Abstract
We extracted the DNA from the Jomon skeletal remains and amplified a part of mitochondrial DNA sequences by using the polymerase chain reaction method. The methodology concerning the extraction, purification and PCR amplification of ancient DNA from the skeletal remains were assessed critically for further application.
The materials used were three skeletal remains iscovered in Chiba Prefecture, that is femur (Imaizumi 1), skull (Imaizumi 2) and tibia (Kusakari). Chronologically, Imaizumi site bel- ongs to the middle phase of Jomon Period and Kusakari Shellmound belongs to the late phase of Jomon period.
Southern hybridization analysis with human Alu repetitive sequences probe revealed that the purified sample from ancient skeletal remains contained very little amount of human DNA and most of the DNA recovered might be of the bacterial or fungal origin.
Amplified mitochondrial DNA sequences by PCR method were V and D-loop regions both containing noncoding sequences that are known to have intraspecific sequence heterogeneity.
Although there was no Hae III restriction polymorphism in PCR amplified region V sequence, nucleotide sequence analysis revealed single substitution T at 8240 to A in one indi- vidual. As for D-loop region, we could not find restriction fragment polymorphism expected by Ava II, Hpa II, Hae III digestion, including three samples from contemporary human. Nucleotide sequence analysis might be needed in this region.
The study of ancient DNA seems to have a number of technical difficulties, but the method described in this article gives considerably stable result for extraction of DNA from human skeletal remains. In conjunction with this method, we used polymerase chain reaction, in which short region of particular gene can be greatly amplified specifically in vitro from extremely small number of DNA molecules. It makes possible to amplify the ancient DNA up to the amount which allows us to apply standard laboratory techniques for further molecular analysis.
More extended work involving numerous individuals from ancient human skeletal remains will bring the valuable information to the field of anthropology.
