Cebula T A, Koch W H
Division of Microbiology, Food and Drug Administration, Washington, DC 20204.
Mutat Res. 1990 Mar;229(1):79-87. doi: 10.1016/0027-5107(90)90010-2.
An improved DNA colony-hybridization method for the rapid characterization of Salmonella typhimurium hisG46 revertants is described. Oligodeoxyribonucleotides (15-mers) complementary to each of 6 possible transition or transversion mutations and an extragenic suppressor mutation, underlying the His+ phenotype, were prepared. Optimal sequence discrimination was achieved by hybridizing 15-mers at the apparent dissociation temperature (Td) for 2 h with chromosomal DNA of revertant colonies affixed to Whatman 541 filters. Subsequent exposure of filters to UVA radiation (320-400 nm) in the presence of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) resulted in cross-linking of perfectly matched probes and target DNA sequences while sequences containing a single base-pair mismatch could be discriminated with a brief denaturing wash. No false negative results were obtained with the new procedure. An analysis of 204 spontaneous and 174 PUVA-induced TA100 revertants is presented.
本文描述了一种改进的DNA菌落杂交方法，用于快速鉴定鼠伤寒沙门氏菌hisG46回复突变体。制备了与His⁺表型所对应的6种可能的转换或颠换突变以及一种基因外抑制突变各自互补的寡脱氧核糖核苷酸（15聚体）。通过在表观解链温度（Td）下将15聚体与固定在Whatman 541滤膜上的回复突变菌落的染色体DNA杂交2小时，实现了最佳的序列区分。随后，在4'-羟甲基-4,5',8-三甲基补骨脂素（HMT）存在的情况下，将滤膜暴露于UVA辐射（320 - 400 nm），导致完全匹配的探针与靶DNA序列交联，而含有单个碱基对错配的序列可通过短暂的变性洗涤加以区分。新方法未获得假阴性结果。本文还对204个自发和174个PUVA诱导的TA100回复突变体进行了分析。
