Mutant Enrichment with 3′-Modified Oligonucleotides A Practical PCR Method for Detecting Trace Mutant DNAs
Mutant Enrichment with 3′-Modified Oligonucleotides A Practical PCR Method for Detecting Trace Mutant DNAs
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DOI：
10.1016/j.jmoldx.2011.07.003
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发表时间：
2011-11-01
影响因子：
4.1
通讯作者：
Ki, Chang-Seok
中科院分区：
文献类型：
--
作者：
Lee, Seung-Tae;Kim, Ji-Youn;Ki, Chang-Seok
许多临床情况需要高灵敏度和可靠的分子测定;然而，由于分子检测方法的固有局限性，实现此类测定仍然是一个挑战。在这里，我们描述了一种简单而廉价的富集技术，我们称之为突变富集与3 '-修饰的寡核苷酸（MEMO）。该方法基于使用3 '-修饰的寡核苷酸引物，其阻断正常等位基因的延伸，但能够延伸突变的等位基因。评价了该技术检测EGFR、KRAS、BRAE、TP 53、JAK 2和NPM 1基因中常见癌症突变的能力。我们使用下游桑格测序实现了10（-2）到10（-6）的灵敏度，这取决于引物的浓度和热力学。MEMO可适用于定量实时PCR平台和其他下游测定。该技术可实际应用于各种医疗情况。（J Mol Diagn 2011，13：657-664. DOI：10.1016/j.jmoldx.2011.07.003）
Many clinical situations necessitate highly sensitive and reliable molecular assays; however, the achievement of such assays remains a challenge due to the inherent limitations of molecular testing methods. Here, we describe a simple and inexpensive enrichment technique that we call mutant enrichment with 3'-modified oligonucleotides (MEMO). The method is based on the use of a 3'-modified oligonucleotide primer that blocks extension of the normal allele but enables extension of the mutated allele. The performance of the technique was evaluated with respect to its ability to detect common cancer mutations in the EGFR, KRAS, BRAE, TP53, JAK2, and NPM1 genes. We achieved sensitivities of 10(-2) to 10(-6) using downstream Sanger sequencing, depending on the concentrations and thermodynamics of the primers. MEMO may be applicable to the quantitative real-time PCR platform and other downstream assays. This technique may be practically applicable to various medical situations. (J Mol Diagn 2011, 13:657-664. DOI: 10.1016/j.jmoldx.2011.07.003)