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DNA Typing Strategy for the Identification of Old Skeletal Remains

Dept. of Forensic MedicineYonsei University College of Medicine

Hwan Young Lee, Ph.D.

Presentation Overview

DNA ExtractionComplete demineralization

DNA recovery using silica-based column

mtDNA AnalysisModified midi- and mini-primers

Quality analysis based on mtDNA phylogeny

STR GenotypingAutosomal-STR and Y-STR

Size-reduced mini-STR

Redundant approach to data generation and analysis

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DNA Extraction and Quantification

DNA from Old Skeletal Remains

Low copy number (LCN) DNA

Highly degraded DNA

Presence of PCR inhibitors

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Sample PreparationThe surface of each bone sample was removed using a dental drill, and the sample was cut into small slices using a dental diamond disk.

Irradiation with UV light

Cleaned bone sample was powdered using 6750 SpexCertiPrep Freezer/Mill (SPEX CertiPrep, NJ)

DNA Extraction from Old Skeletal Remains

Complete demineralization

Large-scale silica-based column extraction

Croat Med J 2007;48:478-85

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Complete Demineralization

Demineralization solution0.5 g bone power

15 ml of 0.5 M EDTA and 0.5% SDS

3 mg Proteinase K

Incubation48 hours at 56℃ in dry incubator

1 hour after additional treatment of 3 mg of Proteinase K

DNA Recovery Using Silica-based Column

2 ml of DNA extractQIAamp® Blood DNA Maxi column

Buffers from QIAquick® PCR purification kit

50 µl of DNA extract

Concentration of DNA extractQIAamp® DNA Mini column

Buffers from QIAquick® PCR purification kit

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Large-scaleSmall-scale*(Yang et al. 1998)

106.1 ± 05.49029.4 ± 19.665

060.6 ± 18.80038.9 ± 04.524

361.4 ± 09.90039.5 ± 06.693

518.5 ± 82.13108.1 ± 09.592

074.1 ± 05.64012.7 ± 00.081

Concentration(pg/µl)Sample Concentration

(pg/µl)

Quantification Using Real-time qPCRQuantifiler® Human DNA Quantification kit

Applied Biosystems 7500 Real-time PCR system

*DNA was extracted using the QIAquick® PCR purification kit after incomplete demineralization of bone powder by small-volume high-concentration of EDTA.

Sequence and Quality Analyses of mtDNA from Old Skeletal Remains

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Primers for Amplification of mtDNA

F015/R259*

F155/R389

F15989/R16251

P11

F16159*/R16410m*

P12

P21

P22F403/R614*

P31

16024 16569/1 340 43873 57416365

F15989/R16153*

M11

F015/R187*

M21

F120*/R285

M22F403/R568

M31

F220/R389

M23F16088*/R16233*

M12

F16159*/R16322m*

M13

F16258*/R16410m*

M14

HV1 HV2 HV3

Midi-primerSet

Mini-primerSet

Lee et al. 2008BioTechniques 44:555-558

Detection of mtDNA Sequence Errors

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mtDNAmanager’s Open Database

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Automatic Estimation of Haplogroups

Along with the data import, simultaneous estimation of the most-probable mtDNA haplogroup is carried out.

Haplogroup-specific Mutation Motifs

Control Region Mutation motifs for more than 400 mtDNA haplogroups

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Quality Analysis of mtDNA Sequences

Accompanying mutations for Expected HG

Clear key diagnostic mutations

Detection of Possible mtDNA Errors

16362C?

150?

N9a1: 16129-16223-16257A-16261-150

16319 missed out?

A5a: 16187-16223-16290-16319-235-523d-524d

16311 missed out M10b? B5b?

Artificial recombination?

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STR Genotyping of DNA from Old Skeletal Remains

STR Typing Using Low Copy Number DNA

LCN DNAAllele drop out

Allele drop-in

Stochastic effect

http://www.cstl.nist.gov/div831/strbase/training.htm

ExtractionAliquotPCR

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PCR Strategy for LCN DNA

Independant set up of PCR

1st amplification

PCR master mix I for 1st DNA extract

PCR master mix II for 2nd DNA extract

2nd amplificationPCR master mix III for 1st DNA extract

PCR master mix IV for 2nd DNA extract

“LCN interpretation rule”Replicate analyses with duplicate results prior to reporting alleles

Scoring of STR Alleles

Peak detection threshold75 ~ 200 RFU

Cut-off minor alleleUnder 15% of peak height of major peak

An allele cannot be scored unless it is observed at least tree times in four PCR reactions

http://www.cstl.nist.gov/div831/strbase/training.htm

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Scoring of STR Alleles

GATA GATAGATAGATAGATA GATA

Forward flanking region Reverse flanking region

AmpFlSTR® MiniFiler™ was used as a complement to the AmpFlSTR® Identifiler®

Y-miniplex plus as a complement to the AmpFlSTR® Yfiler™

In-house miniplex NC01 plus was used to increase the discrimination capacity of the system

Size-reduced Amplicon of mini-STRs

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AmpFlSTR® MiniFiler™

AmpFlSTR® Identifiler®

Y-miniplex plus

http://forensic.yonsei.ac.kr/protocols.htmlBioquest INC

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Y-miniplex plus

AmpFlSTR® Yfiler™

In-house NC01 plus

D14S14349 15

VIC

D22S104511 19

NED

11 17D10S12486FAM

PET TPOX5 14

50bp 100bp 150bp75bp 125bp

LIZ

500 LIZ™-internal lane standard

http://forensic.yonsei.ac.kr/protocols.html

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Improved STR Typing Results

14.312.317.213.911.021Total

17.016.518.015.014.86High

14.812.418.014.612.08Medium

11.68.715.712.16.77Low

Y-miniplexplus

AmpFℓSTR®

YFiler™ KitNC01 plusAmpFℓSTR®

MiniFiler™ KitAmpFℓSTR®

Identifiler® Kit

17 Y-STR loci 18 AS-STR loci 15 AS-STR loci Number of

samples

Sample

quality

Mean number of successfully genotyped STR loci was calculated using AmpFℓSTR® Identifiler® Kit, AmpFℓSTR® MiniFiler™ Kit, NC01plus, AmpFℓSTR® YFiler™ Kit and the Y-miniplex plus in skeletal remains obtained from Korean War victims (n = 21).

Thank you for your attention!

hylee192@yuhs.achttp://forensic.yonsei.ac.kr