Accurate genotyping of fragmented DNA using a toehold assisted padlock probe
2021
Gao, Yanmin | Qiao, Hongyan | Pan, Victor | Wang, Zhaoguan | Li, Jiaojiao | Wei, Yanan | Ke, Yonggang | Qi, Hao
Fragmented DNA from blood plasma, i.e., cell-free DNA, has received great interest as a noninvasive diagnostic biomarker for “point-of-care” testing or liquid biopsy. Here, we present a new approach for accurate genotyping of highly fragmented DNA. Based on toehold-mediated strand displacement, a toehold-assisted padlock probe and toehold blocker were designed and demonstrated with new controllability in significantly suppressing undesired cross-reaction, promoting target recycling and point mutation detection by tuning the thermodynamic properties. Furthermore, toehold-assisted padlock probe systems were elaborately designed for 14 different single-nucleotide variants (SNVs) and were demonstrated to be able to detect low concentration of variant alleles (0.1%). In addition, a target, spanning a narrow sequence window of 29 nucleotides on average is sufficient for the toehold-assisted padlock probe system, which is valuable for the analysis of highly fragmented DNA molecules from clinical samples. We further demonstrated that the toehold-assisted padlock probe, in combination with a unique asymmetric PCR technique, could detect more target SNVs at low allele fractions (1%) in highly fragmented cfDNA. This allows accurate genotyping and provides a new commercial approach for high-resolution analysis of genetic variation.
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