고인골유전자증폭효율이높은PCR 반응조건
Establishment of PCR Reaction Condition for Highly SuccessfulAncient DNA PCR
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고인골의 시료 및 핵산의 양적 제한 때문에 표적핵산의 PCR 증폭이 고인골 핵산 분석에 필수적으로 요 구되는 전제 조건이다. 현대인의 신선한 핵산과는 달리, 사후 보존에 불리한 환경 속에 오랜 시간 동안 방치되어 온 고인골 시료로부터의 PCR 증폭은 성공적이지 못한 경우가 흔하다. 따라서 고인골 DNA에 적합한 PCR 증폭 방법의 시행이 요구된다. 그러나 고대 시료에 적합한 향상된 PCR 증폭 기법에 대한 체계적인 연구가 시행된 보고 이 연구에서는 한국과 몽골의 약 500년에서 3300년 된 PCR 증폭이 어려운 8개 고인골 검체들로부터 광범위한 PCR 조건들을 체계적으로 시도하고 PCR 증폭의 성공율을 비교하였다. PCR 조건들의 증폭율을 비교하기 위한 표 적 핵산으로서는 카피수가 많은 사립체 핵산과 세포내 단일 카피 DNA로서 현대 한국인에서 흔한 Y 염색체 일배 체 그룹으로 알려진 O 일배체 그룹 표지(M175) 핵산을 이용하였고, 성공적인 증폭산물의 형성의 검증은 증폭산물 들의 염기서열을 분석을 통하여 시행하였다. 이 연구를 통하여 고인골에서 핵산을 증폭하기 위한 최적의 PCR 조건을 확립하였고, 결정된 방법은 검사한 모 든 샘플에서 사립체 핵산을 성공적으로 증폭시켰고, M175 Y 염색체 일배체 표지 핵산의 증폭은 검사된 시료수의 50% 성공율을 나타냈지만 시도된 조건들에서 가장 높은 성공율을 나타내었다. 이 결과는 성공적인 고인골 핵산분석을 위한 최적의 PCR 조건으로 분자 유전학적 인류학 연구에 유용하게 쓰일 수 있음을 시사한다.
Abstract
The ancient bone DNA analysis essentially requires PCR amplification of the targeting genes of study due to the limitation of the ancient bone sample and DNA amounts. In contrast to the fresh living human DNA, it is common to face failing in amplifying the poorly preserved ancient DNA after death. Therefore, the optimized PCR methods appropriate for ancient DNA are required. However, there is no report to date that a systemic investigation of enhanced PCR amplification methods suitable for ancient samples has been conducted Approximately 500~3,300-year-old Korean and Mongolian ancient bones that are resistant to PCR were selected and an extensive number of PCR conditions were systematically investigated for the comparison of PCR success rates. For the PCR analysis, a mitochondrial DNA fragment as a multicopy DNA and a M175 Y chromosome biallelic marker DNA fragment as a single copy DNA that is the marker of the prevalent Y haplogroup (haplogroup O) in Korea were targeted. The identity of the amplified products were confirmed by DNA sequencing. Through this study, we established the optimized PCR conditions for the highly successful amplification of ancient bone DNAs. This estabilished method allowed for the successful amplification of mitochondrial DNAs from all the ancient bone samples tested and the amplification by 50% success rates in the amplification of M175 Y chromosome biallelic marker DNA but with the highest success rates. These results demonstrate that the optimized PCR condition will be useful for the promising ancient DNA analysis in the fields of molecular genetic anthropological studies.
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