Oncogene detection at the single cell level.
Oncogene detection at the single cell level.
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作者：
R. Kumar;M. Barbacid
我们描述了在单细胞水平检测致癌基因以及在正常细胞和肿瘤细胞混合群体中阳性鉴定代表性不足的致癌等位基因的策略。通过将聚合酶链反应 (PCR) 技术与液体杂交和凝胶阻滞测定相结合，我们已经能够检测单个细胞（包括一个受精小鼠卵子）中的 H-ras 序列。我们还描述了 PCR 方案的修改，涉及使用不匹配的引物。该程序允许创建新的限制性片段长度多态性（RFLP）诊断特定点突变。这种实验方法使我们能够在存在 10(5) 个正常细胞的情况下检测单个杂合细胞中的 ras 癌基因。
We describe strategies for the detection of oncogenes at the single-cell level and for the positive identification of under-represented oncogenic alleles in mixed populations of normal and tumor cells. By combining the Polymerase Chain Reaction (PCR) technique with a liquid hybridization and gel retardation assay, we have been able to detect H-ras sequences in single cells, including in one fertilized mouse ovum. We also describe a modification of the PCR protocol involving the use of mismatched primers. This procedure allows for the creation of novel Restriction Fragment Length Polymorphisms (RFLP) diagnostic of specific point mutations. This experimental approach has allowed us to detect ras oncogenes in a single heterozygous cell in the presence of 10(5) normal cells.