DNA Typing on Ancient Bone Samples: Case Studies of Pre-Columbian Cuban Aborigines and Members of the Che Guevara’s Guerrilla in Bolivia
E. Riego1, R. Lleonart1,
M.V. Sainz de la Pena2, F. Amaro3, K. Bacallao3, M.
Santlesteban2, F. Riolo4, L. Herrera1, M. Blanco4,
H. Currentl2, A. Puentes, R. Rodriguez5, R. Travieso5,
and J. de la Fuente1
1Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba
2Laboratorio Central de Criminalistica, La Habana, Cuba
3Instituto de Medicina Legal., La Habana, Cuba
4Laboratorio de Investigaciones del Sida, La Habana, Cuba
5Facultad de Biologia, Universidad de La Habana
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The fact that some regions of the DNA are highly polymorphic has allowed the development of the so called DNA typing, one of the most promising techniques for individual identification for forensic purposes and paternity assessment, among other potential applications. The most recent advances have been achieved after the introduction of the Polymerase Chain Reaction (PCR) allowing the amplification and analysis of tiny amounts of DNA from biological samples. Nevertheless, even very small quantities of DNA are extremely difficult to obtain when the samples have been buried for long periods of time. Several procedures have been published which are claimed to yield useful DNA for PCR-based typing, however, the results are somehow very variable.
In this report we show the results of the application of a procedure for DNA extraction which has allowed the successful typing of ancient bone samples. The DNA extraction protocol was based on a proteolytic digestion of decalcified bone powder followed by a spin dialysis/concentration step to concentrate the DNA and eliminate Taq Polymerase inhibitors at the same time.
Successful PCR reactions were obtained in almost all the cases. "Refractory" samples were made amplifiable by the inclusion of acetylated BSA in the reaction.
Casework results are shown including the identification of one of the members of the Che Guevara’s guerilla in Bolivia (bone samples thirty five years old, buried on high temperature/humidity conditions) and a paternity case solved on a 2000 years old Pre-Columbian burial site. While the first case was done using STR typing (HUMCSF1PO, HUMTH01, HUMTPOX, HUMF13A, HUMFES/FPS, HUMF13B, HUMHPRTB, HUMvWA, and HUMLPL), the most ancient bone samples were analyzed by direct DNA sequencing of amplified mitochondrial DNA hypervariable region I. In all cases, strict measures were followed to avoid false positives, given the exquisite sensitivity of the PCR reaction.
As part of the STR analysis, a database containing information on allele frequencies of the Cuban population was created and used to estimate the probability of match.
These technologies, although rather laborious, are extremely effective and are now being used routinely by the forensic scientists in our country for human identification, molecular anthropology matters, etc.
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