Y chromosomes, respectively. Primers, which yield a 212 bp X-specific amplicon and a 218 bp Y-specific product by bracketing the same 6 bp deletion, were also described in the original amelogenin paper (Sullivan et al. 1993) and have been used in conjunction with the D1S80 VNTR system (Budowle et al. 1996).
An advantage with the above approach, i.e., using a single primer set to amplify both chromosomes, is that the X chromosome product itself plays a role as a positive control. This PCR-based assay is extremely sensitive. Mannucci and co-workers were able to detect as little as 20 pg (~3 diploid copies) as well as sample mixtures where female DNA was in 100-fold excess of male DNA (Mannucci et al. 1994).
Other regions of the amelogenin gene have size differences between the X and Y homologues and may be exploited for sex-typing purposes. For example, Eng and co-workers (1994) used a single set of primers that generated a 977 bp product for the X chromosome and a 788 bp fragment for the Y chromosome. In this case, a 189 bp deletion in the Y relative to the X chromosome was used to differentiate the two chromosomes.
A careful study found that 19 regions of absolute homology, ranging in size from 22-80 bp, exist between the human amelogenin X and Y genes that can be used to design a variety of primer sets (Haas-Rochholz and Weiler 1997). Thus, by spanning various deletions of the X and/or Y chromosome, it is possible to generate PCR products from the X and Y homologues that differ in size and contain size ranges that can be integrated into future multiplex STR amplifications.
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