Tabone Tania, Sallmann Georgina, Chiotis Maria, Law Matthew, Cotton Richard
Genomic Disorders Research Centre, St Vincent's Hospital, PO Box 2900, Victoria 3065, Australia.
Nat Protoc. 2006;1(5):2297-304. doi: 10.1038/nprot.2006.352.
This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.
本方案描述了错配化学切割（CCM）方法的使用，以评估DNA区域是否含有突变并对其进行定位。与其他检测短DNA片段中突变且需要高度特异性解链温度的突变检测技术（如单链构象多态性（SSCP）分析、变性高效液相色谱（DHPLC）和变性梯度凝胶电泳（DGGE））相比，CCM具有更高的诊断灵敏度，适用于肿瘤基因中突变的检测，并且可以分析长度小于或等于2 kb的扩增子。为了检测突变，将PCR异源双链体与两种错配特异性试剂一起孵育。羟胺修饰未配对的胞嘧啶，高锰酸钾修饰未配对的胸腺嘧啶。然后将样品与哌啶一起孵育，哌啶在修饰的错配碱基位点切割DNA主链。切割产物通过电泳分离，揭示突变的身份和位置。CCM方法可以有效地检测点突变以及插入和缺失。本方案可在10小时内完成。
