Albitar Adam Z, Ma Wanlong, Albitar Maher
NeoGenomics Laboratories.
NeoGenomics Laboratories;
J Vis Exp. 2017 Mar 29(121):55130. doi: 10.3791/55130.
Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.
在评估治疗后的残留疾病、在治疗期间筛查新出现的耐药突变或患者循环肿瘤细胞较少时，准确检测和鉴定低频突变可能会出现问题。野生型阻断PCR随后进行测序分析，作为检测这些低频突变的方法，具有高灵敏度、灵活性和简便性。通过在新的或先前建立的基于常规PCR的测序检测中添加定制设计的锁核酸寡核苷酸，在1000个野生型等位基因的背景下，可实现约1个突变等位基因的灵敏度（1:1000）。在提取步骤中使用尿嘧啶DNA糖基化酶，可以部分纠正与福尔马林固定石蜡包埋组织中常见的脱氨基事件相关的测序假象。这里优化的方案专门用于检测MYD88突变，但可作为设计任何野生型阻断PCR检测的模板。与其他常用的检测低频突变的方法（包括等位基因特异性PCR和实时定量PCR）相比，野生型阻断PCR检测的优点包括假阳性出现次数更少、灵活性更高且易于实施，以及能够检测已知和未知突变。
