DNA Use in Human Identification Forensic cases -- matching suspect - PDF document

The development of reduced size Y chromosomal STR for genotyping of degraded DNA Myung Jin Park a , Hwan Young Lee a , Ji-Eun Yoo a , Ukhee Chung a , Seung-Chul Kang b , Kyoung-Jin Shin a,b , Jong-Hoon Choi a,b , Woo-Ick Yang a,b , Sang-Ho Cho a,b , Chong-Youl Kim a,b a Department of Forensic Medicine, College of Medicine, Yonsei University b Human Identification Research Institute, Yonsei University DNA Use in Human Identification � Forensic cases -- matching suspect with evidence � Paternity testing -- identifying father � Historical investigations � Missing persons investigations � Mass disasters -- putting pieces back together � Convicted felon DNA databases

Y Chromosomal STRs Y STR Markers � Applications DYS19 DYS389I/II - forensic investigation DYS390 - genealogical purpose DYS391 DYS392 - evolutionary studies DYS393 DYS385 � Advantages to Human Identity Testing DYS388 DYS434 - male component isolated without differential extraction DYS435 - paternal lineages DYS436 DYS437 DYS438 DYS439 ….. Sources of Biological Evidence � Blood DNA degradation: � Semen � If DNA exposed to external environmental for any length of time, degradation can � Saliva occur due to bacterial, biochemical or oxidative process � Urine � Environmental contaminants can also be � Hair commingled with the forensic samples � Teeth � Bone � Tissue

Approaches for Degraded Samples � mtDNA - high copy number per cell - maternal lineages - time-consuming process, low discriminatory power � SNPs - small size, l ower mutation rate - Easier data interpretation (no microvariants or stutter) - How many SNPs = STR - Databases, Platform for SNP typing? � STRs - Highly variable , higher discriminatory power - Rapid processing is attainable STRs Use in Degraded Samples � Poor amplification of the larger sized loci of > 200-250 bp � The yield of complete target fragment is greatly reduced - “Decay Curve”

Commercial Y-STRs kit AmpFlSTR Yfiler PowerPlex Y 200 bp 250 bp 250 bp 300 bp 300 bp 350 bp 350 bp 100 bp 100 bp 150 bp 150 bp 200 bp DYS391 DYS456 DYS389I DYS389I DYS390 DYS439 DYS389II DYS389II 90-118 99-123 148-168 150-170 209-233 203-231 262-286 256-296 DYS438 DYS458 DYS437 DYS19 DYS19 DYS385 DYS392 101-121 133-165 183-199 187-207 232-268 247-307 294-327 DYS393 DYS393 DYS391 DYS390 DYS439 GATA C4 DYS385 DYS392 115-139 104-136 152-160 191-227 204-224 242-262 243-315 303-321 GATA H4 DYS437 DYS438 DYS448 122-146 182-194 228-253 282-318 Redesign of Y-STR Maker Forward flanking region Reverse flanking region GATA GATA GATA GATA GATA GATA Advantages of Approach: � Retains same marker information (database compatibility) � Uses highly polymorphic STR loci (high discriminatory power)

Materials and Methods � New PCR primer design - DYS 390 , DYS 391 , DYS 392 , DYS 385 a/b, DYS 438 , DYS 439 , DYS 448 GATA C 4 (DYS 635 ) (according to Yfiler) additional DYS 446 , DYS 449 - Reference sequences: GeneBank (http://www.ncbi.nlm.nih.gov) - Primer 3 : the PCR product size was made as small as possible � DNA samples - Concordance : 100 buccal swabs samples form unrelated Koreans - Sensitivity : 1 ng, 500 pg, 250 pg, 125 pg, 62 pg, 31 pg, 15 pg of control 9948 male DNA (promega, Madison, MA) - Degraded DNA : A blood DNA treated with 0.01 U of DNase I (NEB, Beverly, MA) for 0 min, 2 min, 5 min, 10 min, 15 min, 20 min, 30 min - Real cases : DNA (>0.01 ng/ µ l) extracted from 10 50- year old bones by modification of the QIAamp protocol by Yang et al . (Am J Phys Anthropol, 105: 539-43) Materials and Methods � Multiplex PCR - Total 10 µ l PCR reaction: 1 µ l of DNA, 1.6 µ l of Gold STR buffer 2.0 U of AmpliTaq Gold polymerase and primers - Cycling condition: 95 ° C 11’; 96 ° C 1’; 94 ° C 30’’, 60 ° C 30’’, 70 ° C 45’’ x 10; 90 ° C 30’’ 60 ° C 30’’ 70 ° C 1’ x 20-25; 60 ° C 30’ using the GeneAmp 9600 � Detection System - ABI prism 310 genetic analyzer, Gene Scan software 3.1, and Genotyper 2.5 software (Applied Biosystems, CA, USA)

Commercial Y-STRs kit AmpFlSTR Yfiler 200 bp 250 bp 300 bp 350 bp 100 bp 150 bp DYS456 DYS389I DYS390 DYS389II 99-123 150-170 209-233 262-286 DYS458 DYS19 DYS385 133-165 187-207 247-307 DYS393 DYS391 DYS439 GATA C4 DYS392 115-139 152-160 204-224 242-262 303-321 GATA H4 DYS437 DYS438 DYS448 122-146 182-194 228-253 282-318 Reduced size Y-STRs Multiplex I 250 bp 300 bp 100 bp 200 bp 350 bp 150 bp DYS392 DYS389I DYS19 DYS389II 106-124 151-171 189-209 263-287 DYS456 DYS458 DYS437 DYS448 91-115 133-165 182-194 220-256 DYS439 GATA C4 DYS449 91-115 147-167 222-258 Multiplex II 250 bp 300 bp 100 bp 200 bp 350 bp 150 bp DYS391 GATA H4 DYS385a/b 93-109 122-146 172-232 DYS393 DYS390 113-137 160-184 DYS438 GATA C4 DYS446 105-130 147-167 218-256

Y-STRs information in this study Multiplex II Multiplex I Y-STR GenBank GenBank Allele Allele Product Size Product Size Size Y-STR GenBank GenBank Allele Allele Product Size Product Size Size locus Accession Allele Range Spread Yfiler Mini-Y set Reduction locus Accession Allele Range Spread Yfiler Mini-Y set Reduction DYS391 152-168 bp 93-109 bp 59 AC011302 11 7-9 16 bp DYS392 AC011745 13 10-16 18 bp 303-321 bp 106-124 bp 197 GATAH4 AC011751 12 8-14 24 bp 122-146 bp 122-146 bp DYS19 AC017019 15 13-18 20 bp 187-207 bp 188-208 bp - DYS385 Z93950 10 8-23 60 bp 247-307 bp 172-232 bp 75 DYS389I AF140635 14 11-16 20 bp 150-170bp 150-170bp - DYS393 AC006152 12 10-16 24 bp 115-139 bp 113-137 bp DYS389II 31 26-32 24 bp 262-286bp 262-286bp DYS390 209-233 bp 160-184 bp 49 AC011289 24 21-27 24 bp DYS456 AC010106 15 12-18 24 bp 99-123 bp 91-115 bp 8 DYS438 228-253 bp 105-130 bp 123 DYS458 AC002531 AC010902 10 16 13-21 9-14 25 bp 32 bp 133-165 bp 133-165 bp - DYS437 DYS446 * AC002992 16 13-16 12 bp 294-334 bp 182-194 bp 218-258 bp 182-194 bp 76 - AC006152 14 11-19 40 bp DYS448 282-318 bp 220-256 bp 62 AC025227 19 17-23 36 bp DYS439 AC002992 13 9-14 20 bp 204-224 bp 117-137 bp 87 GATAC4 G42673 21 19-24 20 bp 242-262bp 147-167 bp 95 (DYS635) DYS449 * 344-380 bp 222-258 bp 122 AC051663 29 26-35 36 bp * Compared to previously reported product size Sensitivity Multiplex I Multiplex II 1ng 500pg 250pg 125pg 62pg 31pg 15pg

Amplification in Artificially Degraded DNA AmpFlSTR Yfiler 0.01U of DNase I 2 5 10 15 20 30 min min min min min min 0 min 2 min 5 min 10 min 15 min Larger sized allele bounded DYS389II, DYS385a/b, GATA C4(DYS635), DYS448 20 min 30 min Amplification in Artificially Degraded DNA Multiplex I Multiplex II 0 min 2 min 20 min GATA C4 20 min 385a 385b 448 389II 5 min 10 min 30 min 30 min 15 min 20 min 30 min

Amplification in the Bone DNA Mini Y-STR set AmpFlSTR Yfiler Case 1 456 389I 19 393 H4 385a/b 391 448 392 437 439 389II C4 438 390 458 Case 2 DYS389II 393 385a/b 456 438 448 392 H4 458 391 19 439 390 389I 437 C4 Y STR Profiling Results in 10 Bone DNAs Yfiler (size) MiniY (Size) Yfiler (%) Mini-Y (%) Drop in DYS392 303-321 106-124 3 (30%) 9 (90%) 0 DYS438 228-253 105-130 6 (60%) 8 (80%) 1 GATA C4 242-262 147-167 7 (70%) 9 (90%) 0 DYS439 204-224 117-137 7 (70%) 9 (90%) 0 DYS385a/b 247-307 172-232 3 (30%) 7 (70%) 1 DYS448 282-318 220-256 6 (60%) 8 (80%) 0 DYS391 152-168 AmpFlSTR Yfiler 93-109 Mini-Y set 7 (70%) 9 (90%) Yfiler+Mini-Y set 0 DYS390 209-233 160-184 7 (70%) 8 (80%) 1 Amplified Loci 108 (67.5 %) 135(83.1 %) 140 (87.5 %) DYS456 99-123 91-115 9 (90%) 8 (80%) 2 DYS393 115-139 113-137 8 (80%) 7 (70%) 2 Total Loci 160 160 160 GATA H4 122-146 122-146 7 (70%) 10 (100%) 1 DYS458 133-165 133-165 7 (70%) 9 (90%) 4 DYS437 182-194 182-194 8 (80%) 9 (90%) 1 DYS389I 150-170 150-170 9 (90%) 8 (80%) 1 DYS19 187-207 188-208 6 (60%) 7 (70%) 1 DYS389II 262-286 262-286 8 (80%) 7 (70%) 0

Conclusion � Redesigned Y-STRs Primer sets, in which amplicon size is kept at minimum, provide an effective tool for degraded forensic samples, as seen from the sensitive study, enzymatically degradation study, and the real case samples. � The use of these primer sets with commercial kits will increase the probability that degraded samples can be typed. � Also they provide a check for the presence of allele drop out due to problem with primer binding of commercial STR sets � Development of new additional mini Y-STRs or Y-SNPs is required because a few Y STR loci cannot be made into more smaller amplicon