EMEL HÜLYA YÜKSELOĞLU, Esra BAYHAN, Gülten RAYİMOĞLU, FATMA ÇAVUŞ YONAR, Kadir DASTAN

Identification with mitochondrial DNA typing from one sperm cell isolated by micromanipulation

Abstract Due to the limited or high degree degradation of nuclear DNA (Deoxyribonucleic acid) content in some cases of sexual assault at forensic cases, successful results in standard genetic profiling techniques are not achieved. The aim of this study is with swab to characterize the male mitotype by mtDNA (mitochondrial DNA) typing from individually collected sperm cells together with physically differentiation of the perpetrator spermatozoa among the victim epithelium cells using micromanipulation technique. In this study, 10 samples of oral swabs were collected for the first step (from 5 volunteer pairs), and vaginal swabs for the second step after the sexual intercourse. In the first step, intra-oral swab specimens were analysed and the mtDNA profiles of the male and female were determined. These profiles were compared to the Revised Cambridge Reference Sequence. For each couple, SNP regions not seen in women present in men were detected. Based on these detected SNP (Single Nucleotide Polymorphism) regions, each couple’s male-specific SSPs (Sequence Specific Priming) were designed. In the second step, DNA isolation was performed by using micromanipulation technique from vaginal swab samples taken from women after sexual intercourse and sperm cells were individually collected. PCR (Polymerase Chain Reaction) was performed using SSPs designed in the first step. Then male mtDNA profiles were obtained. According to the results, the overlapping regions in relatives from the maternal side since SSP is not designed, and not able to be used in cases which relative men are involved are the disadvantages of the method. However, thanks to the micromanipulation technique used in the study, the risk of contamination is reduced and individual samples can be isolated with sperm isolation and sequenced with SSPs designed for the individual, indicating that it is a method that can be prefered in sensitive situations such as pack rape crimes involving more than one person from the same male lineage.

PDF

___

Pereira J, Neves R, Forat S, et al. MTDNA typing of single-sperm cells isolated by micromanipulation. Forensic Sci Genet. 2012;6:228-35.

Meredith M, Bright JA, Cockerton S, et al. Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection. Forensic Sci. Int. Genet. 2012;6:91-6.

Mulero JJ, Chang CW, Calandro LM, et al. Development and validation of the AmpFlSTR1 YfilerTM PCR amplification kit: a male specific single amplification 17Y-STR multiplex system. J Forensic Sci. 51 2006;51:64-75.

Prinz M, Boll K, Baum H, et al. Multiplexing of Y-chromosome specific STRs and performance for mixed samples. Forensic Sci. Int. 1997;85: 209-18.

Sibille I, Duverneuil C, Lorin de la Grandmaison G, et al. Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa. Forensic Sci. Int. 2002;125:212-6.

Sanders CT, Sanchez N, Ballantyne J, et al. Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis. J. Forensic Sci. 2006;51:748-57.

Mayntz-Press KA, Sims LM, Hall A, et al. J. Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples. J Forensic Sci. 2008;53:342-8.

Fujita Y, Kubo S. Application of FTAw technology to extraction of sperm DNA from mixed body fluids containing semen. Leg Med (Tokyo). 2006;8:43-7.

Petit C, Martel-Petit V, Hienne R, Frackowiak S. Single sperm cell isolation by micromanipulation for human identification in sexual assault. Forensic Sci. Int. Genet. Suppl. Ser. 1.2008;1:60-2.

Rousselet F, Mangin P. Mitochondrial DNA polymorphisms: a study of 50 French Caucasian individuals and application to forensic casework. Int J Legal Med. 1998;111:292-8.

Nara A, Harihara S, Iwadate K, et al. Sequence analysis for HV I region of mitochondrial DNA using WGA (Whole Genome Amplification) method. Leg Med (tokyo). 2009;11:115-8.

Tully G, Bâr W, Brinkmann B, Carracedo A, Consideration by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles. Forensic Sci Int. 2001;124:8391.

Coble MD, Just RS, O’Callaghan JE, et al. Single nucleotide polymorphisms over theentiremt DNA genomethatincrease the power of forensic testing in Caucasians. Int J Legal Med. 2004;118: 137-46.

Ballantyne KN, Oven M, Ralf A, et al. MTDNA SNP multiplexes for efficient inference of matrilineal genetic ancestry with in Oceania. Forensic Sci Int Genet. 2012;6:425-36.

Vallone PM, Just RS, Coble MD, et al. A multiplex allele specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int J Legal Med.2004;118:147-57.

Köhnemann S, Pfeiffer H, Application of mtDNA SNP analysis in forensic casework, Forensic Sci Int Genet. 2011;5:216-21.

Foran DR. Relative degradation of nuclear and mitochondrial DNA: an experimental approach. J Forensic Sci. 2006;51:766-70.