Griep M A
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Anal Biochem. 1995 Dec 10;232(2):180-9. doi: 10.1006/abio.1995.0005.
A continuous assay was developed for processive DNA polymerases. The specific enzyme used to develop the assay was the most processive polymerase known, Escherichia coli DNA polymerase III holoenzyme. The assay was based upon the recovery of the intrinsic fluorescence of single-stranded DNA binding protein (SSB) as it was displaced from the DNA template during DNA synthesis. The intrinsic fluorescence of SSB was quenched by as much as 80% when it bound to single-stranded DNA. As the DNA was replicated, SSB was displaced and recovered its fluorescence. The amount of fluorescence recovered was directly proportional to the amount of DNA synthesized and was used to quantitate the rate of DNA synthesis. However, since 50 to 60 nucleotides must be replicated for every SSB tetramer released, the assay is expected to work best for processive DNA polymerases. The only requirement for using this assay with other DNA polymerases is that they be able to synthesize DNA on a template coated with SSB. The replication SSBs do not pose an obstacle to the assay because they all appear to have intrinsic fluorescence that is sensitive to their ssDNA-bound state.
开发了一种用于持续性DNA聚合酶的连续测定法。用于开发该测定法的特定酶是已知的持续性最强的聚合酶，即大肠杆菌DNA聚合酶III全酶。该测定法基于单链DNA结合蛋白（SSB）在DNA合成过程中从DNA模板上被置换时其固有荧光的恢复。当SSB与单链DNA结合时，其固有荧光被淬灭多达80%。随着DNA的复制，SSB被置换并恢复其荧光。恢复的荧光量与合成的DNA量直接成正比，并用于定量DNA合成速率。然而，由于每释放一个SSB四聚体必须复制50至60个核苷酸，因此该测定法预计对持续性DNA聚合酶效果最佳。将该测定法用于其他DNA聚合酶的唯一要求是它们能够在涂有SSB的模板上合成DNA。复制型SSB对该测定法不构成障碍，因为它们似乎都具有对其单链DNA结合状态敏感的固有荧光。
