[PDF] - DNA Use in Human Identification Forensic cases -- matching suspect PDF Document

SLIDE 1

Myung Jin Parka, Hwan Young Leea, Ji-Eun Yooa, Ukhee Chunga, Seung-Chul Kangb, Kyoung-Jin Shina,b, Jong-Hoon Choia,b, Woo-Ick Yanga,b, Sang-Ho Choa,b, Chong-Youl Kima,b

The development of reduced size Y chromosomal STR for genotyping of degraded DNA

aDepartment of Forensic Medicine, College of Medicine, Yonsei University bHuman Identification Research Institute, Yonsei University

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

DNA Use in Human Identification

SLIDE 2

Y Chromosomal STRs

Y

STR Markers DYS19 DYS389I/II DYS390 DYS391 DYS392 DYS393 DYS385 DYS388 DYS434 DYS435 DYS436 DYS437 DYS438 DYS439

…..

Applications

forensic investigation
genealogical purpose
evolutionary studies

Advantages to Human Identity Testing

male component isolated without differential extraction
paternal lineages

Blood Semen Saliva Urine Hair Teeth Bone Tissue

Sources of Biological Evidence

DNA degradation: If DNA exposed to external environmental for any length of time, degradation can

ccur due to bacterial, biochemical or
xidative process

Environmental contaminants can also be commingled with the forensic samples

SLIDE 3

mtDNA - high copy number per cell

maternal lineages
time-consuming process, low discriminatory power

SNPs - small size, lower 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

Approaches for Degraded Samples

Poor amplification of the larger sized loci of >200-250 bp The yield of complete target fragment is greatly reduced

“Decay Curve”

STRs Use in Degraded Samples

SLIDE 4

Commercial Y-STRs kit

100 bp 250 bp 200 bp 150 bp 300 bp 350 bp

DYS389I

150-170

DYS456

99-123

DYS390

209-233

DYS389II

262-286

DYS458

133-165

DYS19

187-207

DYS385

247-307

DYS393

115-139

DYS391

152-160

DYS439

204-224

GATA C4

242-262

DYS392

303-321

GATA H4

122-146

DYS437

182-194

DYS438

228-253

DYS448

282-318

AmpFlSTR Yfiler

100 bp 250 bp 200 bp 150 bp 300 bp 350 bp

PowerPlex Y

DYS391

90-118

DYS389I

148-168

DYS439

203-231

DYS389II

256-296

DYS438

101-121

DYS437

183-199

DYS19

232-268

DYS392

294-327

DYS393

104-136

DYS385

243-315

DYS390

191-227

GATA GATA GATA GATA GATA GATA

Advantages of Approach: Retains same marker information (database compatibility) Uses highly polymorphic STR loci (high discriminatory power)

Forward flanking region Reverse flanking region

Redesign of Y-STR Maker

SLIDE 5

Materials and Methods

New PCR primer design

DYS390, DYS391, DYS392, DYS385a/b, DYS438, DYS439, DYS448

GATA C4 (DYS635) (according to Yfiler) additional DYS446, DYS449

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: 1ng, 500pg, 250pg, 125pg, 62pg, 31pg, 15pg of control

9948 male DNA (promega, Madison, MA)

Degraded DNA: A blood DNA treated with 0.01U of DNase I (NEB, Beverly, MA)

for 0 min, 2 min, 5 min, 10min, 15 min, 20min, 30min

Real cases: DNA (>0.01 ng/µl) 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 µl PCR reaction: 1 µl of DNA, 1.6 µl 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)

SLIDE 6

Commercial Y-STRs kit

100 bp 250 bp 200 bp 150 bp 300 bp 350 bp

DYS389I

150-170

DYS456

99-123

DYS390

209-233

DYS389II

262-286

DYS458

133-165

DYS19

187-207

DYS385

247-307

DYS393

115-139

DYS391

152-160

DYS439

204-224

GATA C4

242-262

DYS392

303-321

GATA H4

122-146

DYS437

182-194

DYS438

228-253

DYS448

282-318

AmpFlSTR Yfiler

100 bp 250 bp 200 bp 150 bp 300 bp 350 bp

Multiplex I

Reduced size Y-STRs

DYS389I

151-171

DYS19

189-209

DYS389II

263-287

DYS456

91-115

DYS458

133-165

DYS437

182-194

100 bp 250 bp 200 bp 150 bp 300 bp 350 bp

Multiplex II

DYS449

222-258

DYS446

218-256

GATA H4

122-146

DYS392

106-124

DYS448

220-256

DYS439

91-115

GATA C4

147-167

DYS391

93-109

DYS385a/b

172-232

DYS393

113-137

DYS438

105-130

GATA C4

147-167

DYS390

160-184

SLIDE 7

62 220-256 bp 282-318 bp

36 bp 17-23 19 AC025227

DYS448

182-194 bp

182-194 bp 12 bp 13-16 16 AC002992 DYS437

133-165 bp

133-165 bp 32 bp 13-21 16 AC010902 DYS458

150-170bp

262-286bp 150-170bp 262-286bp 20 bp 24 bp 11-16 26-32 14 31 AF140635 DYS389I DYS389II

122 222-258 bp 344-380 bp

36 bp 26-35 29 AC051663

DYS449* 95 147-167 bp 242-262bp

20 bp 19-24 21 G42673

GATAC4

(DYS635)

87 117-137 bp 204-224 bp

20 bp 9-14 13 AC002992

DYS439 8

91-115 bp 99-123 bp 24 bp 12-18 15 AC010106 DYS456

188-208 bp

187-207 bp 20 bp 13-18 15 AC017019 DYS19

197 106-124 bp 303-321 bp

18 bp 10-16 13 AC011745

DYS392

Size Reduction Product Size Mini-Y set Product Size Yfiler Allele Spread Allele Range GenBank Allele GenBank Accession Y-STR locus

Y-STRs information in this study

Multiplex I

76 218-258 bp 294-334 bp

40 bp 11-19 14 AC006152

DYS446* 49 160-184 bp 209-233 bp

24 bp 21-27 24 AC011289

DYS390

113-137 bp 115-139 bp 24 bp 10-16 12 AC006152 DYS393

75 172-232 bp 247-307 bp

60 bp 8-23 10 Z93950

DYS385

122-146 bp 122-146 bp 24 bp 8-14 12 AC011751 GATAH4

59 93-109 bp 152-168 bp

16 bp 7-9 11 AC011302

DYS391 123 105-130 bp 228-253 bp

25 bp 9-14 10 AC002531

DYS438

Size Reduction Product Size Mini-Y set Product Size Yfiler Allele Spread Allele Range GenBank Allele GenBank Accession Y-STR locus

Multiplex II

* Compared to previously reported product size

Sensitivity

Multiplex I Multiplex II

1ng 500pg 250pg 125pg 62pg 31pg 15pg

SLIDE 8

Amplification in Artificially Degraded DNA

2 min 5 min 10 min 15 min 20 min 30 min

0.01U of DNase I AmpFlSTR Yfiler

0 min 2 min 5 min 10 min 15 min 20 min 30 min

Larger sized allele bounded DYS389II, DYS385a/b, GATA C4(DYS635), DYS448

2 min 5 min

Multiplex I Multiplex II

0 min 10 min 15 min 20 min 30 min 20 min GATA C4 448 389II 30 min 20 min 30 min 385b 385a

Amplification in Artificially Degraded DNA

SLIDE 9

DYS389II 393 390 389I

385a/b 458 19

456 456 389I 390 393 389II 458 19

385a/b

391

Case 1 Case 2

439

C4 C4

392 392 H4

H4 437 438

437

438

448 448

Amplification in the Bone DNA

AmpFlSTR Yfiler Mini Y-STR set

1 1 1 4 1 2 2 1 1 1 Drop in 7 (70%) 7 (70%) 8 (80%) 9 (90%) 9 (90%) 10 (100%) 7 (70%) 8 (80%) 8 (80%) 9 (90%) 8 (80%) 7 (70%) 9 (90%) 9 (90%) 8 (80%) 9 (90%) Mini-Y (%) 8 (80%) 262-286 262-286 DYS389II 6 (60%) 188-208 187-207 DYS19 9 (90%) 150-170 150-170 DYS389I 8 (80%) 182-194 182-194 DYS437 7 (70%) 133-165 133-165 DYS458 7 (70%) 122-146 122-146 GATA H4 8 (80%) 113-137 115-139 DYS393 9 (90%) 91-115 99-123 DYS456 7 (70%)

160-184 209-233 DYS390

7 (70%)

93-109 152-168 DYS391

6 (60%)

220-256 282-318 DYS448

3 (30%)

172-232 247-307 DYS385a/b

7 (70%)

117-137 204-224 DYS439

7 (70%)

147-167 242-262 GATA C4

6 (60%)

105-130 228-253 DYS438

3 (30%)

106-124 303-321 DYS392

Yfiler (%) MiniY (Size) Yfiler (size)

Y STR Profiling Results in 10 Bone DNAs

160 135(83.1 %) Mini-Y set 160 160 Total Loci 140 (87.5 %) 108 (67.5 %) Amplified Loci Yfiler+Mini-Y set AmpFlSTR Yfiler

SLIDE 10

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