粒線體DNA於身份鑑定之應用

Application of Mitochondrial DNA Analysis in Human Identification

許瑋真(Wei-Chen Hsu) ； 李龍雄(Lung-Hsiung Li) ； 吳柏燕(Po-Yen Wu) ； 趙淑貞(Shu-Chen Chao) ； 陳如珍(Ju-Chen Chen)

《北市醫學雜誌》 5卷2期 (2008/04) Pp. 215-222

https://doi.org/10.6200/TCMJ.2008.5.2.05

摘要

一般身份鑑定首先以國際通用之包含性別的十六基因點位系統為基礎分析，若有親子關係概率不到99.99%或半手足、手足關係鑑定、疑突變等的情況下，為提高鑑別力，除依鑑定當時的需求增作X或Y染色體DNA STR式型別鑑定法和其他新增點位系統外，有時也須加做代表母系遺傳之粒線體D-loop之兩個高變異小區HV Ⅰ、HV Ⅱ鹼基序列的分析，利用PCR技術分別複製粒線體DNA之HV Ⅰ、HV Ⅲ兩段鹼基，以瓊脂電泳法確定PCR產物大小後純化產物，以BigDye Terminator Sequencing Kit進行定序反應並純化後以DNA定序儀分析。本文以三組粒線體DNA鑑定案例，探討粒線體DNA鑑定技術在身份鑑定上的應用及相關注意事項，三組案例分別和Anderson等發表之粒線體DNA序列比較後列出不同的位置與序列，鑑定結論以2003年美國的The Scientific Working Group on DNA Analysis Methods (SWGDAM)發表的Guidelines為準則。分析粒線體DNA時必須考慮各類檢體腐敗DNA降解速度之不同、PCR的抑制物質、DNA的萃取量及品質、特定組織的異質現象、螢光序列分析圖及正、反向引子定序結果比對、排除雜訊干擾、防治污染等，除以基因定序外，低程度的異質現象也可用Denaturing high performance liquid chromatography (DHPLC)、Multiplex Ligation-dependent Probe Amplification (MLPA)等方法分析，粒線體HV Ⅰ、HV Ⅱ鹼基序列的分析可鑑別一個人的母系血緣關係、輔助人類身分鑑定，對於族群分佈、人類起源探討、遠古檢體的分析可作為輔助STR系統的利器。

關鍵字

粒線體DNA ； 異質現象 ； 定序

並列摘要

In general, identification was based on the worldwide-accepted 16-gene-loci system which including sexual genes. If probability of paternity is less than 99.99%, to different relations between half-siblings, siblings and mutation is a challenge. In addition to X or Y chromosomal and DNA STR typing and other newly adopted loci system, we also could make analysis of the two highly varied sequences of HV Ⅰ, HV Ⅱ which are on the D-loop of mitochondrial DNA, the maternal gene substances. We carried out PCR to amplify the mitochondrial DNA, HV Ⅰ and HV Ⅱ. After gel electrophoresis, we purify the PCR product. Then we use BigDye Terminator Sequencing Kit for sequencing, by use of DNA sequencing machine. We present three groups of mitochondrial DNA to study the application of mitochondrial DNA certification technique on identification. This three groups of cases were compared to the Anderson report, and the differences of the mitochondrial DNA sequence were listed. We also used the guidelines which the Scientific Working Group on DNA Analysis Methods (SWGDAM) reported in 2003 for reference. When we analyze mitochondrial DNA, there are some factors should be taken into consideration. They include the variation of the degradement velocity of different sample, the inhibition product of PCR, the amount of DNA extraction, quality of DNA and the heteroplasmy of some tissue. Correct sequencing, comparison of the result of forward and reverse primers sequencing should be done to rule out wrong signals, and prevent contamination. In addition to gene sequencing, low grade heteroplasmy could be dealt by Denaturing high performance liquid chromatography (DHPLC). Multiplex Ligation-dependent Probe Amplification (MLPA). The analysis of mitochondrial HV Ⅰ, HV Ⅱ sequencing is able to identify the maternal sanguinity, then it could assist in the human identification. It also complements the genomic STR system in distribution of population, the origin of human, and analysis for fossil.

並列關鍵字

mitochondrial DNA ； Heteroplasmy ； Sequence