基于切口酶的等温全基因组扩增体系的建立与应用

Establishment and application of isothermal whole genome amplification system based on nicking enzyme

目的 建立依赖切口酶Nt.BstNBI的等温全基因组扩增体系,并对该体系的微量生物物证STR分型效能进行验证。方法 选用切口酶Nt.BstNBI(识别序列为GAGTC)和等温扩增的聚合酶Bst,建立切口酶依赖的扩增体系(NDA)。对DNA样本进行NDA并纯化NDA产物,对NDA前后的样本进行复合扩增和毛细管电泳,检测该方法的有效性、灵敏性。结果 007标准品12个常染色体基因座的扩增效率提高2倍以上,灵敏度约为3 pg/μL;19个Y染色体个基因座的扩增效率提高2倍以上,灵敏度约为6 pg/μL。8个人血样本扩增效率提高2倍以上的常染色体基因座与007标准品一致。结论 本文建立的NDA体系准确性好,能够对包含特定侧翼序列的STR基因座进行有效的线性扩增,对法医微量生物物证的高效扩增检测具有重要意义。

Objective To establish an isothermal whole genome amplification system based on the incision enzyme Nt.BstNBI,and to verify the STR typing efficiency of the established system for trace biological evidence.Methods The notch enzyme Nt.BstNBI whose recognition sequence is GAGTC and isothermal polymerase Bst were selected to establish notch enzyme dependent amplification(NDA).DNA samples were prformed NDA and the NDA products were purified.Perform multiplex amplification and capillary electrophoresis were conducted on before and after NDA,in order to test the effectiveness and sensitivity of this method.Results The amplification efficiencies of 12 autosome loci for 007 standard were more than doubled,and the sensitivity was about 3 pg/μL.The amplification efficiencies of 19 Y chromosome loci were more than doubled,and the sensitivity was about 6 pg/μL.The Autosome loci whose amplification efficiency of 8 human blood samples were more than 2 times higher were consistent with the 007 standard.Conclusion The NDA technology established in this article has good accuracy and can effectively linearly amplify STR loci containing specific flanking sequences,which is of great significance for eficient amplification detection of trace biological evidence in the field of forensic science.