Orita M, Suzuki Y, Sekiya T, Hayashi K
Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.
Genomics. 1989 Nov;5(4):874-9. doi: 10.1016/0888-7543(89)90129-8.
We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.
我们报道了一种用于检测基因组DNA特定序列中碱基变化的快速且灵敏的方法。该技术基于以下事实：通过在聚合酶链反应中使用标记底物，基因组序列的特定区域可同时被有效标记和扩增；并且在非变性聚丙烯酰胺凝胶中，单链核酸的电泳迁移率不仅取决于其大小，还取决于其序列。该过程不涉及限制性酶切、印迹或与探针杂交。我们发现，长度达200个碱基的片段中，大多数单碱基变化都可作为迁移率变化被检测到。通过该技术检测到了RAS癌基因的激活。我们还表明，人类散布的重复序列，即Alu重复序列具有高度多态性。
