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摘要


福馬林為含有40%之甲醛水溶液,為殺菌劑及防腐液來保存生物標本,若經福馬林固定後即會破壞其DNA,影響組織(formalin-fixed tissues)及石蠟包埋之蠟塊組織(formalin-fixed, paraffin-embedded tissues)的DNA品質,其DNA受損程度視組織不同而有差異。福馬林造成DNA裂解的作用主要使蛋白質與蛋白質形成交錯(cross-linking)及蛋白質與DNA形成交錯,或使核酸鹼基彼此形成交錯,經福馬林固定之組織因DNA裂解導致長片段DNA不易複製,例如長片段之STR基因座。本研究目標在於以複合式PCR評估福馬林對組織DNA裂解之影響及STR型別研判之影響。研究方法採取新鮮心肌及主動脈血管以一般福馬林(un-buffered formalin)及磷酸緩衝之中性福馬林(phosphate buffered formalin)分別固定作用1天、2天、3天、1週、2週及4週,採用ABI AmpF1STR(上标 ®)Identifiler(上标 TM)及ABI AmpF1STR(上标 ®) Minifiler(上标 TM)套組分析其STR型別。另分析石蠟包埋組織案例之STR型別及粒線體DNA序列。研究結果顯示隨福馬林固定時間愈久,其DNA萃取量愈少,經一般福馬林固定組織之DNA萃取量少於中性福馬林固定組織之DNA萃取量,經一般福馬林固定4週之心肌組織DNA萃取量少於經固定1週之心肌組織DNA萃取量。另外,石蠟包埋組織之STR 15型出現長片段與短片段型別分佈不平衡現象,使用Minifiler(上标 TM)套組可提升長片段之STR型別檢出率。依研究結果歸納結論:(1)福馬林固定愈久,DNA裂解愈嚴重,DNA含量減少。(2)一般福馬林較中性福馬林易對組織內之DNA產生裂解。(3)彈力纖維可能與主動脈血管較耐於福馬林浸潤有關。(4)蠟包埋組織是可供人身鑑定及DNA鑑定選擇之檢體。

並列摘要


Formalin is a 40% aqueous solution of formaldehyde. It is used as a disinfectant and also a preservative for biological specimens. Tissue sample fixed with formalin adversely affects DNA, and the quality of DNA in formalin-fixed, paraffin-embedded (FFPE) tissues varies from sample to sample. Formaldehyde fixes tissues by irreversibly cross-linking primary amino groups in proteins with nearby nitrogen atoms in protein or DNA through a methylene (CH2) linkage. It is difficult to amplify longer segments such as the larger STR loci from fixed tissues due to DNA fragmentation, and the intensity of DNA degradation depends on the fixative and the fixation time. The goal of this study was to conduct an assessment of DNA derived from formalin-fixed tissues and FFPE tissues for DNA typing using multiplex PCR. Cardiac muscle and aorta from fresh remains were fixed with un-buffered formalin or 4% phosphate buffered formalin for a time period ranging from 1 day to 4 weeks. AmpFlSTR(superscript ®) Identifiler(superscript TM) and AmpFSlTR(superscript ®) MiniFiler(superscript TM) PCR amplification kits developed by Applied Biosystems were used to obtain DNA profiles from fixed tissue samples. STR typing and the mtDNA D-loop sequencing were also performed from three FFPE tissues. The results showed that the DNA recovery rate was inversely proportional to the fixation time. In addition, the amount of DNA extracted from tissues fixed with un-buffered formalin was less than that fixed with phosphate buffered formalin; the amount of DNA extracted from cardiac muscle fixed with un-buffered formalin for 4 weeks was less than that for 1 week. Imbalanced peaks could be seen among the 15 STR loci from FFPE tissues, and AmpFlSTR(superscript ®) MiniFiler(superscript TM) PCR amplification kit could employed to improve the recovery of larger STR loci. Based on our research findings, the conclusions are as follows: (1) The amount of DNA was decreasing with longer fixation time. (2) The DNA degradation rate of tissues fixed with un-buffered formalin was higher than that fixed with phosphate buffered formalin. (3) The biomechanical properties of elastic matrix in aorta may resist formalin penetration. (4) STR analysis is capable of typing FFPE tissue samples, and it can be used for human identification and other forensic applications.

參考文獻


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Hill, CR,Kline, MC,Mulero, JJ(2007).Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits.J Forensic Sci.52(4),870-3.

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