【作者】 尚鑫；

【导师】 任晓冬；

【作者基本信息】 吉林大学 ， 生物工程（专业学位）， 2019， 硕士

【摘要】 随着现代科技的不断进步,DNA技术越来越受到重视。作为能够识别锁定嫌疑人的有力工具,DNA检验技术在刑事案件检验中得到广泛应用在重大疑难案件中发挥了重要作用。陈旧生物样本检验是目前DNA检验技术中需解决的难题,在实际工作中我们发现,陈旧生物样本物证经常出现检验位点检出不全,甚至无法得到有效分型的现象。致使一些重大疑难命案积案使破受到影响陈旧生物样本物证因为生物样本浸润到木质纤维素载体里时问较长,不可避免地受外在条件环境中的光、空气、水分等的影响^[1],现有的生物样本处理和DNA提取方法都对于镶嵌在紧密载体内部的生物样本和木质纤维素载体交联的生物样本处理不足,生物样本无法充分释放,对于保存时间长、蛋白变性程度严重,以及与载体交联严重的生物样本的DNA提取都不理想,有时甚至无法获得有效DNA,因而浪费了宝贵的生物样本和现场检材,影响了案件侦破和关键证据的取得。为了从陈年的含有生物样本的木质纤维素类物证检材中提取出DNA生物样本证据并进行检验,本研究利用高温老化的方法对物证进行老化模拟实验,由于在法医学中,木质纤维素物证检材主要分为纸类和棉布类,所以本研究分别针对两类进行了研究。又因DNA检测所需酸碱条件为碱性,故选取了3种在pH值为9的情况下仍有较高的活性的商品酶,利用酶法,即将纤维素酶^[2]、商品纤维素酶A^[3]和商品纤维素酶B分别针对纸类^[4]木质纤维素物证检材和布类木质纤维素物证检材进行复配,使所需得的生物样本DNA从木质纤维素物证检材被降解形成的纤维松散结构中溶出,成功提取生物样本DNA并利用STR-PCR技术进行检测。经过对选取的三种商品酶的酶活测定与蛋白含量测定,降解纸类木质纤维素物证检材最佳酶复配方案为纤维素酶与碱性商品纤维素酶A蛋白含量比为18.7:17.9,商品纤维素酶B的蛋白含量为3.21 mg/ml,商品纤维素酶B溶解入液体酶后离心取上清后,再与pH值为9的Tris-HCI溶液配置成蛋白浓度为1.66 mg/ml的酶液为最终的针对纸类复配纤维素酶。降解布类木质纤维素物证检材最佳酶复配方案为纤维素酶和碱性商品纤维素酶A蛋白含量比为26.18:10.74,固体商品纤维素酶B蛋白含量为3.21 mg/ml时降解棉布释放还原糖量最多,在此复配方案的基础上,再与pH值为9的Tris-HCI溶液配置成蛋白浓度为1.348mg/ml时,降解棉质物证效果最好。为了检验木质纤维素检材的降解效果,利用扫描电子显微镜,观察检材纤维素的损伤情况。结果显示,扫描电子显微镜显示两种复配酶水解对纸类和布类纤维造成了明显的损伤;有效地解决了陈年含有生物样本的木质纤维素类物证检材物证中所需的生物样本DNA提取这一关键问题;并针对几个陈年案例运用复配酶降解的方法处理后,将所得的生物样本DNA进行STR-PCR分型检验,与未用酶法处理的实验组能够得到效果更好的特征峰,即酶法能有效地降解陈年含有生物样本的木质纤维素类物证检材,从而释放出所需的生物样本DNA以供检验。本研究不仅为陈年积案的侦破提供了强有力的手段,也为法医学的进步发展提供了新的思路和方法。更多还原

【Abstract】 With the continuous advancement of modern technology,DNA technology has received more and more attention.As a powerful tool to identify locked suspects.DNA testing technology has been widely used in criminal case testing and played an important role in major difficult cases.Old biological sample testing is a difficult problem to be solved in current DNA testing technology.In actual work,we found that the evidence of old biological samples often showed incomplete detection of the test sites,and even the phenomenon of effective classification could not be obtained.As a result,some major difficult cases have caused the impact of the old biological samples to be affected by the infiltration of biological samples into the lignocellulosic carrier,which is inevitably affected by the light,air and moisture in the external conditions^[1]..Existing biological sample processing and DNA extraction methods are insufficient for the biological samples crosslinked by the biological sample embedded in the compact carrier and the lignocellulosic carrier,and the biological sample cannot be fully released.For DNA samples with long storage times,severe protein denaturation,and severe cross-linking with carriers,DNA extraction is not ideal,and sometimes even effective DNA is not available,thus wasting valuable biological samples and on-site materials,affecting case detection and The acquisition of key evidence.In order to extract and test DNA biosamples from aged samples of lignocellulosic materials containing biological samples.In this study,the aging simulation experiment was carried out on the physical evidence by means of high temperature aging.Since in the forensic science,the lignocellulosic materials are mainly divided into paper and cotton,this study has studied two categories.Since the acid-base conditions required for DNA detection were alkaline,three commercial enzymes having high activity at pH 9 were selected.Enzymatic method,that is,cellulase^[2],Commercial Cellulase A^[3]and Commercial Cellulase B are respectively compounded on paper^[4]lignocellulosic materials and cloth lignocellulosic materials.The required biological sample DNA is eluted from the loose structure of the fiber formed by degradation of the lignocellulosic material,and the biological sample DNA is successfully extracted and detected by STR-PCR technology.After the enzyme activity measurement and protein content determination of the selected three commercial enzymes,the optimal enzyme compounding scheme for degrading paper lignocellulosic materials is the ratio of cellulase to alkaline Commercial Cellulase A protein of18.7:17.9.The protein content of Commercial Cellulase B was 3.21 mg/ml,and the Commercial Cellulase B was dissolved in liquid enzyme,and the supernatant was centrifuged,and then the enzyme having a protein concentration of 1.66 mg/ml was prepared with a pH of 9 Tris-HCI solution.The liquid is the final cellulase for the paper.The optimal enzyme compounding scheme for degradable fabric lignocellulosic materials is that the ratio of cellulase to alkaline Commercial Cellulase A protein is 26.18:10.74,and the content of solid Commercial Cellulase B protein is 3.21 mg/ml.The sugar content was the highest.On the basis of this compounding scheme,when the protein concentration was 1.348mg/ml with the pH 9 Tris-HCI solution,the degradation of cotton material was the best.In order to examine the degradation effect of the lignocellulosic material,the damage of the cellulose of the sample was observed by a scanning electron microscope.The results showed that scanning electron microscopy showed that the hydrolysis of two kinds of compound enzymes caused obvious damage to paper and cloth fibers;it effectively solved the biological sample DNA required for the evidence of lignocellulosic materials containing biological samples.Extracting this key problem;and using the method of compound enzyme degradation for several old cases,the obtained biological sample DNA was subjected to STR-PCR typing test,and the experimental group not treated with enzymatic method could obtain more effects.A good characteristic peak,that is,an enzymatic method,can effectively degrade a lignocellulosic material test sample containing an old biological sample,thereby releasing the desired biological sample DNA for testing.This research not only provides a powerful means for the detection of old-age accumulative cases,but also provides new ideas and methods for the progressive development of forensic science.更多还原