用AFLP结合免疫纳米磁珠技术富集分离BPDE-DNA片段

ISOLATION AND ENRICHMENT OF BPDE-DNA FRAGMENT BY AFLP COMBINATION WITH IMMUNONANOMAGNETIC BEADS

目的应用AFLP结合免疫纳米磁珠分离技术富集、分离与BPDE相互作用的DNA片段,从而为筛选BPDE致癌相关基因提供条件。方法应用AFLP技术和免疫纳米磁珠分离相结合技术获得差异片段,进行聚丙烯酰胺凝胶电泳及硝酸银染色观察。结果不同的选择性扩增引物组合均可得到清晰的多态性电泳图谱,以P1T2、P1T4、P1T3、P2T1、P3T4、P4T1、P5T2等组合扩增出的条带最多,均在60条左右,多态性丰富。对差异片段进行回收和PCR再扩增,再经琼脂糖凝胶电泳证实有回收产物出现,回收PCR再扩增片段与回收前的片段大小一致,成功富集、回收了差异片段。结论AFLP结合免疫磁珠分离技术可以分离富集BPDE-DNA片段,可作为筛选BPDE致癌相关的特异或易感基因的一种方法。

Objective To isolate and enrich DNA fragment which are interactive with benzo（a）pyrene dyhydrodiol epoxode（BPDE）,so as to provide the basis for screening carcinogenetic or tumor susceptibility gene related to BPDE.Methods Amplified fragment length polymorphism（AFLP） combined with immunonanomagnetic beads isolation were used to enrich and amplify DNA fragments which are interactive with BPDE.Differential fragments were displayed by polyacrylamide gel electrophoresis and silver nitrate staining.Results Distinct electrophoresis pattern was obtained in all AFLP selective amplification primer pairs.Polymorphism of the bands was abundant in electrophoresis pattern with P1T2,P1T4,P1T3,P2T1,P3T4,P4T1,P5T2 primer pairs.Differential bands were successfully collected,the molecular weight of collected DNA fragments was identical to that of DNA fragments before collection by agarose gel electrophoresis.Conclusion AFLP combined with immunonanomagnetic beads isolation technique can be used to isolate and enrich BPDE-DNA fragment.