Extraction and Amplification of mtDNA HVR1 from Old and Degraded Samples of Human Skeletal Collected from Khyber Pakhtunkhwa province of Pakistan

Nasir Ali, Muhammad Ilyas, Habib Ahmad, Shaker Khan, Wajiha Shafique, Ghani ur Rehman, Muhammad Jamil, Nazia Akbar, Shkair ul llah, Ikram Qayum, Gohar Rahman


Background: The study of ancient DNA enables the examination of genetic associations between past and present individuals and populations. The recovery and analysis of human DNA from degraded samples precisely has become a central research tool in various scientific fields’ ranges from ancient DNA to forensics and medical sciences. 

Methods: In the current study, remains of human bones are analyzed that dates back to 1000-1200 BC from different burial sites of Khyber Pakhtunkhwa province of Pakistan. Hypervariable region 1 (HVR1) as a genetic marker was selected. Multiple extractions, qubit assays quantification and cloning of PCR products were performed to get the sequences from the hypervariable region 1 of mtDNA. In this study, we also evaluate the possibility of extracting DNA from degraded human tissues (bones and teeth) such as remains buried in archaeological sites and remains of dead bodies buried for many decades. 

Results: Mitochondrial DNA HVR1 of 2000 years old degraded bone specimens were successfully determined by analyzing haplotype, defining polymorphisms in the hypervariable region 1. The prominent haplogroup found was eastern European R  (63%) that was also found in present day population by previous studies.

Conclusion: The protocol developed in this study could be used for the extraction of DNA from old degraded and ancient bone samples.

Keywords: Ancient DNA; Degraded samples; mtDNA; HVS1; Pakistan   

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