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Performance of a Y-Chromosome Specific STR for Mixed Samples

Mechthild Prinz,1,2 Lutz Roewer3 and Howard Baum1
1Department of Forensic Biology, Office of Chief Medical Examiner, New York City, NY
2 Institute for Forensic Medicine, University of Cologne, 50823 Köln, Germany
3 Institute for Forensic Medicine, Humboldt University Berlin, 10115 Berlin, Germany

One problem connected with the application of PCR for DNA-typing, is the failure to amplify the lower component in DNA mixtures with very unequal ratios of heterogeneous DNA. Though even the presence of nonhuman DNA can inhibit the amplification of the human target sequence, the allelic drop-out for mixed samples is mainly caused by the accumulation of PCR products from alleles of the higher component (Ruano et al. 1991). As could be shown by Gyllenstein et al. (1992) this effect can be avoided by employing allele specific primers. A different approach to this problem, is to use Y-chromosome specific polymorphisms, which should be useful in the majority of sexual assault cases-cases with male perpetrators and female victims-for samples where differential lysis was either unsuccessful or impossible.

The Y-chromosome specific tetrameric STR DYS19 or 27H39 (Roewer et al. 1992) has five alleles in the size range of 186-202 bp, and a PIC of 0.65. No mutations were detected in 100 father son pairs (Santos et al. 1993). The PCR was optimized using the primers developed by Roewer et al. (1992) with primer H39.1 being fluorescently labeled (JOE dye). The PCR products were separated and detected on the ABI 373A DNA sequencer (ABD/PE). In order to compare the results for DYS19, all experiments were repeated for the autosomal locus VWA (Kimpton et al. 1992) also employing JOE-labeled primers.

Due to a stretch of CA repeats 5 from the polymorphic region, the allele peak shows two stutter peaks. 25 pg of male DNA could be detected. The mixture experiments involved multiple amplifications of 5ng of total DNA with declining amounts of the lower component down to 50pg, and addition of different amounts of DNA to 400pg of DNA from a second individual. The maximum that was added, was 160ng of DNA, resulting in a 1:400 ratio. Mixtures were made from DNA from two male individuals, and from one male and one female individual.

For the male/male mixtures, as for the locus VWA the occurrence of stutter peaks 4bp smaller than the adjacent allele, poses a problem for the interpretation of mixtures. At a ratio above 1:9, the allele peaks of the lower component were lower than the stutterpeaks, making interpretation of unknown samples impossible. Above a ratio of 1:20, 1:24, and 1:50, 1:49 respectively, the alleles of the lesser component could not be detected anymore. In the mixture with female DNA the Y-specific peak could be detected for 50pg in a 1:99 mixture. For the 400pg series the Y-specific allele peak does not change in peak height, and can be detected in the 1:400 mixture with female DNA.

The STR DYS19 allows a sensitive detection of the presence of DNA from a male individual even with an extremely high background of female cells. Kreike et al. (1995) described the amplification of a non-polymorphic Y-chromosome locus in mixed samples, and also discussed how this approach avoids DNA competition during the PCR reaction. The DYS19 locus has the advantage, that it provides additional information besides the proof of the presence of male DNA. Even though the power of inclusion is not satisfactory, it can efficiently exclude possible suspects.

Since there is no control result for the presence of the X chromosome, the DYS19 locus should not be used for sex determination of samples. In spite of the presence of the two stutter peaks the allele identification is unambigious; the stutter peaks only pose a problem in mixtures of DNA of two male individuals.

Circumstances where the application of the DYS19 PCR polymorphism would be advantageous are:

  1. Sexual assault cases, with a very low presence of sperm cells, where the differential lysis should not be too stringent, as to avoid loss of sperm DNA;
  2. Very old semen stains, where the majority of sperm cells are suspected to be degraded, and can not be retrieved after differential lysis;
  3. Blood evidence in cases with more than one victim of different gender.

Several further Y-chromosome specific STRs are presently under investigation. A panel of polymorphic Y-chromosome STRs would allow a positive identification of the origin of male DNA in extreme mixtures with female DNA. Another possible application of a panel of Y-chromosome specific PCR polymorphism is a rapid screening of chelex extracts of vaginal swabs without the time consuming differential lysis procedure.

REFERENCES

Gyllenstein U.B., Josefsson A., Schemschat K., Saldeen T. and Petterson U. (1992) DNA typing of material with mixed genotypes using allele-specific enzymatic amplification (polymerase chain reaction). Forensic Sci. Intl. 52:149-160.

Kimpton C., Walton A. and Gill P. (1992) A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1:287.

Kreike J. and Lehner A. (1995) Sex determination and DNA competition in the analysis of forensic mixed stains by PCR. Int. J. Leg. Med. 107:235-238.

Roewer L., Arnemann J., Spurr N.K., Grzeschik K.H. and Epplen J.T. (1992) Simple repeat sequences on the human Y-chromosome are equally polymorphic as their autosomal counterparts. Hum. Genet. 89:389-394.

Ruano G., Brash D.E. and Kidd K.K. (1991) PCR: The first few cycles. Amplifications 7:1-4.

Santos F.R., Pena S.D.J. and Epplen J.T. (1993) Genetic and population study of a Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique. Hum. Genet. 90:655-656.

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