Allison D P, Kerper P S, Doktycz M J, Spain J A, Modrich P, Larimer F W, Thundat T, Warmack R J
Health Sciences Research Division, Oak Ridge National Laboratory, TN 37831-6123, USA.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8826-9. doi: 10.1073/pnas.93.17.8826.
Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.
利用原子力显微镜进行直接成像已被用于识别质粒DNA分子中的特定核苷酸序列。这是通过使用EcoRI（Gln-111）实现的，它是一种限制性内切酶的突变体，其结合亲和力比野生型酶高1000倍，但切割速率常数降低了10^4倍。对具有单个（pBS+）和双个（pGEM-luc和pSV-β-半乳糖苷酶）EcoRI识别位点的ScaI线性化质粒进行了成像，并且将结合的酶定位到50至100个核苷酸的分辨率。这种突变型内切核酸酶对EcoRI结合位点表现出的高亲和力，再加上观察到的低水平非特异性结合，对于通过直接原子力显微镜成像对大型DNA克隆进行物理图谱绘制应该是有价值的。
