SuperSelective primer pairs for sensitive detection of rare somatic mutations.
SuperSelective primer pairs for sensitive detection of rare somatic mutations.
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DOI：
10.1038/s41598-021-00920-4
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发表时间：
2021-11-17
影响因子：
4.6
通讯作者：
Vargas DY
中科院分区：
文献类型：
--
作者：
Kramer FR;Vargas DY
SuperSelective 引物凭借其独特的设计，能够在大量密切相关的野生型 DNA 片段存在的情况下，选择性指数扩增含有体细胞突变的稀有 DNA 片段。然而，当超选择性引物与常规反向引物结合使用时，丰富的野生型片段会发生线性扩增，这可能会导致延迟出现的信号，该信号可能与来自稀有突变片段的延迟出现的信号相混淆。我们发现，使用一对超选择性引物，一个对正链中的目标突变特异，另一个对互补负链中的相同突变特异，但两者都具有与突变互补的3'端核苷酸，显着抑制相关野生型序列的线性扩增，并防止由于DNA聚合酶的错误掺入而产生假突变序列。因此，样品中不存在突变片段不会产生假阳性信号，并且样品中突变片段的存在可以清楚地区分为真阳性信号。超选择性引物对的使用应增强多重 PCR 检测的灵敏度，该检测可识别和定量从癌症患者获得的液体活检中的体细胞突变，从而能够选择靶向治疗、确定其随时间的有效性，并在出现新突变时更换更合适的治疗。
SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.
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影响因子：
3.7
作者：
Vargas, Diana Y.;Kramer, Fred Russell;Marras, Salvatore A. E.
通讯作者：
Marras, Salvatore A. E.
影响因子：
50.3
作者：
Bardelli, Alberto;Pantel, Klaus
通讯作者：
Pantel, Klaus
影响因子：
4.1
作者：
Vargas, Diana Y.;Marras, Salvatore A. E.;Kramer, Fred R.
通讯作者：
Kramer, Fred R.
DOI：
10.1073/pnas.0501946102
发表时间：
2005-06-14
影响因子：
11.1
作者：
Pierce, KE;Sanchez, JA;Wangh, LJ
通讯作者：
Wangh, LJ
DOI：
10.1038/nbt0993-1026
发表时间：
1993-09-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
HIGUCHI, R;FOCKLER, C;WATSON, R
通讯作者：
WATSON, R