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首页> 美国政府科技报告 >DNA Sequence Analysis of Revertants of the 'hisD3052' Allele of 'Salmonella typhimurium' TA98 Using the Polymerase Chain Reaction and Direct Sequencing: Application to 1-Nitropyrene-Induced Revertants
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DNA Sequence Analysis of Revertants of the 'hisD3052' Allele of 'Salmonella typhimurium' TA98 Using the Polymerase Chain Reaction and Direct Sequencing: Application to 1-Nitropyrene-Induced Revertants

机译：使用聚合酶链反应和直接测序法测定'鼠伤寒沙门氏菌'Ta98'hisD3052'等位基因回复体的DNa序列分析：应用于1-硝基芘诱导的回复体

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The study used the polymerase chain reaction (PCR) to speed the processing of revertants of Salmonella typhimurium TA98 for DNA sequence analysis. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 228-bp fragment containing the hisD3052 mutation approximately in the center. Following ultra-filtration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. The deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Misalignment of complementary DNA strands within this repeat may account for this mutation, although the possible formation of Z-DNA within this region may also play a role. Other mutations, mostly deletions but also some complex mutations (insertions/deletions/substitutions), occurred within quasi-palindromic regions of DNA. The potential DNA secondary structures within such regions may mediate the templated production of some of these mutations.

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