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首页> 美国卫生研究院文献>Nucleic Acids Research >Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers.
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Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers.

机译：通过改进的PCR技术检测少数族裔点突变：一种灵敏诊断肿瘤进展标记物的新方法。

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The detection of point mutations correlated with diseases, in enzymatically amplified DNA sequences (Polymerase Chain Reaction), is currently performed by digestion of PCR products when an existing restriction site disappears at least in one allele of the amplified mutated sequence or by allele specific radiolabeled probes in all other cases. These methods are the most sensitive but they cannot detect a mutation if it is present in less than 5% of the studied cells. We describe here a method based on the introduction of an artificial restriction site, using a modified primer during the PCR, which creates a RFLP indicative of the studied mutation. This RFLP is detected by a radiolabeled oligonucleotide probe which is not related to the mutation. Our approach multiplies the sensitivity by a factor of 1000 and it is practical for use in screening purposes and the detection, after treatment, of the residual disease in human malignancies. Using this method we detected 20% more mutations at codon 12 in the Ki ras oncogene in DNA from colorectal cancers that were undetectable with all the previous methods.
机译：当前，当存在的限制性酶切位点至少在扩增的突变序列的一个等位基因中消失时，或者通过等位基因特异性的放射性标记探针，通过消化PCR产物来检测酶促DNA序列中与疾病相关的点突变（聚合酶链反应）在所有其他情况下。这些方法是最敏感的，但如果少于5％的研究细胞中存在突变，它们将无法检测到突变。我们在此描述一种基于人工限制性位点的引入的方法，该方法在PCR期间使用修饰的引物，该引物产生了表示研究突变的RFLP。用与突变无关的放射性标记的寡核苷酸探针检测该RFLP。我们的方法将灵敏度提高了1000倍，非常适合用于筛查目的以及治疗后检测人类恶性肿瘤中的残留疾病。使用这种方法，我们检测到来自大肠癌的DNA的Ki ras癌基因中第12位密码子的突变多20％，而这些突变在以前的方法中都是无法检测到的。

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