DNA Typing Strategy for the Identification of Old Skeletal Remains

Transcription

1 DNA Typing Strategy for the Identification of Old Skeletal Remains Hwan Young Lee, Ph.D. Dept. of Forensic Medicine Yonsei University College of Medicine Presentation Overview DNA Extraction Complete demineralization DNA recovery using silica-based column mtdna Analysis Modified midi- and mini-primers Quality analysis based on mtdna phylogeny STR Genotyping Autosomal-STR and Y-STR Size-reduced mini-str Redundant approach to data generation and analysis 1

2 DNA Extraction and Quantification DNA from Old Skeletal Remains Low copy number (LCN) DNA Highly degraded DNA Presence of PCR inhibitors 2

3 Sample Preparation The 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 Spex CertiPrep Freezer/Mill (SPEX CertiPrep, NJ) DNA Extraction from Old Skeletal Remains Complete demineralization Large-scale silica-based column extraction Croat Med J 2007;48:

4 Complete Demineralization Demineralization solution 0.5 g bone power 15 ml of 0.5 M EDTA and 0.5% SDS 3 mg Proteinase K Incubation 48 hours at 56 in dry incubator 1 hour after additional treatment of 3 mg of Proteinase K DNA Recovery Using Silica-based Column QIAamp Blood DNA Maxi column Buffers from QIAquick PCR purification kit 2 ml of DNA extract Concentration of DNA extract QIAamp DNA Mini column Buffers from QIAquick PCR purification kit 50 µl of DNA extract 4

5 Quantification Using Real-time qpcr Quantifiler Human DNA Quantification kit Applied Biosystems 7500 Real-time PCR system Small-scale* (Yang et al. 1998) Large-scale Sample Concentration (pg/µl) ± ± ± ± ± Concentration (pg/µl) ± ± ± ± ± *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 5

6 Primers for Amplification of mtdna HV1 HV2 HV / Midi-primer Set P11 F15989/R16251 P12 F16159*/R16410m* P21 F015/R259* P22 F155/R389 P31 F403/R614* M11 M21 M31 F15989/R16153* M12 F015/R187* M22 F403/R568 Mini-primer Set F16088*/R16233* M13 F16159*/R16322m* M14 F16258*/R16410m* F120*/R285 M23 F220/R389 Lee et al BioTechniques 44: Detection of mtdna Sequence Errors 6

7 mtdnamanager s Open Database 7

8 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 8

9 Quality Analysis of mtdna Sequences Clear key diagnostic mutations Accompanying mutations for Expected HG Detection of Possible mtdna Errors N9a1: A ? 16362C? missed out? A5a: d-524d missed out M10b? B5b? Artificial recombination? 9

10 STR Genotyping of DNA from Old Skeletal Remains STR Typing Using Low Copy Number DNA LCN DNA Allele drop out Allele drop-in Stochastic effect Extraction Aliquot PCR 10

11 PCR Strategy for LCN DNA LCN interpretation rule Replicate analyses with duplicate results prior to reporting alleles Independant set up of PCR 1 st amplification PCR master mix I for 1 st DNA extract PCR master mix II for 2 nd DNA extract 2 nd amplification PCR master mix III for 1 st DNA extract PCR master mix IV for 2 nd DNA extract Scoring of STR Alleles Peak detection threshold 75 ~ 200 RFU Cut-off minor allele Under 15% of peak height of major peak An allele cannot be scored unless it is observed at least tree times in four PCR reactions 11

12 Scoring of STR Alleles Size-reduced Amplicon of mini-strs Forward flanking region Reverse flanking region GATA GATA GATA GATA GATA GATA 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 12

13 AmpFlSTR MiniFiler AmpFlSTR Identifiler Y-miniplex plus Bioquest INC 13

14 Y-miniplex plus AmpFlSTR Yfiler In-house NC01 plus 6FAM VIC NED PET 50bp 75bp 100bp 125bp 150bp D10S D14S D22S TPOX 5 14 LIZ 500 LIZ -internal lane standard 14

15 Improved STR Typing Results 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). Sample Number of 15 AS-STR loci 18 AS-STR loci 17 Y-STR loci quality samples AmpFlSTR Identifiler Kit AmpFlSTR MiniFiler Kit NC01 plus AmpFlSTR YFiler Kit Y-miniplex plus Low Medium High Total Thank you for your attention! hylee192@yuhs.ac 15