Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Design and Synthesis of Oligonucleotides for Soo-PCR
2.2. Screening Various Taq DNA Polymerases for Soo-PCR Primers with a 3′-End Tailing Structure
2.3. Detection of Low-Abundance SNVs (KRAS G12D, EGFR L858R, and EGFR T790M)
3. Results and Discussion
3.1. Schematic Overview of Soo-PCR
3.2. Screening Taq DNA Polymerases for High Specificity
3.3. Optimization of 3′-End Tailing Length of Soo-PCR Primer for Major Cancer Markers
3.4. Validation of Soo-PCR Sensitivity and Specificity for Low-Abundance SNVs
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Taq DNA Polymerase | PCR Cycling Conditions (°C/Times) | ||||
---|---|---|---|---|---|
Initial Denaturation | 70 Cycles | Final Extension | |||
Denaturation | Annealing | Extension | |||
Ampli Taq | 95/10 min | 95/30 s | 56/30 s | 72/15 s | 72/7 min |
Hot Start Taq | 95/30 s | 95/30 s | 56/30 s | 68/15 s | 68/5 min |
EzWay | 95/15 min | 94/30 s | 56/30 s | 72/15 s | 72/10 min |
Dream Taq | 95/3 min | 95/30 s | 61/30 s | 72/1 min | 72/7 min |
Go Taq | 95/2 min | 95/30 s | 56/30 s | 72/15 s | 72/5 min |
Taq DNA Polymerase | ΔCtMP-PMP | ||||
---|---|---|---|---|---|
Length of 3′-End Tailing Structure (nt) | |||||
0 | 1 | 2 | 3 | 4 | |
Ampli Taq | 0.6 | 9.4 | −45 ** | * | * |
Hot Start Taq | 0.3 | 9.3 | −53.4 ** | * | * |
EzWay | 0.5 | 9.1 | * | * | * |
DreamTaq | 0.6 | 7.1 | 17.4 | * | * |
GoTaq | 0.4 | 8.7 | * | * | * |
Target | ΔCtMP-PMP | ||||
---|---|---|---|---|---|
Length of 3′-End Tailing Structure (nt) | |||||
0 | 1 | 2 | 3 | 4 | |
KRAS G12D | 0.4 | 9.1 | −41.3 ** | * | * |
EGFR L858R | 4.9 | 12.2 | −42.6 ** | * | * |
EGFR T790M | 2.5 | 12.3 | −57.6 ** | * | * |
Horizon cfDNA (HD780) | Droplet Digital PCR | Soo-PCR | ||||||
---|---|---|---|---|---|---|---|---|
Gene | Variant | Copy Number | Expected AF (%) | Actual AF (%) | On/Off | Ct Value | ||
WT | MT | Total | ||||||
100% Wild Type | ||||||||
KRAS G12D | 14,995 | 5 | 15,000 | 0.00 | 0.02 | Off | No amplification | |
EGFR L858R | 15,000 | 0 | 15,000 | 0.00 | 0.00 | Off | No amplification | |
EGFR T790M | 14,993 | 7 | 15,000 | 0.00 | 0.05 | Off | No amplification | |
0.1% Allele Frequency | ||||||||
KRAS G12D | 14,980 | 20 | 15,000 | 0.13 | 0.13 | On | 65.3 | |
EGFR L858R | 14,983 | 17 | 15,000 | 0.10 | 0.11 | On | 68.9 | |
EGFR T790M | 14,980 | 20 | 15,000 | 0.10 | 0.12 | On | 65.7 | |
1% Allele Frequency | ||||||||
KRAS G12D | 14,822 | 178 | 15,000 | 1.30 | 1.17 | On | 63.2 | |
EGFR L858R | 14,870 | 130 | 15,000 | 1.00 | 0.87 | On | 52.8 | |
EGFR T790M | 14,878 | 122 | 15,000 | 1.00 | 0.81 | On | 61.1 | |
5% Allele Frequency | ||||||||
KRAS G12D | 14,094 | 906 | 15,000 | 6.30 | 6.03 | On | 53.4 | |
EGFR L858R | 14,337 | 663 | 15,000 | 5.00 | 4.43 | On | 47.9 | |
EGFR T790M | 14,364 | 636 | 15,000 | 5.00 | 4.24 | On | 47.3 |
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Shin, J.; Jung, C. Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers. Biosensors 2023, 13, 380. https://doi.org/10.3390/bios13030380
Shin J, Jung C. Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers. Biosensors. 2023; 13(3):380. https://doi.org/10.3390/bios13030380
Chicago/Turabian StyleShin, Juny, and Cheulhee Jung. 2023. "Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers" Biosensors 13, no. 3: 380. https://doi.org/10.3390/bios13030380
APA StyleShin, J., & Jung, C. (2023). Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers. Biosensors, 13(3), 380. https://doi.org/10.3390/bios13030380