Sex determination of forensic samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence

Abstract

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 (10-20 ng) of degraded human DNA. 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 was adapted to simultaneously amplify a Y-specific DNA repeat sequence from the DYZ1 locus and an X-specific DNA repeat sequence from the DXS424 locus. 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.

Morphological detection of X- and Y-chromosomes in smears and paraffin-embedded tissues using a non-isotopic in situ hybridization technique (NISH)

“…Recently, in situ hybridization techniques have been described using biotinylated or fluoresceine conjugated X-or Y-chromosome specific probes [7,8,13]. As shown in the present study, these methods can be applied to forensic problems by evaluation of smears or paraffin-embedded tissues.…”

“…In addition to the detection of Barr or Y bodies by staining with Acriflavin or Quinacrin [2,3,6,[9][10][11][12] and an identification of X-and Y-chromosomes by dot-blot hybridization or polymerase chain reaction techniques [1,4,5], the morphological detection of sex-specific chromosomes in interphase nuclei has been described [7,8,13]. The present study reports on a NISH technique using centromere-specific biotinylated DNA probes for chromosomes X and Y in smears and paraffin sections.…”

Sex determination and DNA competition in the analysis of forensic mixed stains by PCR

“…Amplification of the amelogenin gene from both X-and Y-chromosomes is performed in a single assay with one pair of primers and the products, 106 and l l 2 b p fragments, are separated in an 4% agarose or 6% polyacrylamide gel (Mannucci et al 1994). The D Y Z l / D X S 4 2 4 system amplifies an X-specific product (181-199bp) and a Y-specific product (102 bp) in a single assay using 2 primer pairs and separation of the products is in a 4% Nusieve agarose gel (Pfitzinger et al 1993). …”

“…Amplified regions of the X and Y chromosomes are the alphoid repeat region (Witt and Erickson 1989), the zincfinger protein ZFX/ZFY (Aasen and Medrano 1990), DYZ1/DXS424 (Pfitzinger et al 1993) or the amelogenin gene (Sullivan et al 1993;Mannucci et al 1994). The most rapid methods, however, require only electrophoresis of the amplified products in an agarose gel (Witt and Erickson 1989;Pfitzinger et al 1993;Mannucci et al 1994). Amplification of the amelogenin gene from both X-and Y-chromosomes is performed in a single assay with one pair of primers and the products, 106 and l l 2 b p fragments, are separated in an 4% agarose or 6% polyacrylamide gel (Mannucci et al 1994).…”

Brief communication: Multiplex X/Y‐PCR improves sex identification in aDNA analysis

“…For that purpose, a multiplex PCR was used to amplify the amelogenin gene together with two X-and two Y-specific short tandem repeats (STRs). This strategy, of amplifying multiple X-and Y-chromosomal sequences, has been used successfully in forensics (Pfitzinger et al, 1993;Tun et al, 1999;Young et al, 2000;Honda et al, 2001), and is recommended for archaeological investigations (Brown, 1998). The multiplex PCR approach presented here is particularly suitable for application to highly degraded DNA, as the lengths of the products amplified ranged from 91-166 bp due to the new primer design we conducted.…”