摘要
本文报道建立^(32)P后标记方法(^(32)P-Postlabeling Method)以用于检测致癌物-DNA加成物。其基本原理:用核酸酶P1(Nu.P1)完全消化DNA,释出5′单磷酸脱氧核苷(pN),而加成的碱基(Xi),则释出5′磷酸二脱氧核苷(pXipN);经前列腺酸性磷酸酶(PAP)作用后,分别生成N和XipN,然后经T_4多聚核苷酸激酶(PNK)作用,将[γ-^(32)P]ATP的^(32)P转移XipN的5′末端上,生成标记*pXipN而N不被标记。经聚乙烯亚胺(PEI)纤维素薄层纯化、分离和放射自显影,则可确定已加成的碱基,若DNA量已知,则可进行定量分析。在人羊膜上皮细胞FL中,适量苯并(a)芘[B(a)P]处理24b后,可分离到4个加成物斑块,其总相对标记量(Relative Adduct Labeling,RAL)有剂量依赖性关系。
A 32P-postlabeling method that labeles adducted bases specifically are established to detect DNA adducts. In this study, DNA is digested with nuclease P1 and prostatic acid phosphatasc. The normal nucleotides are released as normal nucleosides (N), and the adducted nuclcctidcs are excised as adducted dinucleotides (XipN). The dinucleotides (XipN) but not nuclcosides (N) are converted to 5'-32P-labeled dinucleotides ( pXipN), by T4 polynucleotidc kinasc-catalyzed [32P] phosphate transfer from [y-32P] ATP. Then DNA adducts are purified and separated by thin layer chromatography on polyethylencimine (PEI)-ccllulose sheets, and produced characteristic autoradiographic fingerprints. FL cells are treated with different concentration of benzo(a)pyrene for 24 hours, four adduct spots are found and the total RAL has dose-response relationship.
出处
《癌变·畸变·突变》
CAS
CSCD
1993年第4期30-35,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
省自然科学基金
