DNA Profiling of the Semen Donor in Extended Interval (72 h) Post Coital Cervicovaginal Samples

Transcription

1 DNA Profiling of the Semen Donor in Extended Interval (72 h) Post Coital Cervicovaginal Samples Jack Ballantyne University of Central Florida, Orlando, FL National Center for Forensic Science

2 Problem some rape victims provide vaginal samples > hours after the incident ability to obtain an autosomal STR profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended (> 24-48h difficult, >72 h not normally possible) from classical forensic serology and reproductive biology studies sperm persist in the post-coital vaginal canal up to 3 days after intercourse from the medical literature sperm may be detectable up to 7 days after intercourse in the cervix sperm are few in number after these extended intervals

3 Sperm Loss Over Time 1. vaginal lavage 2. vaginal drainage 3. normal intra-cervicovaginal sperm degradative changes sperm become damaged and fragile 4. below analytical sensitivity of the test 5. during the differential extraction process within the laboratory multiple manipulations of the sample few remaining sperm lyse into female fraction kinetics of the PCR process majority component in an admixture will titrate out critical PCR reagents female/male DNA ratio > 300/1 failure to type the male component

4 Potential Solutions to Problem? use Y-STRs theoretically can detect male in female/male admixtures ignores female component no differential extraction avoids unnecessary sample loss low copy number approach add large quantities of DNA ( ng) to permit adequate sampling of small number of still persisting sperm increased PCR cycle number (34-35 cycles) ensure cervicovaginal sampling low to mid-vaginal sampling may not recover sperm after extended post coital interval

5 Not All Y-STRs Are Made Equal Y chromosome retains high level of sequence homology with the X Different Y-STR primers will possess different degrees of homology with the X chromosome

6 Why the Y Can t Get Rid of the X cross reactivity of a Y-STR locus with homologous X sequences is bad artifacts may confound allelic assignments reduction in sensitivity titration of primers and other critical PCR reagents novel Y-STR multiplexes developed for forensic use need to undergo a series of validation exercises that go beyond simply optimizing the PCR reaction conditions. stringent performance checks on their efficacy required to determine potential confounding effects from female DNA.

7 Multiplex I

8 Multiplex II

9 Autosomal STRs 48h Post-Coital Sample, Sperm Fraction

10 Autosomal STRs 48h Post-Coital Sample, Non-Sperm Fraction

11 Autosomal STRs 48h Post-Coital Sample, No Differential Extraction

12 Multiplex I 48h Post-Coital Sample, Sperm Fraction

13 Multiplex I 48h Post-Coital Sample, No Differential Extraction

14 Multiplex II 48h Post-Coital Sample, Sperm Fraction

15 Multiplex II 48h Post-Coital Sample, No Differential Extraction

16 Non-Sperm Fractions A 24 h 450 ng of Non-Sperm Fraction DNA Yields Y-STR Profiles B 48 h

17 MPI/II Fail at Post Coital Intervals > 72 h despite the development of these systems to exhibit minimal female DNA artifacts loss of signal due to binding of several of the primers from different loci to homologous regions on the X particularly at high copy number develop modified versions of MPI/MPII remove from MPI/II those loci with characteristics that indicate they may interact with very high copy number (HCN) female DNA

18 Screening Strategies for the Development of Modified Versions of MPI/II remove loci which require unusually high primer concentrations or possess low signal intensities indicative of primers binding to homolgous sequences on the X iterative screening against 450 ng of non-differentially extracted DNA from 85 h post coital samples development of 2 multiplexes (MPA/MPB) collectively comprise 11/19 of MPI/II loci

19 SENSITIVITY AND SPECIFICITY OF MPA 30 pg of single source male DNA 3000 fold excess of female DNA

20 Multiplex A 4d Post-Coital Sample, No Differential Extraction

21 Multiplex A 4d Post-Coital Sample, Sperm Fraction

22 SENSITIVITY AND SPECIFICITY OF MPB 30 pg of single source male DNA 3000 fold excess of female DNA

23 Multiplex B 4d Post-Coital Sample, No Differential Extraction

24 Multiplex B 4d Post-Coital Sample, Sperm Fraction

25 Monoplexes 4d Post-Coital Sample, No Differential Extraction

26 Importance of Cervicovaginal Sampling lower to midvaginal sample (24 h) cervicovaginal Sample (48 h)

27 Early Work Conclusions it is possible to obtain the genetic profile of the semen donor in postcoital cervicovaginal samples recovered up to 4 d after intercourse use of carefully selected Y-STR markers chosen for their ability to detect the male donor in an overwhelming quantity of background female DNA no differential extraction ng of input template DNA increased cycle number (34-35 cycles) cervicovaginal sampling such strategies may significantly impact the recovery and processing of rape evidence

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29 Performance of Commercial Y-STR Kits With Extended Interval Post Coital Samples (> 3 Days)

30 Y-STR Kits Tested Applied Biosystems AmpFlSTR Yfiler PCR Amplification Kit Promega PowerPlex Y System Reliagene Y-PLEX 12 In House Multiplex Multiplex 1 (MPI) In House Multiplex Multiplex B (MPB)

31 Post Coital Samples 3 donor couples Post coital cervicovaginal swabs (x2) taken by each female volunteer 3-7 days after separate acts of sexual intercourse 72, 96, 120,144 and 168 h Taken after a 7 day abstention period Negative control taken- pre-coital swab Only data from samples that had a negative precoital result (no male DNA profile) was used

32 Y-STR Multiplex Systems: Post Coital Interval: Up to 3 Days Pair 1 0 hours 12 hours 24 hours 48 hours 72 hours Non-Differential Extraction 300 ng AmpFlSTR Yfiler Multiplex I (+) - Multiplex B Promega PowerPlex Y System Reliagene Y-PLEX (+) (+) (+) Differential Extraction 1 ng Male AmpFlSTR Yfiler Multiplex I Multiplex B Promega PowerPlex Y System Reliagene Y-PLEX (+)

33 5 Y-STR Multiplex Systems: Post Coital Interval: 3 Days

34 5 Y-STR Multiplex Systems: Post Coital Interval: 5 Days

35 Strategies for Extending Post Coital Interval Detection: 3-7 Days Extraction Technique Differential vs. non-differential Post PCR Purification-Minelute Input Volume Maximum for each kit

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39 5 Days Post Coitus- Non- Differential Y-Filer Minelute vs. No Minelute DYS456 DYS389I DYS390 DYS389II DYS456 DYS389I DYS390 DYS389II DYS 458 DYS19 DYS385 DYS 458 DYS19 DYS385 DYS 393 DYS 391 DYS 439DYS 635 DYS 392 DYS 393 DYS 391 DYS DYS 635 DYS Y-GATAH4 DYS437 DYS438 DYS448 Y-GATA H4 DYS437 DYS438 DYS448

40 5 Days Post Coitus Non-Differential PowerPlex Y A No Minelute vs. Minelute B DYS391 DYS389I DYS439 DYS389II DYS391 DYS389I DYS439 DYS389II DYS438 DYS437 DYS19 DYS392 DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385 DYS393 DYS390 DYS385

41 6 Days Post Coitus Non-Differential PowerPlex Y No Minelute vs. Minelute DYS391 DYS389I DYS389II DYS391 DYS389I DYS439 DYS389II DYS438 DYS437 DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385 DYS393 DYS390 DYS385

42 6 Days Post Coitus Differential PowerPlex Y No Minelute vs. Minelute DYS391 DYS389I DYS439 DYS389II DYS391 DYS389I DYS439 DYS389II DYS438 DYS437 DYS19 DYS392 DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385 DYS393 DYS390 DYS385

43 6 Days Post Coitus Non-Differential Y-Filer No Minelute vs. Minelute DYS456 DYS389I DYS390 DYS456 DYS389I DYS390 DYS389II DYS458 DYS385 DYS458 DYS19 DYS385 DYS393 DYS 391 DYS439DYS635 DYS392 DYS393 DYS391 DYS439DYS635 DYS392 Y-GATA H4 DYS437 DYS438 DYS448 Y-GATA H4 DYS437 DYS438 DYS448

44 6 Days Post Coitus Differential Y-Filer No Minelute vs. Minelute DYS456 DYS389I DYS390 DYS389II DYS456 DYS389I DYS390 DYS389II DYS458 DYS19 DYS385 DYS458 DYS19 DYS385 DYS393 DYS391 DYS439 DYS393 DYS391 DYS439 DYS 635 DYS392 DYS437 DYS438 Y-GATA H4 DYS437 DYS438 DYS448

45 5 Y-STR Multiplex Systems: Post Coital Interval: 3 Days Post Coital Interval (Days) Y-STR System Donor Couple (4) (11) - Y-filer (9) (4) (1) (1) (4) (11) - PowerPlex Y (6) (10) (3) Y-Plex (4) NA NA NA NA NA Multiplex I NA NA NA NA NA Multiplex B NA NA NA NA NA

46 Post PCR Purification: Differential vs. Non-Differential & Minelute Y-STR System Y-filer PowerPlex-Y DNA Extraction Post Coital Interval (Days) No Minelute Minelute No Minelute Minelute No Minelute Minelute No Minelute Minelute ND 1 (13) (12) (4) - (1) D (4) (6) (11) + - (1) ND (14) + (1) (11) - - D (9) (10) - (1) ND 1 (5) (7) (1) - (3) (2) (1) - D (4) (6) (10) + - (1) ND (8) + (1) (2) (2) - D (6) (10) - (3)

47 Y-Filer 7 Days Post Coitus: 5μl Extract Amplified No Minelute vs. Minelute 109 DYS DYS391

48 Y-Filer 7 Days Post Coitus: 10μl Extract Amplified No Minelute vs. Minelute DYS456 DYS389I DYS390 DYS389II 7 Days (10 ul) 307 DYS DYS389I 128 DYS390 DYS389II 7 Days (10 ul) + MinElute DYS458 DYS19 DYS385a/b 556 DYS458 DYS DYS385a/b DYS393 DYS391 DYS439 DYS635 DYS DYS DYS391 DYS439 DYS635 DYS392 Y-GATA H4 DYS437 DYS438 DYS448 Y-GATA H4 DYS437 DYS438 DYS448

49 Conclusions Number of DNA profile enhancement strategies employed Cervical brushing Differential versus non-differential DNA extraction Post-PCR purification Standard manufacturers cycling conditions used Y-STR profiles Full profiles routinely 3-4 days after intercourse Profiles 5-6 days after intercourse but mainly partial Use of post PCR purification significantly improved ability to obtain profile, especially from the 5-6 day samples Better profiles with differential lysis (sperm fraction) 8 locus Y-STR profile from 7 day post-coital sample Approaching limit for sperm detection in cervix

50 Future? Increased cycle number Reduced amplimer sizes ( mini-y ) Improved cervicovaginal collection devices Autosomal STRs?

51 Acknowledgements Ashley Hall, PhD Kathy Press, MS Lynn Sims, BS Pam Smith, MS National Institute of Justice

52 Thank you!