HUMARA X-chromosome inactivation assay for detection of female minor component in male/female mixed bloodstains

Analysis of mixed stains from forensic casework by means of standard PCR-based typing of autosomal short tandem repeats (STRs) becomes difficult when the minor component is present at less than one tenth of the concentration of the major component. The human androgen receptor (HUMARA) X-chromosome inactivation assay allows to detect even a small number of female cells in the presence of a high background of male cells in male/female mixed bloodstains. DNA cleavage by means of methyl-sensitive restriction enzyme (HhaI) is followed by typing of the HUMARA locus with the nested PCR technique: methylation of restriction sites on inactivated female X chromosomes allows the efficient amplification of low amounts of endonuclease-resistant female-derived target sequences. In this study, the method attained a 10−3 level of sensitivity in the detection of the female minor component.