Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation.

Abstract

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.