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KMID : 0357820090330020147
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Korean Journal of Legal Medicine
2009 Volume.33 No. 2 p.147 ~ p.152
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Experience on Sequencing of Mitochondrial DNA from 1200 Year Old Bone Using Cloning
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Lee Hye-Young
Seo Seung-Bum
Zhang Ai-Hua
Yi Jin-A
Kim Hye-Yeon
Jung Suk-Bae
Choung Chong-Min
Shin Dong-Hoon
Lee Soong-Deok
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Abstract
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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.
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KEYWORD
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ancient DNA, mtDNA, cloning, sequencing, modified mini-primer set
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