烧骨DNA提取与检测技术的实验研究

Experimental study of DNA extraction and detection in burned bone

目的探索烧骨DNA提取与检测方法。方法常压下以200℃分别对30例成人股骨焚烧1h后,生物冷冻研磨,EDTA脱钙,改良酚-氯仿提取DNA,光度计检测DNA浓度和纯度;5色荧光标记,PCR复合扩增,基因分析仪毛细管电泳,软件收集数据和分析。结果改良酚-氯仿法提取烧骨DNA浓度为(28.5±1.7)ng/μl,16个基因位点基本齐全,相对荧光单位(RFU)>500;传统酚-氯仿法提取烧骨DNA浓度为(4.7±0.8)ng/μl,基因位点检出不全,RFU<500。结论改良酚-氯仿法、PCR复合扩增、基因分析能对烧骨DNA进行有效提取和检测,但操作时要严格规程。

Objective To explore how to extract and detect DNA from burned bone.Methods 30 adult femoral bone were incinerated at 200℃ 1 hour in normal pressure,and then freezed and grinded to be bone meal in the liquid nitrogen before decalcification by ethylenediamine tetraacetic acid（EDTA）.The deoxyribonucleic acid（DNA） was extracted by improved phenol-chloroform,and the luminometer was used to detect the concentration and purity of the purified DNA.Subsequently DNA was five-fluorescent labeled,amplified by PCR,followed by capillary electrophoresis and the data analysis.Results DNA concentration was（28.5±1.7） ng/μl under the improved phenol-chloroform method,and the 16 genetic locus were detected,with the relative fluorescence units（RFU） of more than 500.The DNA concentration was about（4.7±0.8） ng /μl,and some gene cound not be detected under the traditional phenol-chloroform extraction.Conclusions The DNA extraction and detection can be performed in burned bone by the improved phenol-chloroform method,multiplex polymerase chain reaction,and gene locus analysis.