Extraction, PCR amplification and cloning of aDNA from human remains of the Warring States (475-221 B.C.)

Yang Qun, Ozawa Tomowo, Hayashi Seiji and Wang Bo

pp. 13-24, in English, with Chinese abstract

Abstract:

By applying BIO 101 ancient DNA purification reagent set, we extracted DNA from an ancient human bone sample of the Warring States (475-221 B.C., i.e., about 2,200 to 2,400 years old) collected in Xushui, Hebei Province. The DNA extract was subjected to PCR amplifications using a set of a various primers targeting for a variable length of fragments within the D-loop part of the mitochondrial DNA for humans. The PCR experiments show that targeted DNA segments of 150 and more base pairs (bp) in length were not possible to amplify in our study and we obtained a fragment of 101 bp belonging to the mitochondrial D-loop segment. A double PCR amplification was found to be necessary in order to obtain detectable amount of PCR product using agarose gel electrophoresis with ethidium bromide staining. The PCR product was cloned into a vector and multiple clones were sequenced. By comparing the resulting DNA sequences with those of modern and other ancient human DNA sequences from previous studies, it is shown that the PCR amplified DNA was indeed of mitochondrial DNA affinity and most likely of original ancient Chinese people due to unique mutations as compared with those of modern human populations. Special precautions were taken in sample preparation and initial PCR amplifications. Nucleotide comparison of the acquired sequences with those of other homologous ancient human and related DNA sequences and modern human populations are presented. Further experiments to obtain longer DNA sequences are needed in order to make a reliable and accurate population analysis.

Keywords: aDNA (ancient DNA), mitochondrial D-loop, human, Warring States, cloning