Megaplex DNA typing can provide a strong indication of the authenticity of ancient DNA amplifications by clearly recognizing any possible type of modern contamination.

Recent experiments revealed the perfect applicability of megaplex typing by autosomal short tandem repeats (STRs) to degraded DNA. The advantages of megaplex approaches lie in reduced amounts of sample material that are necessary and in remarkable time saving. Furthermore, megaplex typing clearly recognizes possible contaminations and thus has a large potential for indicating authenticity in ancient DNA analysis. This is demonstrated by three examples in which various types of contaminations could clearly be identified as such and even traced back to their origin. This would have been impossible using control samples, due to the sporadic nature of these types of contaminations.

Ancient DNA-typing approaches for the determination of kinship in a disturbed collective burial site.

Several DNA-typing approaches are applied for identification and kinship analysis. Autosomal Short Tandem Repeat (STR) typing produces the genetic fingerprint that is unique to an individual. Y-chromosomal STR typing identifies individuals of the same paternal lineage, and sequence analysis of the hypervariable region of the mitochondrion can identify maternally related individuals. The combined approach of these DNA-typing methods allows the determination of kinship even in complex collective burial situations. In a bronze age collective site, the typing methods were tested for applicability to ancient DNA. For each approach, results were obtained, leading to the conclusion that the determination of kinship is achievable.

STR allelic frequencies in a German skeleton collection.

Chromosomal DNA was isolated from bones from a German skeleton collection (Goslar, 18th century) and detected by PCR. Nine microsatellite regions were amplified by multiplex reactions using the AmpFlSTR Profiler Plus kit and analysed to obtain their allelic distribution. A statistical evaluation of the results revealed no allelic differentiation between the historic sample and a modern German one at each locus.

Ancient DNA profiling by megaplex amplications.

Simultaneous amplification of nine human short tandem repeat (STR) DNA sequences and the amelogenin locus allows reducing to an absolute minimum the amount of sample material that is necessary for genetic identification or kinship analysis. Valuable remains can be studied this way without any visible damage, as is demonstrated by typing the DNA of a tooth root from the Saxon warrior Widukind, who died about 1200 years ago. The broad applicability of the megaplex approach is shown by typing bone and teeth specimens ranging from a few months to 3000 years of age employing AmpFISTR Profiler Plus. Additionally, megaplex STR typing is the method of choice for proving the authenticity of molecular results derived from ancient degraded DNA.

Reconstruction of a historical genealogy by means of STR analysis and Y-haplotyping of ancient DNA.

Archaeological excavations in St Margaretha's church at Reichersdorf, Germany, in 1993 led to the discovery of eight skeletons, so far assumed to be of the Earls of Königsfeld, who used the church as a family sepulchre over a period of seven generations from 1546 to 1749. DNA-based sex testing and analysis of autosomal short tandem repeat systems (STR) was carried out to confirm the assumption of kinship. Since five of the individuals were determined as males, analysis of Y-specific STRs seemed feasible. A comparison of Y-haplotypes revealed that one individual could not be linked to the Königsfeld patrilineage, an observation supported by autosomal STR evidence. Two individuals typed as females posed an identification problem, since supposedly only male members of the family were buried in St Margaretha's. Nevertheless, these individuals could tentatively be identified as members of the House of Königsfeld through genetic fingerprinting.

Amplification of Y-chromosomal STRs from ancient skeletal material.

The adaptation to ancient DNA analysis of a Y-chromosomal STR (short tandem repeat) multiplex comprising the four STR systems DYS19, DYS390, and DYS389I/II shows the suitability of Y-chromosomal STR typing on ancient human remains. A new primer site for the system, DYS389I/II, resulting in products shortened by 94 bp, was chosen to serve the special needs of amplification of ancient DNA. For the first time, it was possible to amplify STR loci of the Y chromosome from historical and prehistorical bones of up to 3000 years old.

[Classification of isolated skeletal elements using aDNA typing]. / Zuordnung isolierter Skelettelemente mittels aDNA-typing.

Analysis of ancient DNA of material found in the Lichtensteinhöhle, a burial site of the Younger Bronze Age has been used for the first time to assign isolated skeletal elements to corresponding individuals. The method involved DNA typing through amplification of five Short Tandem Repeat loci which are also used in forensic genetics for the determination of kinship and identification. From all of the examined bone samples DNA was successfully extracted and amplification by means of Polymerase Chain Reaction could be carried out. For the skeletal elements allelic profiles which are specific for an individual were set up. These profiles made it possible to recognize bones belonging to one individual. Elements which were not from this individual could be excluded with certainty by aDNA analysis.