Danuta Miscicka-Sliwka, Tomasz Grzybowski, Jakub Czarny, and Marcin Wozniak
The Ludwik Rydygier’s University School of Medical Sciences, Forensic Medicine Institute, ul. Curie-Sklodowskiej 9, Bydgoszcz 85-090, Poland

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PCR-based DNA typing using short tandem repeat (STR) loci in combination with high resolution denaturing polyacrylamide gel electrophoresis and automated fluorescence detection has revolutionized forensic casework in recent years. STR systems with hypervariable repeats, are particularly attractive for forensic purposes because of the high discriminating index (DI), often equivalent to the combined DI of simple or compound repeats. The human beta-actin related pseudogene (ACTBP2) [1,2] is one of the most informative but also one of the most complex tetranucleotide markers tested so far. Our sequence data [3] revealed the presence of significant sequence variation between alleles of the same size which has very important implications for the forensic use of ACTBP2 locus. In the alleles ranging in size from 275 to 323 bp, hexamer units AAAAAG or AGAAAG occurred in the repeat region in addition to AAAG repeats. Two alleles (317 and 321 bp) contained two hexamers in the repeat region. There was considerable polymorphism of the hexamer position leading to three different allelic variants of 275 bp, two of 279 bp, two of 287 bp and four of 307 bp. In the first place, the presence of microheterogeneity can make calling of discrete alleles difficult. Thus, methods distinguishing alleles solely by size are not sufficient in ACTBP2 typing. On the other hand, however, length and sequence polymorphism leads to a greater discrimination in human identification. Increasing of informativeness by direct sequencing the alleles from the upper size range would be problematical in casework. Thus, our research effort focuses on the methods allowing maximum utilization of the ACTBP2 sequence variation without direct sequencing. This paper describes the results of automated fluorescent PCR-SSCP analysis performed on 11 allelic variants of the ACTBP2 locus. All allelic variants of the same size have been easily resolved on the basis of their secondary conformation. An interesting application of the method is presented when an individual was homozygous for two distinct alleles of the same size. SSCP analysis combined with automated fluorescence detection provides a rapid and sensitive means of screening small sequence changes in DNA amplified from high microvariation microsatellite loci. Possibilities of the routine implementation of the technique in forensic casework are discussed.

REFERENCES

1.Moos, M., Gallwitz, D., EMBO J. 1983, 2, 757-761

2. Polymeropoulos, M.H., Rath, D.S., Xiao, H., Merril, C.R., Nucleic Acids Res. 1992, 20, 1432

3.Miscicka-Sliwka, D., Grzybowski, T., Electorphoresis 1997, in press.