Research ArticleThe Use and Effectiveness of Triple Multiplex System forCoding Region Single Nucleotide Polymorphism inMitochondrial DNA Typing of Archaeologically ObtainedHuman Skeletons from Premodern Joseon Tombs of Korea

Chang Seok Oh12 Soong Deok Lee13 Yi-Suk Kim4 and Dong Hoon Shin12

1Bioanthropology and Paleopathology Lab Institute of Forensic Science Seoul National University College of Medicine28 Yongon-dong Chongno-Gu Seoul 110-799 Republic of Korea2Department of Anatomy Seoul National University College of Medicine 28 Yongon-dong Chongno-GuSeoul 110-799 Republic of Korea3Department of Forensic Medicine Seoul National University College of Medicine 28 Yongon-dong Chongno-GuSeoul 110-799 Republic of Korea4Department of Anatomy Ewha Womans University School of Medicine 911-1 Mok-6-dong Yangcheon-GuSeoul 158-710 Republic of Korea

Correspondence should be addressed to Dong Hoon Shin cuteminjaegmailcom

Received 19 December 2014 Accepted 16 March 2015

Academic Editor Otto Appenzeller

Copyright copy 2015 Chang Seok Oh et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Previous study showed that East Asian mtDNA haplogroups especially those of Koreans could be successfully assigned by thecoupled use of analyses on coding region SNPmarkers and control region mutation motifs In this study we tried to see if the sametriplemultiplex analysis for coding regions SNPs could be also applicable to ancient samples fromEast Asia as the complementationfor sequence analysis of mtDNA control region By the study on Joseon skeleton samples we know that mtDNA haplogroupdetermined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control regioncan assign Considering that ancient samples in previous studies make no small number of errors in control region mtDNAsequencing coding region SNP analysis can be used as good complimentary to the conventional haplogroup determinationespecially of archaeological human bone samples buried underground over long periods

1 Introduction

Ancient DNA (aDNA) analysis is very important for under-standing the origin and evolution of mankind in historyOf various aDNA studies analysis on mitochondrial DNA(mtDNA) is one of the best methods to know the phylogenyof archaeologically obtained human samples mtDNA showsmaternal haplotype lineage that is passed down throughouteach generation without changing and reshuffling of DNAIn fact it could be analyzed successfully even in case wherenuclear DNA (nDNA) is degraded seriously [1]

Recently about the determination of East Asian mtDNAhaplogroups Lee et al [2] showed that KoreanmtDNA couldbe allocated into 15 haplogroups by two different multiplexsystems for 21 coding region SNP markers and one deletionmotif As Koreans do have many D4 subhaplogroups thethird set of PCR multiplex systems was also used for defin-ing them in much detail Authors showed that East AsianmtDNA haplogroups especially those of Koreans couldbe successfully assigned by the multiplex analysis systemthey developed [2] As many previously published worksexhibited that some of mtDNA data from degraded samples

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 850648 7 pageshttpdxdoiorg1011552015850648

2 BioMed Research International

were not sufficiently authentic the establishment of moretools for detecting possible sequence errors looks valuable toconcerned researches

Themultiplex systemwas originally designed for mtDNAanalysis of the degraded samples frequently met in the fieldof forensic science [2] However the technique looks verysuggestive to the anthropologists in East Asia as well Likeforensic scientists the biological anthropologists always triedto analyze the aDNA that is seriously degraded remainingin archaeological human samples by small amounts There-fore sequencing errors in aDNA analysis have always beenthe researchersrsquo concern Since archaeological and forensicDNA typing share common subjects to be considered formaking their studies on the degraded samples more suc-cessful and authentic many experimental methods devel-oped for forensic science have been also applied to aDNAresearches

In this respect we wonder if the mtDNA typing by useof coding region SNP analysis could be also applicable toancient samples from archaeological sites in East Asia Asthe technique was proven to be time- cost- and targetDNA-saving [2] it can be used as a good complimentaryto conventional aDNA sequencing if both methods couldshow well-matching results from archaeologically obtainedsamples However regretfully enough there were not anyprevious researches on how perfectly this multiplex systemcan be applied to archaeologically obtained human sam-ples buried underground for several hundred to thousandyears

For the past several years we tried to build a skeletalseries consisting of human bones collected from 16th to 18thcentury Joseon tombs in South Korea Our previous reportson the collection have revealed information concerning thehealth and disease status of premodern Korean people [3ndash9]Using the same human skeleton collection we undertook theexperiments to compare haplogroup-directed data made bytwo different methods conventional control region sequenc-ing and analysis of coding region SNPmarkers It determineswhether the analysis of the triple multiplex system for codingregion SNP analysis like the forensic cases could be alsouseful for the mtDNA analysis of hundred-year-old humanbones from archaeological sites

2 Materials and Methods

Human skeletons (119899 = 11) collected from 16th to 18th centuryKorean tombs (Joseon Dynasty) were used in this studySex determination was made on the basis of morphologicaldifferences manifest in the pelvic bone by the examination ofgreater sciatic notch preauricular sulcus ischiopubic ramussubpubic angle subpubic concavity and ventral arc [10 11]Considered ancillary indicators for sex determination wereskull structures specifically the nuchal crest the mastoidprocess the supraorbital margin the glabella and the mentaleminence [12 13] Age was also estimated by auricular-surface degeneration of the hipbone based on the degree oftransverse organization granularity apical activity retroau-ricular area degeneration and auricular-surface porosity [14]

The age was accordingly categorized into eight phases 1-2young adult (20ndash35 years old) 3ndash6middle-aged (36ndash50 yearsold) and 7-8 old adult (over 50 years old)

The femur fragments from the skeletal remains wereused for aDNA analysis in this study The surfaces of thebones were removed using a sterilized knife after which theywere exposed to UV irradiation for 20min and subsequentlyimmersed in 54 (wv) sodium hypochlorite After the sam-ples were washed with distilled water and absolute ethanolthey were air-dried and pulverized to a fine powder using aSPEX 6750 FreezerMill (SPEX SamplePrep Metuchen NJ)[15 16] Bone powder (05 g) was incubated in 1mL of lysisbuffer (EDTA 50mM pH 80 1mgmL of proteinase K SDS1 01M DTT) at 56∘C for 24 h Total DNA was extractedwith an equal volume of phenolchloroformisoamyl alcohol(25 24 1) and then was treated with chloroformisoamylalcohol (24 1) DNA isolation and purification were per-formed using a QIAmp PCR purification kit (Qiagen HildenGermany)The purifiedDNAwas eluted in 50120583L of EB buffer(Qiagen) [17ndash20]

During sampling or lab work we always wore protectiongloves masks gowns and head caps Our aDNA lab facilitieswere set up in accordance with the protocol of Hofreiter et al[21] The rooms for aDNA extraction or PCR preparationwere physically separated from our main PCR lab The DNAextractionPCR preparation rooms were equipped with nightUV irradiation isolated ventilation and a laminated flowhood The other procedures for authentic aDNA analysissuggested by Hofreiter et al [21] were also followed byus

Three multiplex PCR systems used in this study wereoriginally designed to detect 21 SNPs and a 9-bp deletionmotif [2] and the sizes of each PCR amplicon were originallydesigned below 200 bp for increasing success yields duringDNA typing with degraded samples By multiplex PCR reac-tions I and II major 15 haplogroups could be detected fromEast Asian samples Briefly multiplex I reaction consistedof primers for eight different SNP loci (s4491 s5417 s7642s8793 s8794 s10397 s10400 and s14668) typing the hap-logroups M9 N9 M11 M10 A D5 M and D4 respectivelyMultiplex II reaction also tested seven SNP loci (s3970 s4833s4883 s7196 s8281-8289d s9824 and s12705) for decision ofthe haplogroups R9 G D M8 B M7 and R respectivelyHaplogroup D4 one of the most frequent haplogroups inEast Asian population was further subdivided into D4 D4aD4b D4e D4g D4h and D4j by seven SNP loci (s3010s14979 s8020 s11215 s8701 s5048 and s11696) of multiplexIII reaction [2 22ndash25]

Multiplex PCR amplificationwas done in a 20 120583L reactionvolume containing 40 ng of template DNA AmpliTaq Gold360 Master Mix (Life Technologies USA) and appropriateconcentrations of each primer Thermal cycling was con-ducted on a PTC-200 DNA engine (MJ Research) 95∘C for10min 45 cycles of 95∘C for 20 s 58∘C for 20 s and 72∘Cfor 30 s and a final extension at 72∘C for 10min To purifyPCR products 5 120583L of the PCR products was treated with1 120583L of ExoSAP-IT (catalogue number 78201 USB Cleveland

BioMed Research International 3

OH USA) at 37∘C for 45min After that the enzyme wasinactivated by incubation at 80∘C for 15min

We used twenty-two single base extension (SBE) primersrecommended by Lee et al [2] SBE reactionswere carried outusing a SNaPshotKit (AppliedBiosystemsUSA) according tothe manufacturerrsquos instructions Thermal cycling conditionsfor SBE were as follows denaturation at 96∘C for 10 secannealing at 50∘C for 5 sec extension at 60∘C for 30 secSBE was performed using a PTC-200 DNA Engine (Bio-RadLaboratories Hercules CA) For postextension treatmentreaction mixtures were mixed with 10 unit of shrimp alka-line phosphatase (SAP) incubated at 37∘C for 45min andfollowed by heat inactivation at 80∘C for 15minThe reactantswere analyzed by an ABI PRISM 3100 Genetic Analyzer(Applied Biosystems USA) using GeneMapper ID softwarev321 (Applied Biosystems USA) SNP scoring at each locuswas confirmed by sequencing two samples for each of theobserved alleles

We also did direct sequencing of mtDNA control regionof the samples By sequencing of hypervariable regions I IIand III we could get haplotype of the bones and furtherdetermined haplogroups of them The results could be com-pared with haplogroup determination by coding region SNPanalysis on the same samples Briefly after quantification wasdone by NanoDrop ND-1000 Spectrophotometer (ThermoFisher Scientific MA USA) 40 ng of aDNA was mixed withpremix containing 1X AmpliTaq Gold 360 Master Mix (LifeTechnologies USA) and 10 pmol of each primer (IntegratedDNA Technology USA) PCR conditions used in this studywere as follows predenaturation at 94∘C for 10min 45 cyclesof denaturation at 94∘C for 30 sec annealing at 50∘C for30 sec extension at 72∘C for 30 sec final extension at 72∘C for10min PCR amplification was performed using a PTC-200DNA Engine (Bio-Rad Laboratories Hercules CA) Primersets used for this study were as follows for 267-bp HV1AF15971 (51015840-TTA ACT CCA CCA TTA GCA CC-31015840) andR16237 (51015840-TGT GTG ATA GTT GAG GGT TG-31015840) for 267-bp HV1B F16144 (51015840-TGA CCACCTGTAGTACATAA-31015840)and R16410 (51015840-GAGGAT GGTGGT CAAGGGAC-31015840) for226-bpHV2A F015 (51015840-CACCCTATTAACCACTCACG-31015840) and R240 (51015840-TAT TATTATGTCCTACAAGCA-31015840) for235-bp HV2B F155 (51015840-CTA TTA TTT ATC GCA CCT-31015840)and R389 (51015840-CTG GTT AGG CTG GTG TTA GG-31015840) for167-bp HV3 F403 (51015840-TCT TTT GGC GGT ATG CAC TTT-31015840) and R569 (51015840-GGT GTA TTT GGG GTT TGG TTG-31015840)[26]

The PCR products were separated on 25 agarose gelstained with ethidium bromide and then isolated using aQiagen gel extraction kit (Qiagen Germany) The sequenc-ing of each amplicon was performed by ABI Prism 3100Genetic Analyzer (Applied Biosystems USA) using ABIPrism BigDye Terminator Cycle Sequencing Ready ReactionKit (AppliedBiosystemsUSA)TheobtainedDNAsequenceswere compared with the revised Cambridge ReferenceSequence (rCRS accession number NC 012920) to identifythe sequence differences between themThe resultant controlregion mutation motifs were imported into program mtD-NAmanager (httpmtmanageryonseiackr) with which

most Korean mtDNA haplotypes can be automatically clas-sified into East Asian mtDNA haplogroups and their subhap-logroups [27] or another web-based program for mtDNAhaplogroup analysis (httpdnajameslickcommthap) [2829]

In order to guard against any modern DNA contam-ination of ancient samples the mtDNA profiles of all ofthe researchers involved in this study were determined(with the permission of the Institutional Review Board ofSeoul National University H-0909-049-295)They were thencomparedwith themtDNAprofiles from the Joseon skeletonsto rule out the possibility of modern DNA contamination

3 Results

The sex and age of the samples determined in this studyare summarized in Table 1 By direct sequencing of mtDNAcontrol region every Joseon skeleton could be assigned torelevant existing haplo- or subhaplogroups of mtDNA Inmultiplex PCR analyses to detect 21 SNP markers in mtDNAcoding region eight (multiplex I) and seven (multiplexes IIand III) primer extension peaks for different haplogroupscould be observed Most samples exhibited no missing orextra assignment peaks in the results (Figures 1 and 2)

When the coding region SNP typing data were furthercompared with control region direct sequencing resultswell-matching patterns can be observed between the out-comes of two methods The mtDNA haplogroup expectedby coding region SNP could fall successfully within thesame haplogroup that control region sequencing could make(Table 1 Figure 1) However as far as haplogroup subcladesare concerned the results of direct sequencing on controlregion mutation motif were far better than SNP markeranalysis on coding region The haplogroup subclades of thecases numbers 024 034 036 038 040 045 100 and 238 weremuch successfully determined in direct sequencing of controlregion than in coding region SNP analysis (Table 1 Figure 2)

The absence of modern DNA contamination couldbe confirmed by the comparison of mtDNA haplotypesobtained from the current Joseon skeleton and participatingresearchersrsquo samples Aswe did not see any identical sequencebetween them (Table 1) mtDNA obtained from Joseon sam-ples should have been the endogenous DNA of the ancientpeople but not the outcome of modern DNA contaminationfrom researchers

4 Discussion

Coding region SNP analysis attracts forensic scientistsrsquo inter-est because it is a time- cost- and target DNA-savingmethodfor mtDNA analysis of degraded samples [2 30ndash35] Multi-plex PCR system for coding region SNPused in this studywasoriginally designed for the detection of major haplogroupsof forensic samples from East Asia In the study the triplemultiplex system on coding region SNP markers was proven

4 BioMed Research International

Table 1 Sequencing analysis result of mtDNA coding and control region

Subject Sex AgeHypervariable region Haplogroup

(control region)Haplogroup

(coding region)HVI (15991ndash16390) HVII (034ndash369) HVIII(423ndash548)

004 Male Old 16223T 16235G 16290T 73G 235G 263G3151C rCRS A A

005 Female Young16171G 16223T 16248T16298C 16327T 16344T16357C

73G 248d 263G3151C 489C M8 M8

024 Male Middle 16129A 16223T 16309G16362C

73G 152C 263G3091C 3151C

489C533G D4a1b1 D4a

034 Male Old16189C 16232A 16249C16304C16311C 16344T

73G 263G 310C 489C M1a3b1 M

036 Male Old 16150T 16183C 16185T16189C

73G 151T 197G263G 3151C 546G B4d3 B

038 Male Old 16129A 16223T 16362C 73G 194T 263G3151C 489C D4b2b D4b

040 Male Middle 161881C 161931C 16223T16311C

73G 263G 3091C3151C 489C M41015840101584067 M

045 Male Old16223T 16234T 16311C16316G16362C

73G 263G 3091C3151C 489C M9a1a1c1b1 M9

100 Male Middle16111T 16229A 16257A16261T16362C

73G 150T 263G3092C 3151C rCRS N9a N9

225 Male Middle 16209C 16223T 16362C 73G 195C 234G263G 3151C 489C D4 D4

238 Female Old 16223T 16295T 16319A 73G 146C 263G3091C 3151C

489C513d 514d M7c1a3 M7

Researcher 1 mdash mdash 16172C 16174T 16223T16362C

73G 263G 3091C3151C mdash D4g mdash

Researcher 2 mdash mdash16183C 16189C 16220C16254G16298C 16362C

73G 248d 263G3101C mdash F3b mdash

Researcher 3 mdash mdash16129A 16182C 16183C16189C16232A 16249C 16304C16311C 16344T

73G 152C 248d263G 3101C mdash D6c mdash

to be very useful for analysis on forensic materials [2]Briefly when they tried to do the coding region SNP-based mtDNA analysis on the long bone or molar samplesfrom about 50-year-old skeletal remains of war victims inthe Korean War (1950ndash1953) mtDNA haplogroup couldbe determined successfully even with a limited volume ofdegraded DNA [2] In fact as the triple multiplex systemmakes very high success rate in haplogroup determinationthe combined consideration of coding region SNP markersand control region polymorphism can become a very usefultool for the genetic analysis of degraded samples collectedfrom East Asian populations

This technique is also very suggestive for anthropolo-gists who deal with several hundred- to thousand-year-old

samples from archaeological sites Most of the archaeologicalhuman bones were maintained under the worst preservationconditions for a long while To make matters worse it isvery hard for researchers to get authentic outcomes fromthe ancient samples because only the limited volume of thesamples can be allowed for the analysis of archaeologicallyimportant cases We therefore admit that mtDNA analyseswith highly degraded ancient samples are sometimes toorisky because sequencing errors commonly occurred duringanalysis and they could not be corrected easily by a duecourse of repeated experiments with a sufficient amount ofsamples

In this regard if another time- cost- and sample-savingmtDNA analysis could be also established for the assignment

BioMed Research International 5

10400A

14668G

10397A4491C

8793G7642C

8794C5417C

4883G 4833A9824A

7196A

12705T

3970G 9-bp deletion

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

HV1A HV1B HV2A HV2B HV3

100200300400500

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

GT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

TCTT

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

C

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

-G

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS0005rCRS0005rCRS0005rCRS0005

rCRS0005rCRS0005rCRS0005

rCRS0005

15991

1

16111

121

16231

221

16351

341

35

1

155

121

275

241

424

1

16110

120

16230

220

16350

340

16390

380

154

120

274

240

369

337

548

125

(c) Sequencing result of control region

Figure 1 Analysis on sample number 005 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceHaplogroups (M8) determined by coding region SNP analysis (a) and control region direct sequencing ((b)(c)) are the same for this case

of ancient skeleton samples to relevant existing haplogroupsit will become a convenient method indeed for coun-terchecking the possible errors hidden in the conventionalmtDNA sequencing Our current results showed that mosthaplogroup results determined by coding region SNP analysison Joseon archaeological samples can fall successfully withinthe same haplogroups that were decided by control regionsequencing analysis In fact the results confirm coding regionSNP analysisrsquo error-screening role in the mtDNA haplogroupdetermination even for the ancient samples

However as for the current ancient samples wemust alsoadmit some technical limits of haplogroup determinationbased on coding region SNP analysis Briefly a coding regionSNP analysis on a few samples did not show the subhap-logroup results as completely as observed in the analysis ofcontrol region mutation motifs This means that multiplexSNP analysis could not completely replace the conventionalcontrol region mtDNA sequencing at least for the ancientcases from archaeological fields in South Korea Even soconsidering coding region SNP analysisrsquo superb potential

for reconfirmation of haplogroups determined by mtDNAcontrol region sequencing in time- cost- and target DNA-saving manner the use of this method can be expedient formaking mtDNA haplogroup determination of archaeologicalsamples much authentic

5 Conclusion

Obtaining authentic mtDNA outcomes from archaeologi-cal bone samples still remains a significant challenge toconcerned researchers The identification of possible errorsin conventional sequencing as quickly as possible is thussignificant for authentic mtDNA analysis of archaeologicallyobtained samples In this study we can show that mtDNAhaplogroup determination can also be successfully carriedout by coding region SNP analysis in a time- cost- and targetDNA-saving manner Although the mtDNA subhaplogroupscould not be determined by the coding region SNP analysis ascompletely seen in the control region sequencing the formercan be used as good supplementary to the latter for making

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

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Microbiology

2 BioMed Research International

were not sufficiently authentic the establishment of moretools for detecting possible sequence errors looks valuable toconcerned researches

Themultiplex systemwas originally designed for mtDNAanalysis of the degraded samples frequently met in the fieldof forensic science [2] However the technique looks verysuggestive to the anthropologists in East Asia as well Likeforensic scientists the biological anthropologists always triedto analyze the aDNA that is seriously degraded remainingin archaeological human samples by small amounts There-fore sequencing errors in aDNA analysis have always beenthe researchersrsquo concern Since archaeological and forensicDNA typing share common subjects to be considered formaking their studies on the degraded samples more suc-cessful and authentic many experimental methods devel-oped for forensic science have been also applied to aDNAresearches

In this respect we wonder if the mtDNA typing by useof coding region SNP analysis could be also applicable toancient samples from archaeological sites in East Asia Asthe technique was proven to be time- cost- and targetDNA-saving [2] it can be used as a good complimentaryto conventional aDNA sequencing if both methods couldshow well-matching results from archaeologically obtainedsamples However regretfully enough there were not anyprevious researches on how perfectly this multiplex systemcan be applied to archaeologically obtained human sam-ples buried underground for several hundred to thousandyears

For the past several years we tried to build a skeletalseries consisting of human bones collected from 16th to 18thcentury Joseon tombs in South Korea Our previous reportson the collection have revealed information concerning thehealth and disease status of premodern Korean people [3ndash9]Using the same human skeleton collection we undertook theexperiments to compare haplogroup-directed data made bytwo different methods conventional control region sequenc-ing and analysis of coding region SNPmarkers It determineswhether the analysis of the triple multiplex system for codingregion SNP analysis like the forensic cases could be alsouseful for the mtDNA analysis of hundred-year-old humanbones from archaeological sites

2 Materials and Methods

Human skeletons (119899 = 11) collected from 16th to 18th centuryKorean tombs (Joseon Dynasty) were used in this studySex determination was made on the basis of morphologicaldifferences manifest in the pelvic bone by the examination ofgreater sciatic notch preauricular sulcus ischiopubic ramussubpubic angle subpubic concavity and ventral arc [10 11]Considered ancillary indicators for sex determination wereskull structures specifically the nuchal crest the mastoidprocess the supraorbital margin the glabella and the mentaleminence [12 13] Age was also estimated by auricular-surface degeneration of the hipbone based on the degree oftransverse organization granularity apical activity retroau-ricular area degeneration and auricular-surface porosity [14]

The age was accordingly categorized into eight phases 1-2young adult (20ndash35 years old) 3ndash6middle-aged (36ndash50 yearsold) and 7-8 old adult (over 50 years old)

The femur fragments from the skeletal remains wereused for aDNA analysis in this study The surfaces of thebones were removed using a sterilized knife after which theywere exposed to UV irradiation for 20min and subsequentlyimmersed in 54 (wv) sodium hypochlorite After the sam-ples were washed with distilled water and absolute ethanolthey were air-dried and pulverized to a fine powder using aSPEX 6750 FreezerMill (SPEX SamplePrep Metuchen NJ)[15 16] Bone powder (05 g) was incubated in 1mL of lysisbuffer (EDTA 50mM pH 80 1mgmL of proteinase K SDS1 01M DTT) at 56∘C for 24 h Total DNA was extractedwith an equal volume of phenolchloroformisoamyl alcohol(25 24 1) and then was treated with chloroformisoamylalcohol (24 1) DNA isolation and purification were per-formed using a QIAmp PCR purification kit (Qiagen HildenGermany)The purifiedDNAwas eluted in 50120583L of EB buffer(Qiagen) [17ndash20]

During sampling or lab work we always wore protectiongloves masks gowns and head caps Our aDNA lab facilitieswere set up in accordance with the protocol of Hofreiter et al[21] The rooms for aDNA extraction or PCR preparationwere physically separated from our main PCR lab The DNAextractionPCR preparation rooms were equipped with nightUV irradiation isolated ventilation and a laminated flowhood The other procedures for authentic aDNA analysissuggested by Hofreiter et al [21] were also followed byus

Three multiplex PCR systems used in this study wereoriginally designed to detect 21 SNPs and a 9-bp deletionmotif [2] and the sizes of each PCR amplicon were originallydesigned below 200 bp for increasing success yields duringDNA typing with degraded samples By multiplex PCR reac-tions I and II major 15 haplogroups could be detected fromEast Asian samples Briefly multiplex I reaction consistedof primers for eight different SNP loci (s4491 s5417 s7642s8793 s8794 s10397 s10400 and s14668) typing the hap-logroups M9 N9 M11 M10 A D5 M and D4 respectivelyMultiplex II reaction also tested seven SNP loci (s3970 s4833s4883 s7196 s8281-8289d s9824 and s12705) for decision ofthe haplogroups R9 G D M8 B M7 and R respectivelyHaplogroup D4 one of the most frequent haplogroups inEast Asian population was further subdivided into D4 D4aD4b D4e D4g D4h and D4j by seven SNP loci (s3010s14979 s8020 s11215 s8701 s5048 and s11696) of multiplexIII reaction [2 22ndash25]

Multiplex PCR amplificationwas done in a 20 120583L reactionvolume containing 40 ng of template DNA AmpliTaq Gold360 Master Mix (Life Technologies USA) and appropriateconcentrations of each primer Thermal cycling was con-ducted on a PTC-200 DNA engine (MJ Research) 95∘C for10min 45 cycles of 95∘C for 20 s 58∘C for 20 s and 72∘Cfor 30 s and a final extension at 72∘C for 10min To purifyPCR products 5 120583L of the PCR products was treated with1 120583L of ExoSAP-IT (catalogue number 78201 USB Cleveland

BioMed Research International 3

OH USA) at 37∘C for 45min After that the enzyme wasinactivated by incubation at 80∘C for 15min

We used twenty-two single base extension (SBE) primersrecommended by Lee et al [2] SBE reactionswere carried outusing a SNaPshotKit (AppliedBiosystemsUSA) according tothe manufacturerrsquos instructions Thermal cycling conditionsfor SBE were as follows denaturation at 96∘C for 10 secannealing at 50∘C for 5 sec extension at 60∘C for 30 secSBE was performed using a PTC-200 DNA Engine (Bio-RadLaboratories Hercules CA) For postextension treatmentreaction mixtures were mixed with 10 unit of shrimp alka-line phosphatase (SAP) incubated at 37∘C for 45min andfollowed by heat inactivation at 80∘C for 15minThe reactantswere analyzed by an ABI PRISM 3100 Genetic Analyzer(Applied Biosystems USA) using GeneMapper ID softwarev321 (Applied Biosystems USA) SNP scoring at each locuswas confirmed by sequencing two samples for each of theobserved alleles

We also did direct sequencing of mtDNA control regionof the samples By sequencing of hypervariable regions I IIand III we could get haplotype of the bones and furtherdetermined haplogroups of them The results could be com-pared with haplogroup determination by coding region SNPanalysis on the same samples Briefly after quantification wasdone by NanoDrop ND-1000 Spectrophotometer (ThermoFisher Scientific MA USA) 40 ng of aDNA was mixed withpremix containing 1X AmpliTaq Gold 360 Master Mix (LifeTechnologies USA) and 10 pmol of each primer (IntegratedDNA Technology USA) PCR conditions used in this studywere as follows predenaturation at 94∘C for 10min 45 cyclesof denaturation at 94∘C for 30 sec annealing at 50∘C for30 sec extension at 72∘C for 30 sec final extension at 72∘C for10min PCR amplification was performed using a PTC-200DNA Engine (Bio-Rad Laboratories Hercules CA) Primersets used for this study were as follows for 267-bp HV1AF15971 (51015840-TTA ACT CCA CCA TTA GCA CC-31015840) andR16237 (51015840-TGT GTG ATA GTT GAG GGT TG-31015840) for 267-bp HV1B F16144 (51015840-TGA CCACCTGTAGTACATAA-31015840)and R16410 (51015840-GAGGAT GGTGGT CAAGGGAC-31015840) for226-bpHV2A F015 (51015840-CACCCTATTAACCACTCACG-31015840) and R240 (51015840-TAT TATTATGTCCTACAAGCA-31015840) for235-bp HV2B F155 (51015840-CTA TTA TTT ATC GCA CCT-31015840)and R389 (51015840-CTG GTT AGG CTG GTG TTA GG-31015840) for167-bp HV3 F403 (51015840-TCT TTT GGC GGT ATG CAC TTT-31015840) and R569 (51015840-GGT GTA TTT GGG GTT TGG TTG-31015840)[26]

The PCR products were separated on 25 agarose gelstained with ethidium bromide and then isolated using aQiagen gel extraction kit (Qiagen Germany) The sequenc-ing of each amplicon was performed by ABI Prism 3100Genetic Analyzer (Applied Biosystems USA) using ABIPrism BigDye Terminator Cycle Sequencing Ready ReactionKit (AppliedBiosystemsUSA)TheobtainedDNAsequenceswere compared with the revised Cambridge ReferenceSequence (rCRS accession number NC 012920) to identifythe sequence differences between themThe resultant controlregion mutation motifs were imported into program mtD-NAmanager (httpmtmanageryonseiackr) with which

most Korean mtDNA haplotypes can be automatically clas-sified into East Asian mtDNA haplogroups and their subhap-logroups [27] or another web-based program for mtDNAhaplogroup analysis (httpdnajameslickcommthap) [2829]

In order to guard against any modern DNA contam-ination of ancient samples the mtDNA profiles of all ofthe researchers involved in this study were determined(with the permission of the Institutional Review Board ofSeoul National University H-0909-049-295)They were thencomparedwith themtDNAprofiles from the Joseon skeletonsto rule out the possibility of modern DNA contamination

3 Results

The sex and age of the samples determined in this studyare summarized in Table 1 By direct sequencing of mtDNAcontrol region every Joseon skeleton could be assigned torelevant existing haplo- or subhaplogroups of mtDNA Inmultiplex PCR analyses to detect 21 SNP markers in mtDNAcoding region eight (multiplex I) and seven (multiplexes IIand III) primer extension peaks for different haplogroupscould be observed Most samples exhibited no missing orextra assignment peaks in the results (Figures 1 and 2)

When the coding region SNP typing data were furthercompared with control region direct sequencing resultswell-matching patterns can be observed between the out-comes of two methods The mtDNA haplogroup expectedby coding region SNP could fall successfully within thesame haplogroup that control region sequencing could make(Table 1 Figure 1) However as far as haplogroup subcladesare concerned the results of direct sequencing on controlregion mutation motif were far better than SNP markeranalysis on coding region The haplogroup subclades of thecases numbers 024 034 036 038 040 045 100 and 238 weremuch successfully determined in direct sequencing of controlregion than in coding region SNP analysis (Table 1 Figure 2)

The absence of modern DNA contamination couldbe confirmed by the comparison of mtDNA haplotypesobtained from the current Joseon skeleton and participatingresearchersrsquo samples Aswe did not see any identical sequencebetween them (Table 1) mtDNA obtained from Joseon sam-ples should have been the endogenous DNA of the ancientpeople but not the outcome of modern DNA contaminationfrom researchers

4 Discussion

Coding region SNP analysis attracts forensic scientistsrsquo inter-est because it is a time- cost- and target DNA-savingmethodfor mtDNA analysis of degraded samples [2 30ndash35] Multi-plex PCR system for coding region SNPused in this studywasoriginally designed for the detection of major haplogroupsof forensic samples from East Asia In the study the triplemultiplex system on coding region SNP markers was proven

4 BioMed Research International

Table 1 Sequencing analysis result of mtDNA coding and control region

Subject Sex AgeHypervariable region Haplogroup

(control region)Haplogroup

(coding region)HVI (15991ndash16390) HVII (034ndash369) HVIII(423ndash548)

004 Male Old 16223T 16235G 16290T 73G 235G 263G3151C rCRS A A

005 Female Young16171G 16223T 16248T16298C 16327T 16344T16357C

73G 248d 263G3151C 489C M8 M8

024 Male Middle 16129A 16223T 16309G16362C

73G 152C 263G3091C 3151C

489C533G D4a1b1 D4a

034 Male Old16189C 16232A 16249C16304C16311C 16344T

73G 263G 310C 489C M1a3b1 M

036 Male Old 16150T 16183C 16185T16189C

73G 151T 197G263G 3151C 546G B4d3 B

038 Male Old 16129A 16223T 16362C 73G 194T 263G3151C 489C D4b2b D4b

040 Male Middle 161881C 161931C 16223T16311C

73G 263G 3091C3151C 489C M41015840101584067 M

045 Male Old16223T 16234T 16311C16316G16362C

73G 263G 3091C3151C 489C M9a1a1c1b1 M9

100 Male Middle16111T 16229A 16257A16261T16362C

73G 150T 263G3092C 3151C rCRS N9a N9

225 Male Middle 16209C 16223T 16362C 73G 195C 234G263G 3151C 489C D4 D4

238 Female Old 16223T 16295T 16319A 73G 146C 263G3091C 3151C

489C513d 514d M7c1a3 M7

Researcher 1 mdash mdash 16172C 16174T 16223T16362C

73G 263G 3091C3151C mdash D4g mdash

Researcher 2 mdash mdash16183C 16189C 16220C16254G16298C 16362C

73G 248d 263G3101C mdash F3b mdash

Researcher 3 mdash mdash16129A 16182C 16183C16189C16232A 16249C 16304C16311C 16344T

73G 152C 248d263G 3101C mdash D6c mdash

to be very useful for analysis on forensic materials [2]Briefly when they tried to do the coding region SNP-based mtDNA analysis on the long bone or molar samplesfrom about 50-year-old skeletal remains of war victims inthe Korean War (1950ndash1953) mtDNA haplogroup couldbe determined successfully even with a limited volume ofdegraded DNA [2] In fact as the triple multiplex systemmakes very high success rate in haplogroup determinationthe combined consideration of coding region SNP markersand control region polymorphism can become a very usefultool for the genetic analysis of degraded samples collectedfrom East Asian populations

This technique is also very suggestive for anthropolo-gists who deal with several hundred- to thousand-year-old

samples from archaeological sites Most of the archaeologicalhuman bones were maintained under the worst preservationconditions for a long while To make matters worse it isvery hard for researchers to get authentic outcomes fromthe ancient samples because only the limited volume of thesamples can be allowed for the analysis of archaeologicallyimportant cases We therefore admit that mtDNA analyseswith highly degraded ancient samples are sometimes toorisky because sequencing errors commonly occurred duringanalysis and they could not be corrected easily by a duecourse of repeated experiments with a sufficient amount ofsamples

In this regard if another time- cost- and sample-savingmtDNA analysis could be also established for the assignment

BioMed Research International 5

10400A

14668G

10397A4491C

8793G7642C

8794C5417C

4883G 4833A9824A

7196A

12705T

3970G 9-bp deletion

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

HV1A HV1B HV2A HV2B HV3

100200300400500

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

GT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

TCTT

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

C

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

-G

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS0005rCRS0005rCRS0005rCRS0005

rCRS0005rCRS0005rCRS0005

rCRS0005

15991

1

16111

121

16231

221

16351

341

35

1

155

121

275

241

424

1

16110

120

16230

220

16350

340

16390

380

154

120

274

240

369

337

548

125

(c) Sequencing result of control region

Figure 1 Analysis on sample number 005 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceHaplogroups (M8) determined by coding region SNP analysis (a) and control region direct sequencing ((b)(c)) are the same for this case

of ancient skeleton samples to relevant existing haplogroupsit will become a convenient method indeed for coun-terchecking the possible errors hidden in the conventionalmtDNA sequencing Our current results showed that mosthaplogroup results determined by coding region SNP analysison Joseon archaeological samples can fall successfully withinthe same haplogroups that were decided by control regionsequencing analysis In fact the results confirm coding regionSNP analysisrsquo error-screening role in the mtDNA haplogroupdetermination even for the ancient samples

However as for the current ancient samples wemust alsoadmit some technical limits of haplogroup determinationbased on coding region SNP analysis Briefly a coding regionSNP analysis on a few samples did not show the subhap-logroup results as completely as observed in the analysis ofcontrol region mutation motifs This means that multiplexSNP analysis could not completely replace the conventionalcontrol region mtDNA sequencing at least for the ancientcases from archaeological fields in South Korea Even soconsidering coding region SNP analysisrsquo superb potential

for reconfirmation of haplogroups determined by mtDNAcontrol region sequencing in time- cost- and target DNA-saving manner the use of this method can be expedient formaking mtDNA haplogroup determination of archaeologicalsamples much authentic

5 Conclusion

Obtaining authentic mtDNA outcomes from archaeologi-cal bone samples still remains a significant challenge toconcerned researchers The identification of possible errorsin conventional sequencing as quickly as possible is thussignificant for authentic mtDNA analysis of archaeologicallyobtained samples In this study we can show that mtDNAhaplogroup determination can also be successfully carriedout by coding region SNP analysis in a time- cost- and targetDNA-saving manner Although the mtDNA subhaplogroupscould not be determined by the coding region SNP analysis ascompletely seen in the control region sequencing the formercan be used as good supplementary to the latter for making

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

OH USA) at 37∘C for 45min After that the enzyme wasinactivated by incubation at 80∘C for 15min

We used twenty-two single base extension (SBE) primersrecommended by Lee et al [2] SBE reactionswere carried outusing a SNaPshotKit (AppliedBiosystemsUSA) according tothe manufacturerrsquos instructions Thermal cycling conditionsfor SBE were as follows denaturation at 96∘C for 10 secannealing at 50∘C for 5 sec extension at 60∘C for 30 secSBE was performed using a PTC-200 DNA Engine (Bio-RadLaboratories Hercules CA) For postextension treatmentreaction mixtures were mixed with 10 unit of shrimp alka-line phosphatase (SAP) incubated at 37∘C for 45min andfollowed by heat inactivation at 80∘C for 15minThe reactantswere analyzed by an ABI PRISM 3100 Genetic Analyzer(Applied Biosystems USA) using GeneMapper ID softwarev321 (Applied Biosystems USA) SNP scoring at each locuswas confirmed by sequencing two samples for each of theobserved alleles

We also did direct sequencing of mtDNA control regionof the samples By sequencing of hypervariable regions I IIand III we could get haplotype of the bones and furtherdetermined haplogroups of them The results could be com-pared with haplogroup determination by coding region SNPanalysis on the same samples Briefly after quantification wasdone by NanoDrop ND-1000 Spectrophotometer (ThermoFisher Scientific MA USA) 40 ng of aDNA was mixed withpremix containing 1X AmpliTaq Gold 360 Master Mix (LifeTechnologies USA) and 10 pmol of each primer (IntegratedDNA Technology USA) PCR conditions used in this studywere as follows predenaturation at 94∘C for 10min 45 cyclesof denaturation at 94∘C for 30 sec annealing at 50∘C for30 sec extension at 72∘C for 30 sec final extension at 72∘C for10min PCR amplification was performed using a PTC-200DNA Engine (Bio-Rad Laboratories Hercules CA) Primersets used for this study were as follows for 267-bp HV1AF15971 (51015840-TTA ACT CCA CCA TTA GCA CC-31015840) andR16237 (51015840-TGT GTG ATA GTT GAG GGT TG-31015840) for 267-bp HV1B F16144 (51015840-TGA CCACCTGTAGTACATAA-31015840)and R16410 (51015840-GAGGAT GGTGGT CAAGGGAC-31015840) for226-bpHV2A F015 (51015840-CACCCTATTAACCACTCACG-31015840) and R240 (51015840-TAT TATTATGTCCTACAAGCA-31015840) for235-bp HV2B F155 (51015840-CTA TTA TTT ATC GCA CCT-31015840)and R389 (51015840-CTG GTT AGG CTG GTG TTA GG-31015840) for167-bp HV3 F403 (51015840-TCT TTT GGC GGT ATG CAC TTT-31015840) and R569 (51015840-GGT GTA TTT GGG GTT TGG TTG-31015840)[26]

The PCR products were separated on 25 agarose gelstained with ethidium bromide and then isolated using aQiagen gel extraction kit (Qiagen Germany) The sequenc-ing of each amplicon was performed by ABI Prism 3100Genetic Analyzer (Applied Biosystems USA) using ABIPrism BigDye Terminator Cycle Sequencing Ready ReactionKit (AppliedBiosystemsUSA)TheobtainedDNAsequenceswere compared with the revised Cambridge ReferenceSequence (rCRS accession number NC 012920) to identifythe sequence differences between themThe resultant controlregion mutation motifs were imported into program mtD-NAmanager (httpmtmanageryonseiackr) with which

most Korean mtDNA haplotypes can be automatically clas-sified into East Asian mtDNA haplogroups and their subhap-logroups [27] or another web-based program for mtDNAhaplogroup analysis (httpdnajameslickcommthap) [2829]

In order to guard against any modern DNA contam-ination of ancient samples the mtDNA profiles of all ofthe researchers involved in this study were determined(with the permission of the Institutional Review Board ofSeoul National University H-0909-049-295)They were thencomparedwith themtDNAprofiles from the Joseon skeletonsto rule out the possibility of modern DNA contamination

3 Results

The sex and age of the samples determined in this studyare summarized in Table 1 By direct sequencing of mtDNAcontrol region every Joseon skeleton could be assigned torelevant existing haplo- or subhaplogroups of mtDNA Inmultiplex PCR analyses to detect 21 SNP markers in mtDNAcoding region eight (multiplex I) and seven (multiplexes IIand III) primer extension peaks for different haplogroupscould be observed Most samples exhibited no missing orextra assignment peaks in the results (Figures 1 and 2)

When the coding region SNP typing data were furthercompared with control region direct sequencing resultswell-matching patterns can be observed between the out-comes of two methods The mtDNA haplogroup expectedby coding region SNP could fall successfully within thesame haplogroup that control region sequencing could make(Table 1 Figure 1) However as far as haplogroup subcladesare concerned the results of direct sequencing on controlregion mutation motif were far better than SNP markeranalysis on coding region The haplogroup subclades of thecases numbers 024 034 036 038 040 045 100 and 238 weremuch successfully determined in direct sequencing of controlregion than in coding region SNP analysis (Table 1 Figure 2)

The absence of modern DNA contamination couldbe confirmed by the comparison of mtDNA haplotypesobtained from the current Joseon skeleton and participatingresearchersrsquo samples Aswe did not see any identical sequencebetween them (Table 1) mtDNA obtained from Joseon sam-ples should have been the endogenous DNA of the ancientpeople but not the outcome of modern DNA contaminationfrom researchers

4 Discussion

Coding region SNP analysis attracts forensic scientistsrsquo inter-est because it is a time- cost- and target DNA-savingmethodfor mtDNA analysis of degraded samples [2 30ndash35] Multi-plex PCR system for coding region SNPused in this studywasoriginally designed for the detection of major haplogroupsof forensic samples from East Asia In the study the triplemultiplex system on coding region SNP markers was proven

4 BioMed Research International

Table 1 Sequencing analysis result of mtDNA coding and control region

Subject Sex AgeHypervariable region Haplogroup

(control region)Haplogroup

(coding region)HVI (15991ndash16390) HVII (034ndash369) HVIII(423ndash548)

004 Male Old 16223T 16235G 16290T 73G 235G 263G3151C rCRS A A

005 Female Young16171G 16223T 16248T16298C 16327T 16344T16357C

73G 248d 263G3151C 489C M8 M8

024 Male Middle 16129A 16223T 16309G16362C

73G 152C 263G3091C 3151C

489C533G D4a1b1 D4a

034 Male Old16189C 16232A 16249C16304C16311C 16344T

73G 263G 310C 489C M1a3b1 M

036 Male Old 16150T 16183C 16185T16189C

73G 151T 197G263G 3151C 546G B4d3 B

038 Male Old 16129A 16223T 16362C 73G 194T 263G3151C 489C D4b2b D4b

040 Male Middle 161881C 161931C 16223T16311C

73G 263G 3091C3151C 489C M41015840101584067 M

045 Male Old16223T 16234T 16311C16316G16362C

73G 263G 3091C3151C 489C M9a1a1c1b1 M9

100 Male Middle16111T 16229A 16257A16261T16362C

73G 150T 263G3092C 3151C rCRS N9a N9

225 Male Middle 16209C 16223T 16362C 73G 195C 234G263G 3151C 489C D4 D4

238 Female Old 16223T 16295T 16319A 73G 146C 263G3091C 3151C

489C513d 514d M7c1a3 M7

Researcher 1 mdash mdash 16172C 16174T 16223T16362C

73G 263G 3091C3151C mdash D4g mdash

Researcher 2 mdash mdash16183C 16189C 16220C16254G16298C 16362C

73G 248d 263G3101C mdash F3b mdash

Researcher 3 mdash mdash16129A 16182C 16183C16189C16232A 16249C 16304C16311C 16344T

73G 152C 248d263G 3101C mdash D6c mdash

to be very useful for analysis on forensic materials [2]Briefly when they tried to do the coding region SNP-based mtDNA analysis on the long bone or molar samplesfrom about 50-year-old skeletal remains of war victims inthe Korean War (1950ndash1953) mtDNA haplogroup couldbe determined successfully even with a limited volume ofdegraded DNA [2] In fact as the triple multiplex systemmakes very high success rate in haplogroup determinationthe combined consideration of coding region SNP markersand control region polymorphism can become a very usefultool for the genetic analysis of degraded samples collectedfrom East Asian populations

This technique is also very suggestive for anthropolo-gists who deal with several hundred- to thousand-year-old

samples from archaeological sites Most of the archaeologicalhuman bones were maintained under the worst preservationconditions for a long while To make matters worse it isvery hard for researchers to get authentic outcomes fromthe ancient samples because only the limited volume of thesamples can be allowed for the analysis of archaeologicallyimportant cases We therefore admit that mtDNA analyseswith highly degraded ancient samples are sometimes toorisky because sequencing errors commonly occurred duringanalysis and they could not be corrected easily by a duecourse of repeated experiments with a sufficient amount ofsamples

In this regard if another time- cost- and sample-savingmtDNA analysis could be also established for the assignment

BioMed Research International 5

10400A

14668G

10397A4491C

8793G7642C

8794C5417C

4883G 4833A9824A

7196A

12705T

3970G 9-bp deletion

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

HV1A HV1B HV2A HV2B HV3

100200300400500

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

GT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

TCTT

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

C

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

-G

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS0005rCRS0005rCRS0005rCRS0005

rCRS0005rCRS0005rCRS0005

rCRS0005

15991

1

16111

121

16231

221

16351

341

35

1

155

121

275

241

424

1

16110

120

16230

220

16350

340

16390

380

154

120

274

240

369

337

548

125

(c) Sequencing result of control region

Figure 1 Analysis on sample number 005 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceHaplogroups (M8) determined by coding region SNP analysis (a) and control region direct sequencing ((b)(c)) are the same for this case

of ancient skeleton samples to relevant existing haplogroupsit will become a convenient method indeed for coun-terchecking the possible errors hidden in the conventionalmtDNA sequencing Our current results showed that mosthaplogroup results determined by coding region SNP analysison Joseon archaeological samples can fall successfully withinthe same haplogroups that were decided by control regionsequencing analysis In fact the results confirm coding regionSNP analysisrsquo error-screening role in the mtDNA haplogroupdetermination even for the ancient samples

However as for the current ancient samples wemust alsoadmit some technical limits of haplogroup determinationbased on coding region SNP analysis Briefly a coding regionSNP analysis on a few samples did not show the subhap-logroup results as completely as observed in the analysis ofcontrol region mutation motifs This means that multiplexSNP analysis could not completely replace the conventionalcontrol region mtDNA sequencing at least for the ancientcases from archaeological fields in South Korea Even soconsidering coding region SNP analysisrsquo superb potential

for reconfirmation of haplogroups determined by mtDNAcontrol region sequencing in time- cost- and target DNA-saving manner the use of this method can be expedient formaking mtDNA haplogroup determination of archaeologicalsamples much authentic

5 Conclusion

Obtaining authentic mtDNA outcomes from archaeologi-cal bone samples still remains a significant challenge toconcerned researchers The identification of possible errorsin conventional sequencing as quickly as possible is thussignificant for authentic mtDNA analysis of archaeologicallyobtained samples In this study we can show that mtDNAhaplogroup determination can also be successfully carriedout by coding region SNP analysis in a time- cost- and targetDNA-saving manner Although the mtDNA subhaplogroupscould not be determined by the coding region SNP analysis ascompletely seen in the control region sequencing the formercan be used as good supplementary to the latter for making

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 1 Sequencing analysis result of mtDNA coding and control region

Subject Sex AgeHypervariable region Haplogroup

(control region)Haplogroup

(coding region)HVI (15991ndash16390) HVII (034ndash369) HVIII(423ndash548)

004 Male Old 16223T 16235G 16290T 73G 235G 263G3151C rCRS A A

005 Female Young16171G 16223T 16248T16298C 16327T 16344T16357C

73G 248d 263G3151C 489C M8 M8

024 Male Middle 16129A 16223T 16309G16362C

73G 152C 263G3091C 3151C

489C533G D4a1b1 D4a

034 Male Old16189C 16232A 16249C16304C16311C 16344T

73G 263G 310C 489C M1a3b1 M

036 Male Old 16150T 16183C 16185T16189C

73G 151T 197G263G 3151C 546G B4d3 B

038 Male Old 16129A 16223T 16362C 73G 194T 263G3151C 489C D4b2b D4b

040 Male Middle 161881C 161931C 16223T16311C

73G 263G 3091C3151C 489C M41015840101584067 M

045 Male Old16223T 16234T 16311C16316G16362C

73G 263G 3091C3151C 489C M9a1a1c1b1 M9

100 Male Middle16111T 16229A 16257A16261T16362C

73G 150T 263G3092C 3151C rCRS N9a N9

225 Male Middle 16209C 16223T 16362C 73G 195C 234G263G 3151C 489C D4 D4

238 Female Old 16223T 16295T 16319A 73G 146C 263G3091C 3151C

489C513d 514d M7c1a3 M7

Researcher 1 mdash mdash 16172C 16174T 16223T16362C

73G 263G 3091C3151C mdash D4g mdash

Researcher 2 mdash mdash16183C 16189C 16220C16254G16298C 16362C

73G 248d 263G3101C mdash F3b mdash

Researcher 3 mdash mdash16129A 16182C 16183C16189C16232A 16249C 16304C16311C 16344T

73G 152C 248d263G 3101C mdash D6c mdash

to be very useful for analysis on forensic materials [2]Briefly when they tried to do the coding region SNP-based mtDNA analysis on the long bone or molar samplesfrom about 50-year-old skeletal remains of war victims inthe Korean War (1950ndash1953) mtDNA haplogroup couldbe determined successfully even with a limited volume ofdegraded DNA [2] In fact as the triple multiplex systemmakes very high success rate in haplogroup determinationthe combined consideration of coding region SNP markersand control region polymorphism can become a very usefultool for the genetic analysis of degraded samples collectedfrom East Asian populations

This technique is also very suggestive for anthropolo-gists who deal with several hundred- to thousand-year-old

samples from archaeological sites Most of the archaeologicalhuman bones were maintained under the worst preservationconditions for a long while To make matters worse it isvery hard for researchers to get authentic outcomes fromthe ancient samples because only the limited volume of thesamples can be allowed for the analysis of archaeologicallyimportant cases We therefore admit that mtDNA analyseswith highly degraded ancient samples are sometimes toorisky because sequencing errors commonly occurred duringanalysis and they could not be corrected easily by a duecourse of repeated experiments with a sufficient amount ofsamples

In this regard if another time- cost- and sample-savingmtDNA analysis could be also established for the assignment

BioMed Research International 5

10400A

14668G

10397A4491C

8793G7642C

8794C5417C

4883G 4833A9824A

7196A

12705T

3970G 9-bp deletion

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

HV1A HV1B HV2A HV2B HV3

100200300400500

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

GT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

TCTT

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

C

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

-G

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS0005rCRS0005rCRS0005rCRS0005

rCRS0005rCRS0005rCRS0005

rCRS0005

15991

1

16111

121

16231

221

16351

341

35

1

155

121

275

241

424

1

16110

120

16230

220

16350

340

16390

380

154

120

274

240

369

337

548

125

(c) Sequencing result of control region

Figure 1 Analysis on sample number 005 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceHaplogroups (M8) determined by coding region SNP analysis (a) and control region direct sequencing ((b)(c)) are the same for this case

of ancient skeleton samples to relevant existing haplogroupsit will become a convenient method indeed for coun-terchecking the possible errors hidden in the conventionalmtDNA sequencing Our current results showed that mosthaplogroup results determined by coding region SNP analysison Joseon archaeological samples can fall successfully withinthe same haplogroups that were decided by control regionsequencing analysis In fact the results confirm coding regionSNP analysisrsquo error-screening role in the mtDNA haplogroupdetermination even for the ancient samples

However as for the current ancient samples wemust alsoadmit some technical limits of haplogroup determinationbased on coding region SNP analysis Briefly a coding regionSNP analysis on a few samples did not show the subhap-logroup results as completely as observed in the analysis ofcontrol region mutation motifs This means that multiplexSNP analysis could not completely replace the conventionalcontrol region mtDNA sequencing at least for the ancientcases from archaeological fields in South Korea Even soconsidering coding region SNP analysisrsquo superb potential

for reconfirmation of haplogroups determined by mtDNAcontrol region sequencing in time- cost- and target DNA-saving manner the use of this method can be expedient formaking mtDNA haplogroup determination of archaeologicalsamples much authentic

5 Conclusion

Obtaining authentic mtDNA outcomes from archaeologi-cal bone samples still remains a significant challenge toconcerned researchers The identification of possible errorsin conventional sequencing as quickly as possible is thussignificant for authentic mtDNA analysis of archaeologicallyobtained samples In this study we can show that mtDNAhaplogroup determination can also be successfully carriedout by coding region SNP analysis in a time- cost- and targetDNA-saving manner Although the mtDNA subhaplogroupscould not be determined by the coding region SNP analysis ascompletely seen in the control region sequencing the formercan be used as good supplementary to the latter for making

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

10400A

14668G

10397A4491C

8793G7642C

8794C5417C

4883G 4833A9824A

7196A

12705T

3970G 9-bp deletion

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

HV1A HV1B HV2A HV2B HV3

100200300400500

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

GT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

TCTT

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

C

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

-G

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS0005rCRS0005rCRS0005rCRS0005

rCRS0005rCRS0005rCRS0005

rCRS0005

15991

1

16111

121

16231

221

16351

341

35

1

155

121

275

241

424

1

16110

120

16230

220

16350

340

16390

380

154

120

274

240

369

337

548

125

(c) Sequencing result of control region

Figure 1 Analysis on sample number 005 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceHaplogroups (M8) determined by coding region SNP analysis (a) and control region direct sequencing ((b)(c)) are the same for this case

of ancient skeleton samples to relevant existing haplogroupsit will become a convenient method indeed for coun-terchecking the possible errors hidden in the conventionalmtDNA sequencing Our current results showed that mosthaplogroup results determined by coding region SNP analysison Joseon archaeological samples can fall successfully withinthe same haplogroups that were decided by control regionsequencing analysis In fact the results confirm coding regionSNP analysisrsquo error-screening role in the mtDNA haplogroupdetermination even for the ancient samples

However as for the current ancient samples wemust alsoadmit some technical limits of haplogroup determinationbased on coding region SNP analysis Briefly a coding regionSNP analysis on a few samples did not show the subhap-logroup results as completely as observed in the analysis ofcontrol region mutation motifs This means that multiplexSNP analysis could not completely replace the conventionalcontrol region mtDNA sequencing at least for the ancientcases from archaeological fields in South Korea Even soconsidering coding region SNP analysisrsquo superb potential

for reconfirmation of haplogroups determined by mtDNAcontrol region sequencing in time- cost- and target DNA-saving manner the use of this method can be expedient formaking mtDNA haplogroup determination of archaeologicalsamples much authentic

5 Conclusion

Obtaining authentic mtDNA outcomes from archaeologi-cal bone samples still remains a significant challenge toconcerned researchers The identification of possible errorsin conventional sequencing as quickly as possible is thussignificant for authentic mtDNA analysis of archaeologicallyobtained samples In this study we can show that mtDNAhaplogroup determination can also be successfully carriedout by coding region SNP analysis in a time- cost- and targetDNA-saving manner Although the mtDNA subhaplogroupscould not be determined by the coding region SNP analysis ascompletely seen in the control region sequencing the formercan be used as good supplementary to the latter for making

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

10400A14668T

10397G4491C

8793T

7642C 8794C5417C

4883T4833A

9824T

7196G

12705T3970C

9-bp deletion

3010T14979A

8020A

11215G8701C

5048T11696G

20 30 40 50 60 70

20 30 40 50 60 70

20 30 40 50 60 70

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

10000

8000

6000

4000

2000

0

(a) Coding region

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

Sam

ple

NC

HV1A HV1B HV2A HV2B HV3

100200300400500

(b) Control region

CAAAGCTAAGATTCTAATTTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTTGGGTACCACCCAAGTATTGACTCACCCATCAACAACCGCTATGTATTTCGTACATTACTGCCAG

CCACCATGAATATTGTACGGTACCATAAATACTTGACCACCTGTAGTACATAAAAACCCAATCCACATCAAAACCCCCTCCCCATGCTTACAAGCAAGTACAGCAATCAACCCTCAACTA

AT

TCACACATCAACTGCAACTCCAAAGCCACCCCTCACCCACTAGGATACCAACAAACCTACCCACCCTTAACAGTACATAGTACATAAAGCCATTTACCGTACATAGCACATTACAGTCAA

ATCCCTTCTCGTCCCCATGGATGACCCCCCTCAGATAGGG

GGAGCTCTCCATGCATTTGGTATTTTCGTCTGGGGGGTATGCACGCGATAGCATTGCGAGACGCTGGAGCCGGAGCACCCTATGTCGCAGTATCTGTCTTTGATTCCTGCCTCATCCTAT

G

TATTTATCGCACCTACGTTCAATATTACAGGCGAACATACTTACTAAAGTGTGTTAATTAATTAATGCTTGTAGGACATAATAATAACAATTGAATGTCTGCACAGCCACTTTCCACACA

TG

GACATCATAACAAAAAATTTCCACCAAAccccccctccccc-GCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAAC

C

TAACAGTCACCCCCCAACTAACACATTATTTTCCCCTCCCACTCCCATACTACTAATCTCATCAATACAACCCCCGCCCATCCTACCCAGcacacacacaccgctgctaaccccataccccgaac

C

rCRS 159910038 1rCRS 161110038 121rCRS 162310038 221rCRS 163510038 341

rCRS 350038 1rCRS 1550038 121rCRS 2750038 241

rCRS 4240038 1

369

336

16390

380

16110

120

16230

220

16350

340

154

120

274

240

548

125

C

(c) Sequencing result of control region

Figure 2 Analysis on sample number 038 (a) SNP analysis of mtDNA coding region by a SNaPshot Kit ((b) (c)) Direct sequencing analysisonmtDNA control region (b) Electrophoresis of PCR amplicons (c) Direct sequencing result rCRS revised Cambridge Reference SequenceAlthoughmtDNA haplogroup expected by coding region SNP analysis could fall successfully within the same haplogroup that control regionsequencing couldmake haplogroup subclademade by SNP analysis (D4b)was not as successful as seen in control region sequencing (D4b2b)

the mtDNA typing of archaeological human bones muchauthentic

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Seoul National UniversityHospital (SNUH) research fund (04-2011-0900 2011-1350)and by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by

the Ministry of Education Science and Technology (2012-0002118)

References

[1] J M Butler Forensic DNA Typing Biology and Technologybehind STR Markers Elsevier Burlington Mass USA 2ndedition 2005

[2] H Y Lee J-E Yoo M J Park U Chung C-Y Kim andK-J Shin ldquoEast Asian mtDNA haplogroup determination inKoreans haplogroup-level coding region SNP analysis andsubhaplogroup-level control region sequence analysisrdquo Elec-trophoresis vol 27 no 22 pp 4408ndash4418 2006

[3] M J Kim C S Oh I S Lee et al ldquoHuman mummifiedbrain from a medieval tomb with lime-soil mixture barrier of

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

the Joseon Dynasty Koreardquo International Journal of Osteoar-chaeology vol 18 no 6 pp 614ndash623 2008

[4] D K Kim I S Lee W-L Kim et al ldquoPossible rheumatoidarthritis found in the human skeleton collected from thetomb of Joseon Dynasty Korea dating back to the 1700s ADrdquoInternational Journal of Osteoarchaeology vol 21 no 2 pp 136ndash149 2011

[5] Y S Kim C S Oh S J Lee J B Park M J Kim and D HShin ldquoSex determination of Joseon people skeletons based onanatomical cultural and molecular biological cluesrdquo Annals ofAnatomy vol 193 no 6 pp 539ndash543 2011

[6] D K Kim M J Kim Y Kim et al ldquoLong bone fracturesidentified in the Joseon Dynasty human skeletons of KoreardquoAnatomy amp Cell Biology vol 46 no 3 pp 203ndash209 2013

[7] S S Han K-W Baek M H Shin et al ldquoDental cariesprevalence ofmedieval Korean peoplerdquoArchives of Oral Biologyvol 55 no 7 pp 535ndash540 2010

[8] D H Shin C S Oh Y-S Kim and Y-I Hwang ldquoAncient-to-modern secular changes in Korean staturerdquoAmerican Journal ofPhysical Anthropology vol 147 no 3 pp 433ndash442 2012

[9] J Beom E J Woo I S Lee et al ldquoHarris lines observed inhuman skeletons of Joseon Dynasty Koreardquo Anatomy amp CellBiology vol 47 no 1 pp 66ndash72 2014

[10] T W Phenice ldquoA newly developed visual method of sexing theos pubisrdquoAmerican Journal of Physical Anthropology vol 30 no2 pp 297ndash301 1969

[11] W M Krogman and M Y Iscan The Human Skeleton inForensic Medicine Charles CThomas Publisher Springfield IllUSA 2nd edition 1986

[12] J E Buikstra and D H Ubelaker Standards for Data Collectionfrom Human Skeletal Remains vol 44 of Arkansas Archaeo-logical Survey Research Series Arkansas Archeological SurveyFayetteville Ark USA 1994

[13] D H Ubelaker Human Skeletal Remains Excavation AnalysisInterpretation vol 2 of Manuals on Archeology TaraxacumWashington DC USA 3rd edition 1999

[14] C O Lovejoy R S Meindl T R Pryzbeck and R PMensforthldquoChronological metamorphosis of the auricular surface of theilium a new method for the determination of adult skeletal ageat deathrdquoAmerican Journal of Physical Anthropology vol 68 no1 pp 15ndash28 1985

[15] D H OrsquoRourke M G Hayes and S W Carlyle ldquoAncientDNA studies in physical anthropologyrdquo Annual Review ofAnthropology vol 29 pp 217ndash242 2000

[16] N Rohland and M Hofreiter ldquoAncient DNA extraction frombones and teethrdquo Nature Protocols vol 2 no 7 pp 1756ndash17622007

[17] D Y Yang B Eng J S Waye J C Dudar and S R SaundersldquoImproved DNA extraction from ancient bones using silica-based spin columnsrdquoThe American Journal of Physical Anthro-pology vol 105 no 4 pp 539ndash543 1998

[18] M J Casas E Hagelberg R Fregel J M Larruga and AM Gonzalez ldquoHuman mitochondrial DNA diversity in anarchaeological site in al-Andalus genetic impact of migrationsfrom North Africa in Medieval Spainrdquo American Journal ofPhysical Anthropology vol 131 no 4 pp 539ndash551 2006

[19] M J Blow T Zhang T Woyke et al ldquoIdentification of ancientremains through genomic sequencingrdquo Genome Research vol18 no 8 pp 1347ndash1353 2008

[20] S Calvignac S Hughes C Tougard et al ldquoAncient DNAevidence for the loss of a highly divergent brown bear clade

during historical timesrdquo Molecular Ecology vol 17 no 8 pp1962ndash1970 2008

[21] M Hofreiter D Serre H N Poinar M Kuch and S PaaboldquoAncient DNArdquo Nature Reviews Genetics vol 2 no 5 pp 353ndash359 2001

[22] T Kivisild H V Tolk J Parik et al ldquoThe emerging limbs andtwigs of the East Asian mtDNA treerdquo Molecular Biology andEvolution vol 19 no 10 pp 1737ndash1751 2002

[23] Q-P Kong Y-G Yao M Liu et al ldquoMitochondrial DNAsequence polymorphisms of five ethnic populations fromnorthern Chinardquo Human Genetics vol 113 no 5 pp 391ndash4052003

[24] Q-P Kong H-J Bandelt C Sun et al ldquoUpdating the EastAsian mtDNA phylogeny a prerequisite for the identificationof pathogenic mutationsrdquo Human Molecular Genetics vol 15no 13 pp 2076ndash2086 2006

[25] M Tanaka VMCabrera AMGonzalez et al ldquoMitochondrialgenome variation in Eastern Asia and the peopling of JapanrdquoGenome Research vol 14 no 10 pp 1832ndash1850 2004

[26] M M Holland and E F Huffine ldquoMolecular analysis of thehuman mitochondrial DNA control region for forensic identitytestingrdquo in Current Protocols in Human Genetics vol 147chapter 14 unit 147 2001

[27] H Y Lee I Song E Ha S-B Cho W I Yang and K-JShin ldquomtDNAmanager a Web-based tool for the managementand quality analysis of mitochondrial DNA control-regionsequencesrdquo BMC bioinformatics vol 9 no 1 article 483 2008

[28] E D Aulicino Genetic Genealogy The Basics and BeyondAuthorHouse Bloomington Ind USA 2013

[29] D Kennett DNA and Social Networking A Guide to Genealogyin the Twenty-First Century The History Press London UK2012

[30] H J Bandelt P Lahermo M Richards and V MacaulayldquoDetecting errors in mtDNA data by phylogenetic analysisrdquoInternational Journal of LegalMedicine vol 115 no 2 pp 64ndash692001

[31] H-J Bandelt L Quintana-Murci A Salas and V MacaulayldquoThe fingerprint of phantom mutations in mitochondrial DNAdatardquo The American Journal of Human Genetics vol 71 no 5pp 1150ndash1160 2002

[32] H J Bandelt A Salas and S Lutz-Bonengel ldquoArtificial recom-bination in forensic mtDNA population databasesrdquo Interna-tional Journal of Legal Medicine vol 118 no 5 pp 267ndash2732004

[33] P Forster ldquoTo err is humanrdquo Annals of Human Genetics vol 67no 1 pp 2ndash4 2003

[34] Y G Yao C M Bravi and H J Bandelt ldquoA call for mtDNAdata quality control in forensic sciencerdquo Forensic Science Inter-national vol 141 no 1 pp 1ndash6 2004

[35] A Salas A Carracedo VMacaulayM Richards andH-J Ban-delt ldquoA practical guide to mitochondrial DNA error preventionin clinical forensic and population geneticsrdquo Biochemical andBiophysical Research Communications vol 335 no 3 pp 891ndash899 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology