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Korean Journal of Legal Medicine 2009;33(2):147-152.
Published online November 30, 2009.
Experience on Sequencing of Mitochondrial DNA from 1200 Year Old Bone Using Cloning.
Hye Young Lee, Seung Bum Seo, Ai Hua Zhang, Jina Yi, Hye Yeon Kim, Suk Bae Jung, Chong Min Choung, Dong Hoon Shin, Soong Deok Lee
1Department of Forensic Medicine, Seoul National University College of Medicine, Seoul, Korea. sdlee@snu.ac.kr
2Department of Anatomy, Seoul National University College of Medicine, Seoul, Korea.
3Institute of Forensic Medicine, Seoul National University College of Medicine, Seoul, Korea.
4Department of Archaeology, The Korean National University of Cultural Heritage, Buyeo, Korea.
5Division of DNA analysis, National Institute of Scientific Investigation, Seoul, Korea.
Abstract
Ancient bones have undergone natural decomposition and have been exposed to external environment for long period. Ancient DNA from old bone is usually fragmented. In addition, various kinds of inhibitors are co-extracted. All these may inhibit proper sequencing reaction. Cloning is regarded as the standard method when sequencing aDNA. When cloning, each clone from the same sample may not be of same sequence, and to exact consensus sequence may be difficult. Here we present our experience on 1200 year old bone from Russia, Primorsky Kray area. We have tried to sequence for HV I, II region of mtDNA using modified mini-primer set, which consisted of 7 set to cover the HV I, II. We cloned the PCR product and sequenced all the clones. Amplification efficiency and subsequent success rates were different for each mini primer set. Loci of variation that differ from consensus sequences were rather frequent, and the pattern were variable depending on sample. Except major polymorphic sites that are important when haplogroup designation, 16129 was the most frequent site that was discarded when extracting haplogroup designation.
Key Words: ancient DNA, mtDNA, cloning, sequencing, modified mini-primer set
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