence was then performed against the Cambridge reference sequence (Anderson et a1. 1981). The only variance observed in a hand comparison with the Cambridge D loop sequence in the area sequenced was a G to A transition at np 16303, this created an RsaI site loss. This analysis was performed for comparison ofthe Beothuk sequences in this hypervariable region to previously published results (Appendix A). The D loop was sequenced two times from different PCR products for verification. After the amplification of the Beothuk mtDNA markers was completed, the D- Loop region of the mtDNA of the investigator was amplified using the D-Loop region PCR primers. This was performed in order to determinate if contamination had occurred. The PCR product was extracted from an agarose gel (not shown) and sequencing was obtained. Hand analysis indicated the investigator did not have the same G to A transition at np 16303 as the Beothuk individual (Table 9). 85 Table 8: Sequencing ofInvestigator's D-Loop region Investigator's D- 16008 5' -TTTAAACTATTCTCTGTTCTTTCATGGGGA Loop Region AGCAGATTGGGTACCACCCAAGTATTGACTC ACCCATCAACAACCGCTATGTATTTCGTACAT TACTGCCAGCCACCATGAATATTGTACGGTAC CATAAATACTTGAACCTGTAGTACATAAAAAC CCAATCCACATCAAAACCCCCTCCCCATGCTTA CAAGCAAGTACAGCAATCAACCCTCAACTATC ACACATCAACTGCAACTCCAAAGCCACCCCTCA CCCACTAGGATACCAACAAACCTACCCACCCTT AACA *G*TACATAGTACATA-3' No changes from the Cambridge reference sequence. np 16303 where the G to A transition occu"ed in the Beothuk individual is indicated in red and with asterisks. EUROPEAN MARKER The European marker, AluI -7025 was PCR amplified and run on a gel (not shown). A smear around 100 bp (the expected size ofthe PCR product) was extracted from the gel and sent to be sequenced (Davis Sequencing). According to Davis sequencing, the signal was too weak for sequencing. Optimization of the PCR reaction would be required in order to sequence this marker. ANALYSIS OF THE RSAI RESTRICTION SITE LOSS AT NP 16303 The loss of the RsaI restriction site at np 16303 of the D loop of the mtDNA has been observed in various haplotypes. The Tibetan haplotypes AS142-A145, AS147, and AS154 also have this transition (Torroni et al.1994b). In addition, these haplotypes do not have the HindI and HpaI sites at np 12406. The Beothuk individual was only analyzed for the Native American mtDNA sites. Therefore, these sites were not examined. These Tibetan haplotypes are found at a high frequency in southeastern Asian 86 populations. AS154 does not fall into a haplotype category. AS142-145 and 147 are placed in Haplogroup F (Fig 21). Figure 21 depicts distance-based phylogenetic analysis showing the closeness of the genetic relationship of the Tibetan haplotypes to each other. The rationale of phylogenetic analysis is that the more mutations required to change one sequence into the other, the more unrelated the sequences and the lower the probability that they share a common ancestor. The length of a branch on the phylogenetic tree is proportional to the number of sequence changes in the branch. The nodes (where the branches meet) are representative of a common genetic ancestor. The further to the left the branch is (the closer to the node), the more its sequence is in consensus with the genetic ancestor. Conversely, the further the branch is to the right of the node, the greater the number of sequence changes from the genetic ancestor. Figure 21 indicates that Haplogroup F, which the Tibetans haplotypes with the G to A transition at np 16303 belong to, and Haplogroup B, which the Beothuk individual belongs to, have a common ancestor (denoted in the figure by a blue asterisk). It is possible the G to A transition at np 16303 occurred before the divergence of the two haplogroups. 87 Figure 21: Tibetan Phylogenetic Tree (Torroni et al. 1994b) A phylogenetic tree including 42 Tibetans, 106 Asian, and 34 Siberian haplotypes. A-G are the six major haplogroups observed in Tibetans. The numbers at the end of the branch represent haplotypes. The further the branch from the node, the greater the amount of mutational events that have occurred since divergence from the common ancestor (represented by the node). The blue asterisk denotes a common ancestor of Haplotype F and Haplotype B. 517 --- A1 AS56
! AS156 = 2!'_06 .- -- AS109 Odelj f---- A522 i . ASS9 -. AS119 AS158 7 At,:,. i AS8B ASI22 ; - - AS92 - ASIOB i -. - . AS113 AS114 AS38 ! ---= A 1 . ASISI i
-Lin.......-.- 522 -- -- AS117 A5155 - ASSl -- AS94 --------- AS34 L----- --- __ 55 Af1"OSISl 56 -------. 51 ... AFRICAN OUTGROUP ------_ .... 88 A519 AS29 A5142 AS3I5 ___ AS102 89 In addition, this transition was observed in haplotypes 5, 13, 31-34, 74, 76, 98, 108, and 111 (Torroni et al. 1994a). Nine of these mtDNA haplotypes clustered together on a phylogeny tree (arrow on Fig 22). All of these haplotypes were of Caucasian individuals that had Alzheimer's disease or Parkinson's disease. These individuals were grouped in Haplogroup H. The genetic relationships on this tree are interpreted in the same manner as Fig 21. Again, the Beothuk individual was not successfully analyzed for the additional marker, AluI-7025, just the Native American mtDNA markers. The Beothuk individual had a transition that was observed in the most common Caucasian haplogroup, Haplogroup H. In addition, all of the individuals in the haplotypes in this study that had the G to A transition at np 16303 also had Alzheimer's or Parkinson's disease. It is a possibility that this transition is a marker for these diseases. In that case, it could further be possible that the Beothuk individual tested carried the genetic marker for or even had Alzheimer's or Parkinson's disease. Siberian mtDNA Haplotypes S6, S7, and S20 also have the loss ofthe RsaI site at np 16303 (Torroni et al. 1993b). Like the Beothuk individual, these haplotypes do not have the np 663 HaeIH site. In addition, they have the np 13259 HincH and np 5176 AluI site in common with the Beothuk individual (the 9-bp deletion was not analyzed in the Siberian study). The S20 is an Evenk Haplotype that has also been seen in Koreans and Hans. The S6 and S7 are Nivkh and Udegey Haplotypes. These haplotypes are similar to those observed in the Japanese. S6 and S7 are not in any of the Native American haplogroups. In the phylogenetic tree (which was constructed in the same manner as the previous phylogenetic trees described) in Fig 23, Haplotypes S6 and S7 are grouped together. Their common ancestor branches directly off of the African outgroup. From 90 Figure 22: Phylogenetic Tree of Caucasian mtDNAs (Torroni et al. 1994a). A phylogenetic tree including 34 Siberian (S) and 106 Asian (AS) haplotypes. Four of the Native American haplotypes are represented, A, B, C, and D .. The numbers at the end of the branch represent haplotypes. The further the branch from the node, the greater the amount of mutational events that have occurred since divergence from the common ancestor (represented by the node). The arrow indicates those haplotypes that have the G to A transition at np 16303. . i , I I --.,.--Itt I H .-r-=--- , r- 11,_ : ! __ II .. __ _ '--_."" ';., 1.1: '- __ --t-.IIIII----...-;;--.... L:.:.r--'-- 41' '':'; 1.- ---- 11' . - 11 b"-rf....-- !II' ,--;'ii' I'f' L", .!IIII.IJJ: .all J ;.r was IL1 , .. ., 11 I iif III .. I - III - l __ --""'""-- tl" r' ::::: .. I,. ...: u: . ____ r.----- i-i,_ -___ __ ; If'
92 Figure 23: Siberian Phylogenetic Tree (Torroni et al. 1993b). A phylogenetic tree including Siberian (S) and Asian (AS) haplotypes. Four ofthe Native American haplotypes are represented, A, B, C, and D. . The numbers at the end of the branch represent haplotypes. The further the branch from the node, the greater the amount of mutational events that have occurred since divergence from the common ancestor (represented by the node). The blue asterisk denotes the early divergence from the common ancestor with the Native American haplogroups. The green asterisk denotes the divergence ofHaplogroups A and B before which the G to A transition at np 16303 could have occurred. 93 AS18 L_-===.:;;o.=,--- AS81 A8102 S5 ___ African outgroup this analysis, it appears that Haplotypes S6 and S7 diverged very early on from the common ancestor of the Native American haplogroups (see blue asterisk on Fig 23). 94 This opens up the possibility that the Beothuk may have been remnants of a very ancient group of people. In addition, Haplotype S20 is in Haplogroup A. This indicates that there is a possibility that the G to A transition at np 16303 occurred before the divergence ofHaplogroups A and B (see green asterisk on Figure 23). Lastly, European Haplotypes 15, 17 and 67 also have the loss ofthe RsaI site at np 16303. Haplotype 15 was made up of two Swedes and one Basque individual. Haplotype 67 was made up of one Basque individual. Torroni et al. (1994) suggested that these were Haplogroup H-founder root haplotypes commonly found in Spain and among the Basques. As previously mentioned, this transition was also observed in the European Haplogroup H haplotypes that had Alzheimer's and Parkinson's disease. This indicates that there is a possibility that there was some European admixture in the Beothuk. It would be necessary to perfonn analysis of European mtDNA markers to draw further comparisons to European mtDNA haplotypes. Due to the nature of maternal inheritance in mtDNA, it is more likely that this G to A transition at np 16303 occurred in the founding, Native American haplotype. However, as previously described, some paternal recombination has been observed. One possible scenario is that, at some point in time, admixture between a woman belonging to Native American Haplogroup B and a Caucasian male carrying the G to A transition at np 16303 occurred. This is more likely than admixture of a Native American male with a Caucasian female due to the large amount of recombination that would have had to of occurred in order for the female to have offspring with the Native American markers described for Haplogroup B. However, this scenario would be possible, but unlikely, over a number of generations of admixture solely with the Beothuk beyond the initial Caucasian mother. APPENDICES A & B-COMPILATION OF PUBLISHED mtDNA ANALYSES. The published results of previous studies of mtDNA polymorphisms were 95 difficult to compare because they were reported in different formats. The charts were made in order to facilitate the analysis of the Beothuk polymorphisms. In addition, these tables will be used in future studies in the laboratory involving other Native American groups. The tables were designed so that an individual's polymorphisms, once determined, could be entered into a row, and all haplotypes and haplogroups that have the same polymorphisms as the subject of the study would be immediately identified. Results were interpreted from studies (those studies marked with a superscript reference letter in the References section were used) and entered into an EXCEL (Microsoft Office 2000) chart. The first chart contains a listing of restriction site polymorphisms (Appendix A). The top row is the site of the restriction site polymorphism. The appearance of a "1" denotes the presence of a restriction site (or deletion). The appearance of a "0" denotes the absence of a restriction site (or deletion). Those polymorphisms shared with the Beothuk individual are highlighted. The second chart contains D-Loop sequence differences (Appendix B). CONCLUSIONS The purpose of this project was to detennine the mtDNA haplogroup of the Beothuk chief, Nonosabasut. It was hypothesized that the Beothuk Indian would belong to one of the Native American mtDNA haplogroups. The Beothuk Indians have been largely overlooked in the discussion over the founding of the Americas due to the lack of knowledge about their origins. Through this research, some information about the origins of the Beothuk can be inferred. Perhaps the interest and additional questions this study raises will lead to future studies involving a larger sample size ofBeothuk individuals' remams. In this study, the mtDNA markers (HaeIII-663, HincII-13259, AluI-5176, and the 9-bp deletion) that definitively place an individual into a Native American Haplogroup were examined for the Nonosabasut, the Beothuk representative. It was detennined that the Beothuk individual had the AluI-5176 and 9-bp deletion polymorphisms. In addition, he had HincII-13259 heteroplasmy. Nonosabasut did not have the HaeIII-663 polymorphism. The cumulative result is that Nonosabasut belongs to Native American Haplogroup B. In addition, the mtDNA D-Loop region ofNonosabasut was analyzed. The analysis indicated a G to A transition at np 16303. Through comparison with previously published D-Loop sequences, it was detennined that this transition is primarily found in Caucasians. In addition, to our knowledge, this transition has never been reported in a 97 Native American. This indicated that there may be some Caucasian admixture with the Beothuk. Additional studies should be performed on older Beothuk remains and on European mtDNA markers to determine the age and the origin of this possible admixture. Also, the G to A transition at np 16303 is a transition that appears to have occurred very early in divergence. This supports the belief held by some that the Beothuk were the remains of an archaic group of people. There are many possibilities for future studies using the Beothuk DNA libraries. It would be interesting to look for any ofthe polymorphisms that are definitive for European Haplogroups. In addition, the HincII-13259 polymorphism of this individual could be studied more in depth. Several digestions could be performed using HindI. The remaining 210 bp band could be collected from three or four gels using the GenElute EtBr Spin Columns. Then, an ethanol precipitation could be performed in order to concentrate the PCR product. The concentrated product could be digested with AluI to see is any cut product is apparent on a gel. Also, this PCR product could be sequenced to look for any additional A luI sites. It would also be interesting to obtain a sequence for the D-Loop region being amplified along with the 440 bp PCR product to determine if there are two copies of a portion of the D Loop due to homoplasmic insertion. The creation of a library from the ancient DNA proved to be successful. With the creation of two libraries, all of the markers of interest could be analyzed. In future studies, it would be plausible to create as many as four or five libraries. In addition, the library method proved useful when analyzing irreplaceable DNA. It allowed for reamplification as needed. Therefore, ample ancient DNA was available for optimization of reactions without exhausting the supply of DNA. 98 The methods optimized in this study are currently being used in Dr. Carolyn Vann's laboratory for a study on the Miami Indians. The study is also using ancient DNA extracted from teeth. The Miami Indians have struggled to gain recognition as an American Indian Tribe by the Federal Government. If the molecular study currently being performed shows that they belong to a Native American Haplogroup, it may have important ramifications for the Miami Indians. Along with recognition as an Indian tribe, Federal recognition would give them rights and opportunities to preserve their unique heritage. With the continuing improvements in sequencing, more straight-forward methods of placing individuals in mtDNA haplogroups should be considered for future studies. One method that has been used successfully is amplification of the entire mtDNA using overlapping fragments and nested PCR. Primers are designed to overlapping segments of DNA between 300 and 400 bp long. A second PCR is performed using primers internal to the first set of primers. The PCR products are then sequenced. When the sequences are obtained, the entire mtDNA sequence can be constructed using the overlapping sequences. Technology in molecular biology is rapidly evolving. 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