Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers.

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5' unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine.

Highly Sensitive Enrichment of Low-Frequency Variants by Hairpin Competition Amplification.

Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.

Feasibility of Methylated CLIP4 in Stool for Early Detection of Colorectal Cancer: A Training Study in Chinese Population.

BACKGROUND: Early detection of colorectal cancer (CRC) and precancerous lesion is vitally important for mitigating CRC morbidity and mortality. Aberrant DNA methylations in certain promoter regions have been identified to be closely associated with CRC development and progression, suggesting their potential as diagnostic biomarkers for early detection. In this study, we evaluated the performance of methylated CLIP4 in stool specimens as a potential biomarker for CRC detection. METHODS: A total of 321 subjects out of 365 enrolled participants were included in the final analysis, including 154 CRC patients, 23 advanced adenoma (AA) patients, 49 small polyp (SP) patients, and 95 healthy controls. CLIP4 methylation level was examined by qPCR with bisulfite converted DNA purified from approximately 5 g stool specimen. RESULTS: Methylated CLIP4 test showed high sensitivities of 78.3% (95% CI: 55.8%-91.7%) and 90.3% (95% CI: 84.2%-94.3%) for detecting AA and CRC, respectively, with a specificity of 88.4% (95% CI: 79.8%-93.8%). CLIP4 methylation level discriminated AA and CRC patients from control subjects with area under the curve values of 0.892 (95% CI: 0.795-0.988) and 0.961 (95% CI: 0.938-0.983). Further analysis indicated no significant difference in sensitivities among different ages, genders, stages, locations, sides, tumor sizes and differentiation statuses. CONCLUSIONS: Methylated CLIP4 showed a strong potential as a noninvasive biomarker for early CRC detection.

Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures.

Intramolecular secondary structures within templates have been shown to lower PCR performance. Whereas many approaches have been developed to mitigate such impairment on PCR, their effects can vary greatly depending on template sequences. Here we present a novel, universally effective approach to improve PCR performance involving specifically designed oligonucleotides called disruptors. A disruptor contained three functional components, an anchor designed to initiate template binding, an effector to disrupt intramolecular secondary structure, and a 3' blocker to prevent its elongation by DNA polymerase. A functional mechanism for a disruptor to improve PCR efficiency was proposed where anchor first binds to template followed by effector-mediated strand displacement to unwind intramolecular secondary structure. Such a mechanism was consistent with the observation that anchor played a more critical role for disruptor function. As an example of potential disruptor applications, inverted terminal repeat sequences of recombinant adeno-associated virus vectors were successfully amplified in the presence of disruptors despite their well-known reputation as some of the most difficult templates for PCR amplification and Sanger sequencing due to their ultra-stable T-shaped hairpin structures. In stark contrast, both DMSO and betaine, two PCR additives routinely used to facilitate PCR amplification and Sanger sequencing of GC-rich templates, did not demonstrate any improving effect.

Stability, integrity, and recovery rate of cellular nucleic acids preserved in a new liquid-based cytology medium.

BACKGROUND: Liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening. The cervical swab specimens collected in LBC media can also be used for additional analyses including high-risk HPV (HR-HPV) test, DNA methylation analysis, and HPV E6/E7 mRNA test. METHODS: The stability, integrity, and recovery rate of cellular DNA and RNA after storage at different conditions were evaluated by a quantitative real-time PCR (qPCR) based HR-HPV test, reverse transcription qPCR (RT-qPCR), and agarose gel electrophoresis. Cervical swab specimens collected in a newly developed LBC medium, VersaMedium, and ThinPrep PreservCyt medium were processed on Hologic ThinPrep 5000 instrument. RESULTS: Cervical exfoliative cells fixed by VersaMedium exhibited good cellular morphology with intact membranes and delineated chromatin structures. Cellular DNA preserved in VersaMedium exhibited high level of stability at both room temperature and 4°C, and remained mostly intact at 4°C for up to 28 days. Cellular RNA preserved in VersaMedium maintained higher level of stability and integrity at 4°C than at room temperature. VersaMedium also showed no apparent adverse effect on the recovery rate of nucleic acids. CONCLUSION: In addition to maintaining cellular morphology, when stored at 4°C, VersaMedium preserves cellular nucleic acids and PreservCyt medium without noticeable adverse effects on the recovery rate during purification. Therefore, VersaMedium is an appropriate LBC medium for the collection and preservation of cervical swab specimens. And VersaMedium preserved cellular nucleic acids are of such high quality that they are suitable for HR-HPV qPCR test and RT-qPCR analyses.