Summary
The detection of restriction fragment length polymorphisms (RFLP) (1) in DNA extracted from forensic samples remains impossible in a significant number of cases due to deterioration and contamination of the biological material and the extremely low quantities of DNA isolated. The polymerase chain reaction (PCR) is a recent and particularly convenient method for analysing and typing very small amounts (10–20ng) of degraded human DNA (2, 3). DNA analysis at the level of a few cells present in forensic samples such as bloodstains, semen stains, vaginal swabs and head hair bulbs now appears possible using DNA amplification. A PCR protocol [4, 5] was adapted to simultaneously amplifiy a Y-specific DNA repeat sequence from the DYZ1 locus [6] and an X-specific DNA repeat sequence from the DXS424 locus [7]. The co-amplified Y-specific DNA fragment (102 bp) and X-specific DNA fragments (181–199 bp) were visualized on an ethidium bromide-stained 4% agarose gel. The male or female type of the amplified DNA extracted from blood samples, bloodstains, semen stains, vaginal swabs, brain tissue and 1, 2, 5, or 10 head hair bulbs was determined.
Zusammenfassung
Der Nachweis von Restriktionsfragmentlängen-Polymorphismen (RFLP's) (1) aus DNA, welche von forensischen Proben extrahiert wurden, bleibt häufig unmöglich wegen Verschlechterung, Kontamination des biologischen Materials and extrem geringer Mengen, welche isoliert werden können. Die Polymerase-Kettenreaktion (PCR) ist eine neue und ganz besonders geeignete Methode, um sehr kleine Mengen (10–20 ng) degradierter DNA zu analysieren und zu typisieren (2, 3). DNA-Analysen auf der Ebene weniger Zellen in forensischen Proben wie Blutspuren, Samenspuren, Scheidenabstrichen und Haarwurzeln erscheint nunmehr möglich mit Hilfe der DNA-Amplifikation. Ein PCR-Protokoll (4, 5) wurde adaptiert, um gleichzeitig eine Y-spezifische DNA-Sequenz vom DYZ1-Locus (6) und eine X-spezifische DNA-Sequenz vom DXS424-Locus (7) zu amplifizieren. Das co-amplifizierte y-spezifische DNA-Fragment (102 Bp) und das X-spezifische DNA-Fragment (181-199 Bp) wurden mit einem Ethidiumbromid gefärbten 4% igen Agarosegel sichtbar gemacht. Der männliche oder weibliche Typ der amplifizierten DNA, welche von Blutproben, Blutspuren, Spermaspuren, Vaginalabstrichen, Hirngewebe und 1, 2, 5 oder 10 Haarwurzeln extrahiert worden war, wurde bestimmt.
Similar content being viewed by others
References
Jeffreys AJ, Wilson V, Thein SL (1985) Hypervariable “minisatellite” regions in human DNA. Nature 314:67–73
Higuchi R, von Beroldingen CH, Sensabaugh GF, Erlich HA (1988) DNA typing from single hairs. Nature 332:543–546
Hochmeister MN, Budowle B, Jung J, Borer UV, Comey CT, Dirnhofer R (1991) PCR-based typing of DNA extracted from cigarette butts. Int J Leg Med 104:229–233
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
Nakahori Y, Mitani K, Yamada M, Nakagome Y (1986) A human Y-chromosome specific repeated DNA family (DYZI) consists of a tandem array of pentanucleotides. Nucleic Acids Res 14:7569–7580
Luty JA, Guo Z, Willard HF, Ledbetter DH, Ledbetter S, Litt M (1990) Five polymorphic microsatellite VNTRs on the human X chromosome. Am J Hum Genet 46:776–783
Saiki R, Bugawan T, Horn G, Mullis K, Erlich H (t1986) Analysis of enzymatically amplified B-globin and HLA-DQ. Alpha DNA with allele specifc oligonucleotide probe. Nature 324:163–166
Boerwinkle E, Xiong W, Fourest E, Chan L (1989) Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B 3' hypervariable region. Proc Natl Acad Sci USA 86:212–216
Budowle B, Chakraborty R, Giusti A, Eisenberg A, Allen R (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet 48:137–144
Wong Z, Wilson V, Patel I, Povery S, Jeffreys AJ (1987) Characterisation of a panel of highly variable minisatellites cloned from human DNA. Ann Hum Genet 51:269–288
Gill P (1987) A new method for sex determination of the donors of forensic samples using a recombinant DNA probe. Electrophoresis 8:35–38
Fukuschima H, Hasekura H, Nagai K (1988) Identification of male bloodstains by the dot blot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe. J Forensic Sci 33:621–627
Fattorini P, Caccio S, Gustincich S, Wolfe J, Altamura BM, Graziosi G (1991) Sex determination and species exclusion in forensic samples with probe CY97. Int J Leg Med 104:247–250
Kobayashi R, Nakauchi H, Nakahori Y, Nakagome Y, Matsuzawa, S (1988) Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique. J Forensic Sci 3:613–620
Kobayashi R, Matsuzawa S (1989) Sex determination of bloodstains by the use of a ribonucleic acid (RNA) probe. J Forensic Sci 5:1078–1081
Ludes B, Mangin P, Chaumont AJ (1991) Stability of DNA in brain cortex after long post mortem periods. In: Berghaus G, Brinkmann B, Rittner C, Staak M (eds) DNA-technology and its forensic application. Springer, Berlin Heidelberg New York, pp 187–191
Gill P, Lygo JE, Fowler SJ, Werrett DJ (1985) Forensic application of DNA “fingerprints”. Nature 318:577–579
Giusti A, Baird M, Pasquale S, Balazs I, Glassberg J (1986) Application of deoxyribonucleic acid (DNA) polymorphisms to the analysis of DNA recovered from sperm. J Forensic Sci 31:409–417
Lo YMD, Patel P, Wainscoat JS, San Pietro M, Gillmer MDG, Fleming KA (1989) Prenatal sex determination by DNA amplification from materal peripheral blood. Lancet II:1363–1365
Pascal O, Aubert D, Gilbert E, Moisan JP (1991) Sexing of forensic samples using PCR. Int J Leg Med 104:205–207
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pfitzinger, H., Ludes, B. & Mangin, P. Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence. Int J Leg Med 105, 213–216 (1993). https://doi.org/10.1007/BF01642796
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01642796