Abstract STRs have become almost the exclusive tool of genetic scientists in forensic typing work. Consequently, large numbers of samples are genotyped and the detection of rare abnormalities is to be expected. We found rare losses of alleles, also known as drop-out, at the two STR loci D13S317 and CD4. Drop-out at D13S317 was acci- dentally found in typing of suspects in a murder case and three other examples of drop-out were found at locus CD4 during paternity testing. The lost alleles reappeared when alternative PCR primer pairs were used. Sequences of lost alleles were characterised at the molecular level after cloning. Variations were found in the primer sequences and these are believed to prevent amplification or to re- duce amplification yield and to be the origin of the allele drop-out. Keywords Short tandem repeat · D13S317 · CD4 · Allele drop-out · Primer sequence variation Introduction DNA typing with short tandem repeat systems (STRs) has become the major methodology to obtain individual ge- netic profiles and is widely used in criminal and paternity investigations. PCR amplification of these polymorphic repeat loci is used routinely to establish large numbers of genotypes. Amplification depends on hybridisation with locus-specific primer sequences enclosing the repeat. Considering the polymorphic nature of the human genome, variations could be expected to occur in primer se- quences. These polymorphisms could affect efficient primer/template interaction and change the product yield of the PCR reaction. D13S317 is part of commercial DNA profiling kits and is one of the 13 core STRs typed in the CODIS system al- lowing interlaboratory comparison of genotypes. Allele drop-out causes a heterozygote to be falsely recorded as homozygous, so that when searching data banks, the result may be interpreted as non-concordant. Allele drop-out in a paternity test, as found here for STR locus CD4, could be incorrectly interpreted as an exclu- sion. With CD4, the product yield of the PCR reaction varied and seemed to depend on amplification conditions, but with D13S317 loss of an allele was constant. Since both observations could lead to misinterpretation, we de- cided to investigate the underlying mechanisms of these allele drop-outs. Variation of product yield for the CD4 locus has been observed and studied before (Watanabe et al. 1998) and was associated with a polymorphism in the forward primer sequence. Materials The following STR profiling kits were used: AmpFlSTR Profiler (Perkin Elmer Biosystems, Foster City, Calif.) and Gamma STR Multiplex (Promega, Madison, Wis.). Synthesised primer-pairs for amplification and for sequencing are indicated in Fig. 1. PCR prod- ucts from homozygotes were sequenced directly. PCR products from heterozygotes were cloned in pGEM-T easy (Promega) and clones were selected at random and sequenced with T7 and SP6 primers. If necessary, additional clones were sequenced to obtain both alleles. Sequencing was done on an ABI PRISM 310 Genetic Analyzer using the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems). For CD4, the nomenclature in the literature is confusing and we tried to respect the recommendations drawn up by the ISFH DNA commission. The first repeat motif (TTTTC) on the coding strand [Genome Data Base (GDB) accession no. M86525, GenBank no. 180121] was chosen. In their studies of the CD4 locus, this nomen- clature was also used by Urquhart et al. (1994), Casarino et al. (1996) and Watanabe et al. (1998). Edwards et al. (1991) defined the repeat motif as (CTTTT) and Glock et al. (1995) as (AAAAG) L. Boutrand · B. Egyed · S. Füredi · N. Mommers · G. Mertens · A. Vandenberghe Variations in primer sequences are the origin of allele drop-out at loci D13S317 and CD4 Int J Legal Med (2001) 114 : 295–297 © Springer-Verlag 2001 Received: 2 June 2000 / Accepted: 19 September 2000 SHORT COMMUNICATION L. Boutrand · A. Vandenberghe () Laboratory of Human Molecular Genetics, Faculty of Pharmacy, University Claude Bernard Lyon I, 8 avenue Rockefeller, 69008 Lyon Cedex 08, France Tel.: +33-478-777231, Fax: +33-478-777568 B. Egyed · S. Füredi Department of Biology, Institute for Forensic Sciences, 1903 Budapest, Hungary N. Mommers · G. Mertens Antwerp Blood Transfusion Center, 2650 Edegem, Belgium