고인골 유전자 증폭 효율이 높은 핵산 분리 방법
Development of Ancient DNA Isolation Method for Improved PCR Amplification
1
조회
0
다운로드
4
인용
0
참고문헌
키워드
초록
고인골 핵산(DNA)분석은 인류학 및 고고학에서 인류의 진화 및 계통발생학적 연구에 널리 이용되고 있다. 그러나 이러한 분석을 위한 고인골 핵산 추출은 보존이 잘 안된 고인골 시료에서 성공적이지 못한 경우가 흔하다. 이 연구에서는 이러한 고인골로부터 핵산 분석의 성공율을 높이기 위하여 이온교환수지를 이용한 고순도 핵산 추출법을 시도하였다. 이 방법과 기존에 고인골 핵산 추출법으로 쓰여온 GENECLEAN 키트 (Qbiogene)와 Qiaquick 컬럼(Qiagen)방법을 이용하여 한국과 몽골의 약 500년에서 5000년 된 12개 고인골 검체들로부터 핵산 분석의 성공율을 비교하였다. 핵산 분석 방법으로서 사립체 핵산, 남성 결정 표지 핵산 및 한국인에서 흔한 Y 염 색체 일배체그룹 O 표지(M175) 핵산 증폭 방법을 이용하였다. 이 연구에서 개발된 방법은 기존 방법들에 비하여 핵산분석의 성공율을 현저히 증가시켰다. GENECLEAN 키 트 이용 방법으로 사립체 핵산 증폭은 두 시료에서만 가능하였고, 남성 결정 표지 핵산과 M175 표지 핵산의 증폭 은 모든 검체에서 없었다. Qiaquick 컬럼 방법은 9개 시료에서 사립체 핵산 증폭, 9개 시료에서 남성 결정 표지 핵산 증폭, 6개 시료에서 M175 표지 핵산 증폭이 가능하였다. 이 연구에서 개발된 방법은 모든 시료에서 사립체 핵산 증폭, 10개 시료에서 남성 결정 표지 핵산 증폭, 8개 시료에서 M175 표지 핵산 증폭이 가능하였다. 이 결과는 이온교환수지를 이용한 핵산 추출법이 고인골 핵산분석을 위해 향상된 핵산 분리법으로서 인류학 연구에서 유용하게 쓰일 수 있음을 시사한다.
Abstract
Ancient DNA analyses are widely used for evolutionary and phylogenetic study of mankind in anthropology and archeology. However, the DNA extraction from particularly poorly preserved ancient human samples is often unsuccessful in these analyses. In the present study, to improve the success rate of ancient DNA analysis, we introduced a high grade ancient DNA purification method using ion-exchange columns. We compared the success rate of ancient DNA analysis of this new method with that of the two methods that have been used for ancient DNA extraction, GENECLEAN kit (Qbiogene) and Qiaquick column (Qiagen). Twelve ancient bone samples from Korea and Mongolia that are about 500 to 5,000 years old by an archeological estimation were used. As the DNA analysis methods, polymerase chain reaction (PCR) methods for the amplification of a mitochondrial DNA HV1 segment, a male sex determination marker DNA and M175 marker DNA that is used for the determination of O haplogroup of Y chromosome that is reportedly a common one in modern Korean people. The method developed in this study remarkably increased the success rate of DNA analysis compared with the other two methods. Using the GENECLEAN kit, only two samples were amplifiable for the mitochondrial DNA, no sam-ples for the male sex determination marker and M175 marker DNAs. Using the Qiaquick columns, nine samples were amplifiable for mitochondirial DNA, nine samples for male sex determination marker and six samples for M175 marker. The developed method allowed for the amplification of mitochondrial DNA from all samples, male sex determination marker from eight samples and M175 marker from eight samples. The results demonstrate that ion-exchange columns can be useful for the improved ancient DNA extraction in anthropology and archeology.
참고문헌 (31)
- Calacal GC, "Fungal DNA challenge in human STR typing of bone samples", 50, pp.1394~1401 (2005)
- Cattaneo C, "Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences", 42, pp.1126~1135 (1997)
- Cattaneo C, "A simple method for extracting DNA from old skeletal material", 74, pp.167~174 (1995)
- Edson SM, "Naming the dead-confronting the realities of rapid identification of degraded skeletal remains forensic", 16, pp.63~90 (2004)
- Evison MP, "Extraction of single-copy nuclear DNA from forensic specimens with a variety of postmortem histories", 42, pp.1032~1038 (1997)
- Faerman M, "Sex identification of archaeological human remains based on amplification of the X and Y amelogenin alleles", 167, pp.327~332 (1995)
- Fischer GF, "An HLA class-II allele frequent in Eskimos and Amerindians is found in the Tyrolean Ice Man", 65, pp.363~369 (2001)
- Hagelberg E, "Isolation and characterization ofDNA from archaeological bone", 244, pp.45~50 (1991)
- H?nni C, "Isopropanol precipitation removes PCR inhibitors from ancient bone extracts", 23, pp.881~882 (1995)
- H？ss M, "DNA extraction from Pleistocene bonesby a silica-based purification method", pp.3913~3914 (1993)
- H?ss M, "DNA damage and DNA sequence retrieval from ancient tissues", 24, pp.1304~1307 (1996)
- Hunter P, "Ancient DNA research goes nuclear : A new technique to extract sequence data from nuclear DNA may reveal exciting new insights into evolution and phylogeny", 7, pp.136~139 (2006)
- Jobling MA, "The human Y chromosome: an evolutionary marker comes of age", 4, pp.598~612 (2003)
- Kalmar T, "A simple and efficient method for PCR amplifiable DNA extraction from ancient bones", 28(E67) (2000)
- Kemp BM, "Use of bleach to eliminate contaminating DNA from the surface of bones and teeth", 154, pp.53~61 (2005)
- Keyser-Tracqui C, "Methods for the study of ancient DNA", 297, pp.253~264 (2005)
- Kolman CJ, "Ancient DNA analysis of human populations", 111, pp.5~23 (2000)
- Kumar SS, "Brief communication: discouraging prospects for ancient DNA from India", 113, pp.129~133 (2000)
- Kurosaki K, "Individual DNA identificationfrom ancient human remains", 53, pp.638~643 (1993)
- Mulligan CJ, "Isolation and analysis of DNA from archaeological, clinical, and natural history specimens", 395, pp.87~103 (2005)
- Neeser D, "Sex determination of forensic samples by simultaneous PCR amplification of a-satellite DNA from both the X and Y chromosomes", 40, pp.239~241 (1995)
- O?rourke DH, "Ancient DNA: methods, progress, and perspectives", 8, pp.557~571 (1996)
- P狎bo S, "Genetic analyses from ancient DNA", 38, pp.645~679 (2004)
- Poinar HN, "Amino acid racemization and the preservation of ancient DNA", 272, pp.864~866 (1996)
- Prado VF, "Extraction of DNA from human skeletal remains: practical applications in forensic sciences", 14, pp.41~44 (1997)
- Rohland N, "Comparison and optimization of ancient DNA extraction", 42, pp.343~352 (2007)
- Tebbe CC, "Interference of humic acids and DNAextracted directly from soil in detection and transformationof recombinant DNA from bacteria and a yeast", 59, pp.2657~2665 (1993)
- Underhill PA, "The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations", 65, pp.43~62 (2001)
- Walsh PS, "Chelex 100 as a mediumfor simple extraction of DNA for PCR-based typing fromforensic material", 10, pp.506~513 (1991)
- Yang DY, "Technical note: improved DNA extraction from ancient bones using silica-based spin columns", 105, pp.539~543 (1998)
- Zaporozhenko EV, "Method for rapid DNA extraction from bacterial communities of different soils", 75, pp.127~134 (2006)