Kindstar Global Technology Inc.;
Yongzhou Central Hospital.
J Vis Exp. 2024 Aug 23(210). doi: 10.3791/65647.
When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.
在肿瘤治疗后监测微小残留病灶（MRD）时，与在治疗过程中检测耐药突变和循环肿瘤细胞突变相比，对检测下限的要求更高。传统的 Sanger 测序的野生型突变检测率为 5%-20%，因此其检测下限无法满足相应的要求。为了克服这一问题，已经报道了一些野生型阻断技术，包括阻断物置换扩增（BDA）、不可延伸的锁核酸（LNA）、热点特异性探针（HSSP）等。这些技术使用特定的寡核苷酸序列来阻断野生型或识别野生型，然后将其与其他方法结合，防止野生型扩增和突变扩增，从而具有高灵敏度、灵活性和便利性等特点。本方案使用 BDA，一种与 Sanger 测序相结合的野生型阻断 PCR，来优化 RHOA G17V 低频体细胞突变的检测，检测灵敏度可达 0.5%，可为血管免疫母细胞性 T 细胞淋巴瘤的 MRD 监测提供依据。
