Y chromosomal STRs in forensics

Transcription

1 Haplotype and mutation analysis for newly suggested Y-STRs in Korean father-son pairs Yu Na Oh, Hwan Young Lee, Eun Young Lee, Eun Hey Kim, Woo Ick Yang, Kyoung-Jin Shin Department of Forensic Medicine Yonsei University College of Medicine Y chromosomal STRs in forensics u Useful for the investigation of sexual assault cases and deficiency paternity testing of male offspring u 17 Y-STR loci included in the AmpFlSTR Yfiler PCR amplification kit commonly used u Continuous efforts to achieve higher discrimination led to adding new markers to the Yfiler 17 Y-STRs u Reliable estimates of haplotype and mutation analysis required for non-erroneous interpretation of the genetic profiles 1

2 Newly suggested Y-STR loci Commercial kit 17 STRs + DYS576 DYS481 DYS549 DYS533 DYS570 DYS STRs + DYS627* DYS576 DYS460 DYS518* DYS449* DYS570 DYS533 DYF387S1* DYS481 * RMY-STR DYS460 DYS385a/b DYF387S1 DYS627 DYS391 DYS518 DYS449 2

3 Aim of the study v Development of Y-STR multiplex PCR system analyzing New single copy Y-STR from PowerPlex Y23 Rapidly mutating (RM) Y-STR from Yfiler plus simultaneously üfinal amplicon size < 350 bp ü Sensitivity able to genotype of 100 pg of genomic DNA ü Specificity to analyze mixed DNA with [M:F=1:1000] ratio ü Construction of allelic ladder for accurate genotyping Haplotype and mutation analysis in Koreans Method (1) u Collect information and design optimal primer set for 14 Y-STRs ü 6 Y-STRs for PowerPlex Y23: DYS481, DYS533, DYS549, DYS643, DYS570 and DYS576 ü 4 RM Y-STRs: DYF387S1, DYS449, DYS518 and DYS627 ü DYS460, DYS391 and DYS385a/b u Development of multiplex PCR system (Euplex Y14) u Construction of allelic ladder, bin set and panel for genotyping u Checking mutation on primer binding area using 100 Korean male samples 3

4 Optimizing multiplex PCR system u Multiplex PCR test - Check unexpected primer interference - Adjusting intervals between adjacent loci - Remove incomplete adenylation - Peak height balancing u Reaction Mixture PCR component Volume dh Gold ST*R 10 Buffer Primer Mix 2.0 AmpliTaq Gold (5 U/uL) 2.5 U 0.6 Template DNA (1 ng/ul) 1.0 SUM 10.0 u Thermal Cycle min sec sec 30 cycles sec min 4 forever Allelic size range of 14 Y-STR loci 4

5 Electropherogram of 2800M standard DNA Construction of allelic ladder 5

6 Sensitivity and specificity test [100 pg of 007 DNA] [1 : 1000 mixture] (100 pg of 007 DNA : 100ng of female DNA) Method (2) u Identification of mutations in father-son pairs 363 Korean father-son haplotype transfers in 351 families were analyzed using the developed multiplex PCR system (Euplex Y14). All pairs with a mutation were analyzed using the PowerPlex 16 HS System (Promega) to confirm biological paternity. The protocol was approved by Institutional Review Board of Severance Hospital at Yonsei University. u Statistical analysis Haplotype frequencies and haplotype diversities were estimated according to the Nei s formula. Mutation rates were calculated as the number of mutations divided by the number of allele transmissions. 6

7 Gene diversity 11 Y-STRs in Euplex Y14 Yfiler 17 Y-STRs Locus Gene Diversity Locus Gene Diversity 387S II (C4) I H Haplotype diversity 7

8 Distinctive tri-allelic pattern at DYF387S1 Micro-variant on DYS449, 518, 570, 627 loci 8

9 Gene diversities and mutation estimates for 28 Y-STRs Mutations observed at more than one loci Sample Locus Allele Type of mutation CG29-F DJ022-F DJ064-F SN012-F SN109-F CG29-S DJ022-S DJ064-S SN012-S SN109-S Loss (-1) Gain (+1) Loss (-1) ,20 13,19 Loss (-1) Gain (+1) Gain (+1) 635(C4) Gain (+1) Loss (-1) 635(C4) Loss (-1) 387S1 37,38 37,39 Gain (+1) ,14,15,16 12,14,15,17 Gain (+1) 9

10 Conclusions v Newly suggested 11 Y-STRs with high gene and haplotype diversity values could be successfully used for male DNA analysis in Koreans. v Twenty nine single step mutations were observed and two father-son pairs have mutations at two different loci at 11 Y- STRs with locus-specific mutation rates up to 1.93ⅹ10-2. v DYS449, DYS576 and DYS627 loci showed mutation rate estimates over 1%. However, the mutation rate estimates at DYF387S1, DYS518 and DYS570 were considerably lower in Koreans. Acknowledgements Yu Na Oh Eun Young Lee Hwan Young Lee v This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (No ) 10