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Updated: Apr 24, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique
Feifei Xie1, Jie Huang2, Shoufang Qu2
1Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
Objectives:
The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing.
Design And Methods:
We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods.
Results:
The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing.
Conclusions:
Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.
