Olivas W M, Maher L J
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha.
Biotechniques. 1994 Jan;16(1):128-32.
Conventional methods for mutation detection include Southern hybridization, direct sequencing of PCR products and single-strand conformation polymorphism analysis. We present an additional screening method that employs oligonucleotide-directed DNA triple helix formation to detect mutations within homopurine sequences. The proposed strategy is simple and may be of particular value when screening many DNA samples for changes involving particular homopurine sites. We have applied the method to the analysis of a clinically relevant 8-bp micro-deletion in the human p53 tumor suppressor gene. Affinities of oligonucleotide probes toward radiolabeled wild-type and mutant p53 DNA duplexes were quantitated by electrophoretic mobility shift assays. Recombinant plasmids carrying wild-type or microdeleted forms of the p53 homopurine sites of interest were created. Dimethyl sulfate footprinting was used to verify intended probe specificities. Duplex PCR products amplified from plasmid constructs were directly probed by incubation with labeled oligonucleotides. After electrophoresis and autoradiography, patterns of triple helix formation allowed discrimination between the mutant and wild-type p53 sequences. Direct DNA analysis by triple helix formation may simplify other procedures that normally require DNA denaturation and hybridization.
传统的突变检测方法包括Southern杂交、PCR产物直接测序和单链构象多态性分析。我们提出了一种额外的筛选方法，该方法利用寡核苷酸导向的DNA三链螺旋形成来检测同型嘌呤序列内的突变。所提出的策略很简单，在筛选许多DNA样本以寻找涉及特定同型嘌呤位点的变化时可能具有特殊价值。我们已将该方法应用于分析人类p53肿瘤抑制基因中一个临床相关的8碱基对微缺失。通过电泳迁移率变动分析对寡核苷酸探针与放射性标记的野生型和突变型p53 DNA双链体的亲和力进行了定量。构建了携带感兴趣的p53同型嘌呤位点野生型或微缺失形式的重组质粒。使用硫酸二甲酯足迹法验证预期的探针特异性。从质粒构建体扩增的双链PCR产物通过与标记的寡核苷酸孵育直接进行探针检测。经过电泳和放射自显影后，三链螺旋形成模式能够区分突变型和野生型p53序列。通过三链螺旋形成进行直接DNA分析可能会简化其他通常需要DNA变性和杂交的程序。
