Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
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DOI：
10.1371/journal.pone.0156546
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发表时间：
2016-05-31
期刊：
影响因子：
3.7
通讯作者：
Marras, Salvatore A. E.
中科院分区：
文献类型：
--
作者：
Vargas, Diana Y.;Kramer, Fred Russell;Marras, Salvatore A. E.
我们描述了“超选择性”引物的使用，该引物能够在实时PCR分析中检测和定量与癌症诊断、预后和治疗相关的体细胞突变，可以潜在地分析血液样本（液体活检）中存在的罕见DNA片段。这些脱氧核糖核酸引物的设计既包含一个相对较长的“5’锚定序列”，它与目标DNA片段杂交强烈，也包含一个非常短的、物理上和功能上分离的“3’脚序列”，它与突变目标序列完全互补，但与野生型序列不匹配。即使突变体和野生型之间的差异仅仅是单个核苷酸多态性，在存在1,000,000个野生型片段的情况下，也可以可靠地检测到10个突变片段。多重PCR检测采用一组超选择性引物和相应的一组不同颜色的分子信标探针，可用于不同的突变，虽然发生在不同的细胞，但位于同一密码子的情况。这些非对称实时多重PCR检测包含每个超选择性引物的有限浓度，从而能够通过比较其阈值与样品中存在的参考基因的阈值来同时确定每个突变的丰度。
We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.