TY - JOUR
T1 - Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing
AU - Zhang, Kerou
AU - Song, Ping
AU - Dai, Peng
AU - Zhang, Jinny Xuemeng
AU - Wu, Lucia Ruojia
AU - Cheng, Lauren Yuxuan
AU - Pinto, Alessandro
AU - Kwong, Lawrence
AU - Cabrera, Karina
AU - Wen, Ruoxin
AU - Zhang, David Yu
N1 - Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/4/22
Y1 - 2022/4/22
N2 - Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3′ chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3′ end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.
AB - Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3′ chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3′ end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.
KW - 3′ chemistry-free modification
KW - blocking modification
KW - polymerase chain reaction
KW - secondary structure
KW - variant enrichment
UR - http://www.scopus.com/inward/record.url?scp=85128797544&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85128797544&partnerID=8YFLogxK
U2 - 10.1021/acssensors.2c00183
DO - 10.1021/acssensors.2c00183
M3 - Article
C2 - 35418222
AN - SCOPUS:85128797544
SN - 2379-3694
VL - 7
SP - 1165
EP - 1174
JO - ACS Sensors
JF - ACS Sensors
IS - 4
ER -