Title on the Cover: Xu Zhi, Zhang Fan, Li Hui, Wang Xiaoning, Jin Jianzhong, Jin Li (2005) Primary Research on mtdna from Enshi Cliff Coffin.

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1 Title on the Cover: Xu Zhi, Zhang Fan, Li Hui, Wang Xiaoning, Jin Jianzhong, Jin Li (2005) Primary Research on mtdna from Enshi Cliff Coffin Journal of the Central University for Nationalities 14(2):

2 ) Journal of the CUN(Natural Sciences Edition) May,2005 Vol 14 No 2 1, 1, 1, 2, 1 1 (11, ; 21, ) :, mtdna HVSI, DNA mtdna,, : DNA(mtDNA) ; I(HVSI) ; ; DNA ; (PCR) ; ; :Q986 :A : (2005) , Trapping the past, DNA, DNA,, DNA(ancient DNA,aDNA) 1980,, DNA, DNA [1 ] ;, DNA [2 :1983,Mullis ] (polymerase chain reaction), PCR, DNA [3 ] ;1986 Pggbo,, DNA DNA, DNA,, DNA DNA,, DNA,,, DNA mtdna mtdna DNA ), DNA, DNA,mtDNA DNA, : (1) mtdna [4 ] ; (2), mtdna D2 [5 ] HVSI HVSII ; (3) mtdna, ; (4) ( Effective Population Size) DNA, DNA 10 Y Π Π,mtDNA Π Π ), : : ( ), ( ),, Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved

3 2 : 125, mtdna ;,, mtdna, 1987,Cann Nature 147 mtdna RFLP, [6 ] ;1997, DNA, [7 ], Science 1997,,, :,,,,,,,, 1000 [8 ] , (1) L16016 H16403 L16016 : 5π2ATTCTCTGTTCTTTCATGGG23π H16403 : 5π2ATTGATTTCACGGAGGATGG23π (2) Extracting Buffer [10mM Tris2HCl,015M EDTA,10mgΠmlDTT,015mgΠml K,012 % SDS(pH ) ],Lysis Buffer [8M GuSCN, 20mM EDTA, 10mM Tris2HCl (ph 615), 40gΠl Triton X2100 ], Washing Buffer [6M GuSCN, 10 mm Tris2HCl (ph 615) ], 70 %,, (Milli Q ),,, 015 TBE[45mmolΠL Tris,45mmolΠL,1mmolΠL EDTA],PCR [0125 %(wπv),30 %(vπv),70 %(vπv) ddh 2 O], DL2000, DNA 100,PCR (Takara) (dntp 2mM, 10mM, 5 UΠ l, 10 PCR buffer,mgcl 2 25mM), MicroconYM30 filter (Millipore), Qiaquick PCR Purification Kit (Qiagen) (3) PCR2700 (Applied Biosystems) ;DGW299 ;SCR2300 ; FR2200 ( ) ; SPEX CertiPrep 6750 FreezerΠMill ; Centrifuge 5415 D ( Eppendorf) ;ABI Prism 3100 Genetic Analyzer (Applied Biosystems) 213 (1), 2 5 mm 30min (SPEX CertiPrep 6750 FreezerΠMill), (2) DNA 2 2 2,, DNA ; Boom [9 ] GuSCN, DNA [10 ] : DNA, PCR, - [11 Yang ] : 115g, 315ml,56 C 16h ;8000rpm 5min ;, 2 Lysis Buffer, ; 80ul, 1hr ;12000rpm Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved

126 ) 14 2min ;, 600ul Washing Buffer, ;12000rpm 2min ;, 600ul 70 %, ;12000rpm 2min ;, 600ul, ;12000rpm 2min ;, 65OC ; 400ul Elution Buffer,65OC 1hr ;12000min ;, DNA MicroconYM30 filter (Millipore),

4 126 ) 14 2min ;, 600ul Washing Buffer, ;12000rpm 2min ;, 600ul 70 %, ;12000rpm 2min ;, 600ul, ;12000rpm 2min ;, 65OC ; 400ul Elution Buffer,65OC 1hr ;12000min ;, DNA MicroconYM30 filter (Millipore), Qiaquick PCR Purification Kit (Qiagen) (3) DNA (PCR), I(HVSI),, PCR Tab 1 Prescription of two PCRs Ex Taq Buffer dntps MgCl 2 primer Ex Taq polymerase template ddh 2 O Total 10 2mM 25mM 10uM 5UΠul 5 20ngΠul MilliQ - (ul) (ul) :94 3min (94 30sec 55 50sec 72 50sec) (4) 40cycles 72 5min ENH101 ENH105 ENH108 ENH109 ENH % Fig 1 2 % Electrophoretic photo of several Enshi Cliff Coffin samples PCR, PCR PCR, (5) 214 Bioedit, Network : 5 DNA HVSI rcrs ( ) [12 ] -, DNA, ENH338 ENH105 HVSI 312 1, Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved

2 : 127 2 Tab 2 Haplogroups of Enshi Cliff Coffin samples Haplogroup HVSI Motif ( + 16000) ( ) ENH301 C 126 223 298 327 1000 ENH338 GΠD NA 266A 278 1000 ENH105 M 3 NA 223 1000 ENH212 M8 3 223 298

5 2 : Tab 2 Haplogroups of Enshi Cliff Coffin samples Haplogroup HVSI Motif ( ) ( ) ENH301 C ENH338 GΠD NA 266A ENH105 M 3 NA ENH212 M ENH rcrs HVSI Motif, DNA HVSI Motif ( ), NA GΠD G D,, RFLP ENH108, 313 NETWORK C 2, Median2joining ; ENH301 C ;Out, HVSI YN2 ; GD2 ;WH DNA Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved

6 128 ) 14 DNA [13 14 ], DNA, [13,15 ],, : (1) ( DNA PCR ) ; (2), PCR, PCR, PCR ; (3),,,,,,,, 412 ENH108 HVSI C Fig 2 Network of C Haplogroup, CRS, : (1) 16270, ; (2), ; (3) DNA, ENH108 [16 ], HVSI,,ENH108,,,,, ENH338 ENH105,, [17 ],M 3, 2313 %, ENH105 C G D M8 3 C,, ENH301 HVSI,,, ENH301 C, C,,, [8 ],,,,, 1000,, 3000,, 3000,,, Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved

7 2 : 129 : [ 1 ] HUNAN MEDICAL COLLEGE Study of an ancient cadaver in Mawangtui tomb No 1 of the Han Dynasty in Changsha[M] New York : Ancient Memorial Press, [ 2 ] MULLIS K B, F A FALOONA Specific synthesis of DNA in vitro via a polymerase2catalyzed chain reaction [J ] Methods Enzyml, 1987, 155 : [ 3 ] P BO S Molecular genetic investigations of ancient human remains[j ] Cold Spring Harbor Symp Quant Biol, 1986, 51 : [ 4 ] OLIVO P D, M J VAN DE WALLE, PJ LAIPIS, W W HOUSEWIRTH Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D2loop[J ] Nature, 1983, 306 : [ 5 ] GILES R E, H BLANC, H M CANN, D C WALLACE Nuclear DNA sequences from late Pleistoncene megafauna[j ] Mol Biol Evol, 1999, 16 : [ 6 ] CANN R L, STONEKING M, WILSON A C Mitochondrial DNA and human evolution[j ] Nature, 1987, 323 : [ 7 ] KRINGS M, A STONE, R W SCHMITZ, H KRAINITZKI, M STONEKING, S P BO Neandertal DNA sequences and the origin of modern humans[j ] Cell, 1997, 90 : [ 8 ] [J ] ),1998,16(2) :47-53 [ 9 ] BOOM R, SOL C J A, SALIMANS M M M, JANSEN C L, WERTHEIN VAN DILLEN P M E, VAN DER2NOORDAA J Rapid and simple method for purification of nucleic acids [J ] J ournal of Clinical Microbiology, 1990, 28 : [10 ],,,,,, DNA [J ] ),2003, 12(1) :40-44 [11 ] YANG D Y, ENGB, WAYEJ S, DUDAR J C, SAUNDERS S R Technical note : improved DNA extraction from ancient bones using silica2based spin columns[j ] Am J Phys Anthropol, 1998, 105 : [12 ] ANDREWS R M, KUBACKA I, CHINNERY P F, LIGHTOWLERS R N, TURRNBULL D M, HOWELL N Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA[J ] Nat Genet, 1999, 23 :147 [13 ] HANDT O, HOSS M, et al Ancient DNA : methodological challenges[j ] Experientia, 1994, 50 : [14 ] KOLMAN C J, TUROSS N Ancient DNA analysis of human populations[j ] Am J Phys Anthropol, 2000, 111 :5-23 [15 ] COOPER A, POINAR H N Ancient DNA : do it right or not at all[j ] Science, 2000, 289 :1139 [16 ], [J ],2003,6(48) : [17 ] YONG2GANG YAO, QING2PENG KONG, HANS2J ΒRGEN BANDELT, TOOMAS KIVISILD, YA2PING ZHANG Phylogeographic Differentiation of Mitochondrial DNA in Han Chinese[J ] Am J Hum Genet, 2002,70 : Primary Research on mtd NA from Enshi Cliff Coffin XU Zhi 1, ZHANG Fan 1, LI Hui 1, WANG Xiao2ning 2, J IN Jian2zhong 1, J IN Li 1 (1 Center for Anthropological Studies, School of Life Sciences, Fudan University, Shanghai , China ; 2 Museum of Enshi Tujia&Miao Autonomous Prefecture, Enshi , China) Abstract : The materials in this article is skeleton or tooth samples from Enshi Cliff Coffin After a series of treatments, a part of HVSI sequences on mtdna came to hand and had been amplified And a few experiments were used for ensuring that they were ancient DNAs Then their elementary haplogroups were verified,referring to part of modern human mtdna data base Most of the samples show they belong to south pattern while some North patterns are also found Key words : mitochondrion DNA ( mtdna) ; hypervariable sequence I ( HVSI) ; Cliff Coffin ; ancient DNA ; polymerase chain reaction ( PCR) ; sequencing ; haplogroup Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved [ : ]

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