May 11, 2011

Dong Hoon Shin MD, PhD, MS. Bioanthropology and Paleopathology Lab, Department of Anatomy/Institute of Forensic Science. Seoul National University College of Medicine, 103 Daehak-ro (Yongon-dong), Jongno-gu, Seoul 03080, South Korea. E-mail: cuteminjae@gmail.com(DH Shin); TEL: +82-2-740-8203; FAX: +82-2-745-9528

In general, ancient DNA analysis is the study researches must do very cautiously.

Samples including ancient Ascaris eggs were treated with 1 ml lysis buffer (EDTA 50 mM, pH 8.0; 1mg/ml of proteinase K; SDS 1%; 0.1M DTT) at 56 C for 24 hours. DNA was extracted by phenol/chloroform/isoamyl alcohol (25:24:1). Primers for Ascaris cytochrome b (cyt b), cytochrome c oxidase subunit 1 (COX1), NADH dehydrogenase subunit 1 (NAD1) and internal transcribed spacer 1 (ITS1) gene were made by IDT. The information of primers used is summarized in Table 2.
DNA amplification was done with 25 μl reaction mixture containing AmpliTaq Gold® 360 master mix(Applied Biosystems, Foster City, USA), 10 pmol of each primer. PCR conditions were as follows: pre-denaturation at 94 C for 10 min; 50 cycles of denaturation at 94 C for 45 sec, annealing at 52-65 C for 45 sec, extension at 72 C for 45 sec, and final extension at 72 C for 10 min. The PCR products were separated on 2.5% agarose gel (Invitrogen, USA), and then stained with ethidium bromide. They were photographed using a Vilber Lourmat ETX-20.M equipped with Biocapt software (Vilber Lourmat, France).
Agarose gel electrophoresis was done for the amplified products. We checked if any of specific bands for Ascaris cyt b, COX1, NAD1 and ITS1 genes. Negative controls (extraction controls) were also applied to the electrophoriesis at the same time.
Cloning and sequencing was also done for the amplified PCR products. Briefly, after aDNA in amplified bands was extracted by QIAquick Gel Extraction Kit (Qiagen, Germany), bacterial transformation with amplified DNA product was done using pGEM-T Easy Vector system (Promega, USA). Transformed bacteria were grown in agar plate containing ampicillin (100ug/ml), 0.5 mM IPTG and X-GAL (40 ug/ul) for the next 14 hours. After selected colonies were grown once again in LB media for 12 hours, plasmid was harvested using QIAprep spin miniprep kit (Qiagen, Germany). Sequencing was done on each strand using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, USA) in the 3730xl Automated Sequencer (Applied Biosystems, Foster City, CA).