检测脱氨基速率的超灵敏方法及应用

A Sensitive Assay for the Detection of Cytosine Deamination Rate and Its Application

脱氨基被认为是引起细胞突变的主要因素 ,如果这些脱氨基的产物不被修复 ,将引起转换(transition)突变 .为了理解DNA结构和其化学活性的关系 ,介绍一种新的灵敏的遗传学方法 ,它应用在DNA特定点的脱氨基速率的测定 .这种方法基于M 1 3mp2噬菌体内的 1acZα基因中的CCC脯氨酸密码子的反转突变 ,即每个脱氨基事件表现为在白色菌斑背景中的一个蓝色菌斑 ,其灵敏度可达1 0 5M1 3mp2DNA分子中检验出一个脱氨基事件 .此外 ,该法可以计算脱氨基动力学速率常数和反应活化能 .

Deamination of bases is regarded as the major factor for mutation in DNA and it will induce transition mutation if the products of deamination have not heen repaired. Now a new sensitive genetic assay to determine quanti- tatively deamination of bases in DNA special site has been introduced to understand the relationship between DNA structure and its chemical activities. The assay is based on reversion of a mutant of CCC proline coding sequence which is located in Lac Z α gene of bacteriophage M13mp2 from a colorless to blue plaque phenotype. This approach is highly sensitive; deamination of a single cytosine residue in 105 M13mp2 DNA molecules can be detected. Besides, the kinetic rate constant of the reaction and the activation energy of deamination can be calculated.