Experience on Sequencing of Mitochondrial DNA from 1200 Year Old Bone Using Cloning / 대한법의학회지

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.

Efficiency of Quantification using QuantifilerTM Human DNA Kit in 60 Year Old Bone / 대한법의학회지

Commercial kits usually recommend certain range of the amount of DNA without PCR inhibitors for optimal results. If proper DNA is not used in STR amplification, undesirable results such as enhanced stutter effects, split peaks, allele dropout and poor peak balance could be shown. This phenomenon would happen more frequently in case of bone typing. Therefore, we checked effect of DNA concentration and PCR inhibitors on conventional STR typing results using QuantifilerTM Human DNA Quantification Kit. The 54 bone samples over a 60-year burial period were used in this study. In all samples, the DNA concentration ranged from 0.8 pg/ul to 748 pg/ul and Ct values of IPC that indicates residue of PCR inhibitors ranged from 26.69 to 35.59. The DNA concentration and the number of amplified loci showed positive correlation. The number of loci amplified using Minifiler kit was much more than using Y filer kit. Samples with high Ct value of IPC were not almost amplified with Y filer kit. These results suggested that QuantifilerTM Kit is useful to obtain information for DNA typing.

Three Allele Pattern Detected by STR Typing / 대한법의학회지

STR typing for several loci has been regarded as a standard method for individual identification. The result is rather simple and easy to be interpreted. For each loci one allele for homozygote and two different alleles for heterozygote are expressed as simple peaks. Many peaks more than two suggest unusual situations such as contamination. We experienced two cases of three alleles in two separate samples. Among 15 STR loci typed using AmpFlSTR.. Identifiler.. Kit(Applied Biosystems, Foster City, CA) and Powerplex..16 system (Promega, Madison, WI), three alleles were noted in D8S1179 locus in one sample, in D21S11 locus in another sample. We sequenced all the three amplified alleles through cloning to reveal the structure, and amplified nearby regions to check the extent of three alleles. We present the results here together with review on many alleles in one sample.