DETECTION OF MINORITY POINT MUTATIONS BY MODIFIED PCR TECHNIQUE - A NEW APPROACH FOR A SENSITIVE DIAGNOSIS OF TUMOR-PROGRESSION MARKERS
DETECTION OF MINORITY POINT MUTATIONS BY MODIFIED PCR TECHNIQUE - A NEW APPROACH FOR A SENSITIVE DIAGNOSIS OF TUMOR-PROGRESSION MARKERS
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DOI：
10.1093/nar/17.20.8093
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发表时间：
1989-10-25
影响因子：
14.9
通讯作者：
KITZIS, A
中科院分区：
文献类型：
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作者：
HALIASSOS, A;CHOMEL, JC;KITZIS, A
在酶促扩增的DNA序列（聚合酶链反应）中，与疾病相关的点突变的检测目前通过消化PCR产物（当扩增的突变序列的至少一个等位基因中存在的限制性位点消失时）或在所有其他情况下通过等位基因特异性放射性标记的探针来进行。这些方法是最敏感的，但如果突变存在于少于5%的研究细胞中，则无法检测到突变。我们在这里描述了一种方法的基础上引入一个人工的限制性位点，在PCR过程中使用一个修改的引物，它创建一个RFLP指示研究的突变。该RFLP通过与突变无关的放射性标记寡核苷酸探针检测。我们的方法将灵敏度乘以1000倍，并且对于用于筛查目的和治疗后检测人类恶性肿瘤中的残留疾病是实用的。使用这种方法，我们检测到20%以上的突变，在密码子12 Ki ras癌基因的DNA从结直肠癌是无法检测到的所有以前的方法。
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.