Mechtild Prinz,1,2 Cornelia Schmitt2 and Howard Baum2
1Dept. Forensic Biology, Office of Chief Medical Examiner, New York City,
NY
2Institute for Forensic Medicine, University of Cologne, 50823 Köln, Germany
For various PCR based DNA typing systems, it has been reported that identical alleles show different electrophoretical mobility on native PAGE (polyacrylamide gel electrophoresis) versus agarose and denaturing PAGE (Eng et al. 1994; Moeller et al. 1994). This poses a problem for interlaboratory data comparison and data banking.
The AmpFLP locus Apo B shows a number of subtypes on nondenaturing PAGE, which increase the power of discrimination for this locus. Utilizing three different electrophoretic systems for the separation of the subtype alleles, it could be shown that these subtypes on native polyacrylamide gels were of the same length, and migrated with the regular ladder alleles on agarose and denaturing polyacrylamide gels. In order to detect the molecular basis for this aberrant electrophoretical behavior, two subtypes and the corresponding ladder alleles were sequenced using the primers described by Boerwinkle et al. (1989), dye deoxy terminator cycle sequencing chemistry, and the automated sequencer 373A (ABD/PE).
The Apo B VNTR region consists of seven different types of core repeat units, mostly pure AT repeats with regular interspersion of one C or one G (Desmarais et al. 1993; Ellsworth et al. 1994). Subtype and ladder alleles did not have major differences in their sequence. They displayed the same number of repeat units but differed in number and order of the different type of repeats. Due to the GC interspersions the secondary structure varies for different type of repeat units. Therefore depending on the variation in the region of pure AT repeats, the potential hairpin loop formation for the VNTR sequence differed for subtypes and ladder alleles, which is thought to explain the shift in electrophoretical mobility on native PAGE (see also Prinz et al. submitted.).
REFERENCES
Boerwinkle E., Xiong W., Fourest E. and Chan L. (1989) Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: application to the apolipoprotein B hypervariable region. Proc. Natl. Acad. Sci. U.S.A. 86:212-216.
Desmarais E., Vigneron S., Buresi C., Cambien F., Cambou J.P. and Roizes G. (1993) Variant mapping of the Apo(B) AT rich minisatellite. Dependence on nucleotide sequence of the copy number variations. Instability of the non-canonical alleles. Nucl. Acids Res. 21:2179-2184.
Ellsworth D.E., Shriver M.D. and Boerwinkle E. (1995) Nucleotide sequence analysis of the apolipoprotein B 3VNTR. Hum. Mol. Genet. 4:937-944.
Eng B., Ainsworth P. and Waye J.S. (1994) Anomalous migration of PCR products using nondenaturing polyacrylamide gel electrophoresis: the amelogenin sex-typing system. J. Forensic Sci. 39:1356-1359.
Möller A., Meyer E. and Brinkmann B. (1994) Different types of structural variation in STRs: HumFES/FPS, HumVWA and HumD21S11. Int. J. Leg. Med. 106:319-323.
Prinz M., Schmitt C., Staak M. and Baum H. (submitted) Apo B VNTR subtypes on native polyacrylamide gels are caused by positions of variant repeat units. Int. J. Leg. Med.
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