建立以粒線體DNA檢測混合檢體之個化分析系統

Abstract

在刑事案件中，現場蒐證人員為了釐清犯罪事實，往往會採集多種生物跡證（evidence samples，如血液、尿液等），連同嫌疑人的標準檢體（reference sample，通常是口腔黏膜細胞棉棒）提供給鑑識實驗室進行比對，期望藉由生物技術來證明或排除嫌疑人涉案的可能。雖然DNA技術的發展，autosomal STR (short tandem repeats) 分析已成為最快速且有效的個化鑑定方法。然而刑事證物的來源多樣，常有跡證樣本發生autosomal DNA嚴重裂解而無法分析autosomal STR進行鑑定的情形。為了提高案件證物的檢出鑑別力，鑑識人員可改採粒線體DNA序列分析來協助做個案鑑定。粒線體是透過母系遺傳，因此在個案鑑定上，只能做到母系來源的確認，雖無法達到完全個化分析，惟其區別率仍可高達99%以上。另由於細胞內含有豐富的粒線體DNA，因此在高度腐敗的檢體內，還是能夠藉由分析粒線體DNA的序列而協助個案鑑識。分析粒線體DNA 上control region的HVI（highervariable region）和HVII段，不同母系間之粒線體DNA HVI、HVII序列一般會有數個位點的差異，鑑識人員即依據該等位點的差異進行判斷及分析生物跡證與採自嫌疑人之對照檢體之粒線體DNA是否源自同一母系。 實務上最常使用粒線體序列分析的檢體為吸毒嫌疑人的DNA與呈毒品檢測陽性反應的尿液作比對。常見案例為院檢要求確認陳舊尿液是否為吸毒嫌疑人所排放？該尿液是否為混合檢體？若有，更希望能夠確認第二人的身份。然而，目前即使能從粒線體DNA HVI、HVII核酸序列分析的圖譜直接看出是否含有一人以上的混合DNA樣本，但仍無法有效的辨別檢體的來源。本研究利用目前常用的分生技術，建立一套鑑定流程，以解決混合檢體案件中個化分析的困難：先TA cloning將增幅後的粒線體DNA HVI、HVII核酸序列片段個別選殖至大腸桿菌達到株化目的、colony PCR將之各別增幅、結合高分辨熔解曲線（high resolution melting, HRM）screening判定株化核酸片段種類、最後以核酸定序確認，大量的減少人力、物力及時間。此一流程除了可應用於尿液檢體外，將來亦可應用於其他的混合檢體類別，如石蠟包埋組織塊等，對於提昇鑑識科技極有助益。

For forensic individualitation, characteristics of the crime scene evidence should be compared to that of reference samples from the suspects. Modern biotechnologies are commonly applied to prove/rule out the possibility of suspect’s involvement of the case. Analysis of autosomal short tandem repeats (STR) has been the most efficient and effective method for individualized identification. Nonetheless, the evidence samples are usually collected from various sources and uncertain circumstances, while deteriorated samples give rise to severely-degraded autosomal DNA and hamper autosomal STR analysis. As mitochondrial DNAs are rich in cells, analysis of mitochondrial DNA HVI (higher variable region I) and HVII in the control region is applied in alternative in case of encountering rotten samples or samples containing scanty genomic DNA. The mitochondrial DNAs are maternally inherited. Therefore, the matching in mitochondrial DNA confirms only the maternal origins but not the individualization of the DNA donor in the evidence materials, but power of discrimination can still reach 99%. In practice, mitochondrial DNA analysis has been commonly applied in controversial cases in comparing the DNAs collected from the aged urine and the freshly collected buccal epithelium of the drug-abuse suspect who gave a positive result in the urine drug screening. Upon the request of the court, the forensic laboratory has to identify if the aged urine was expelled by the drug-abuse suspect, or, whether the urine is a mixed sample? Sometimes, the second donor in the mixed sample has to be confirmed. Nevertheless, in the case of finding a mixed DNA sequences while performing traditional PCR/DNA sequencing of mitochondrial HVI/HVII sequence, the source of the DNA could not be identified. The objective of this research is to set up a procedure for individualized- identification within the aged and mixed urine samples. In brief, mitochondrial DNA HVI/ HVII sequence were amplified by PCR, followed by TA cloning to incorporate individual DNA fragment into single E. coli. The cloned DNA of the bacterial colonies were then amplified and grouped by high resolution melting (HRM), and verified by DNA sequencing. Screening and auto-grouping of PCR products derived from TA cloning by HRM help to reduce the cost, time, and man-power in handling large amount of colonies. This procedure will be helpful in individualized-identification of the DNA donor in the mixed samples.