Alonso et al. (2003, 2004; Alonso and Martin, 2005) described a method for nuDNA quantification based on TaqMan-MGB real-time PCR amplification of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and an X-specific fragment (AMGX: 106 bp), making possible not only DNA quantitation but also sex determination. Detection of the specific AMGX fragment (106 bp) and AMGY fragment (112 bp) was achieved using the primer pair sequences previously described (Sullivan et al., 1993) and two fluorogenic MGB probes that specifically detect the AMGX fragment (FAM-labelled) or the AMGY fragment (VIC-labelled). The MGB probes were designed to target the 6 bp X-deletion / Y-insertion segment within the AMG second intron fragment. The method has been applied to the analysis of LCN DNA samples in forensic and ancient DNA studies (Alonso et al., 2004).
A SYBR-green real-time PCR assay of the human amelogenin gene using specific primers to produce a Y-specific fragment of 73 bp and a 3-bp-deleted X-specific fragment of 70 bp was described (Andreasson and Allen, 2003). The assay allows quantification of the nuDNA copy number, but sex determination, which is based on a dissociation curve analysis that displays the different melting temperatures of X- and Y-specific products, has certain limitations for forensic applications and especially for the analysis of mixed male-female forensic samples.
A specific real-time PCR quantification kit based on the TaqMan-MGB detection of a region (61-64 bp) of the SRY locus is also commercially available (Green et al., 2005). The assay, which detects only male DNA, is intended particularly for use in samples with mixed male-female DNA, such as sexual assault evidence, where it may be useful for specific male DNA detection. The kit has also been validated for use in forensic casework according to SWGDAM guidelines.
A sex chromosome TaqMan-MGB assay was designed around a 90 bp deletion on the X chromosome in an X-Y homologous region to target a 77 bp fragment on the human X chromosome and a 167 bp fragment on the human Y chromosome (Walker et al., 2005).
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