Cloning of Complete Geminivirus Genomes by Rolling Circle Amplification (RCA)Zerbini
Methods Mol Biol (2025) 2912, 11-18
Abstract: Rolling circle amplification (RCA) revolutionized geminivirology before high-throughput sequencing. Using the DNA polymerase of phage phi29 and random primers, it is a simple isothermal reaction which will amplify circular DNA without any prior knowledge of its sequence. It is thus ideal to clone complete geminivirus genomes. The procedure outlined here has been used in the author's laboratory for more than 15 years and has allowed the cloning of thousands of full-length genomes. It consists of three parts: first, the RCA reaction itself; second, the analysis of RCA products with restriction enzymes, to identify an enzyme that will cleave the genome at a single site; and third, the ligation of the linearized genome to a plasmid vector, followed by E. coli transformation. The whole procedure will normally take 3 days.© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
A Primer-Regulated Rolling Circle Amplification (RCA) for Logic-Controlled Multiplexed Enzyme AnalysisChai, Sun, Hou
et alACS Appl Bio Mater (2025) 8 (3), 2408-2418
Abstract: DNA-related enzymes are associated with various diseases and have been potential biomarkers for clinical diagnosis. Developing robust and ultrasensitive methods is extremely favorable for the detection of these biomarkers. To this purpose, a primer-regulated rolling circle amplification (RCA) strategy was ingeniously proposed. Briefly, the RCA primer, which was invalidated with 3'-inverted dT (locked state) and unable to initiate an amplification reaction by phi29 DNA polymerase, was embedded with the recognition substrate of the specific enzyme. In the presence of the target, the recognition and cleavage process of the enzyme prompted the release of the 3'-inverted dT and the regeneration of 3'-OH (unlocked state), satisfying the vital prerequisite for RCA. By adopting this programmable and modular design, the recognition substrate can be either single base sites or a specific sequence for different types of enzymes. This also enables us to conduct single or multiple enzyme detection conveniently, relying on a logic-controlled manner including YES, OR, AND, and AND-OR operations. Overall, the proposed strategy is uniquely insightful and provides a universal tool for multiple analyses of diverse DNA-related enzymes.
Whole-genome amplification as a tool to improve bacterial detection by PCR in microbiological samples after endodontic treatmentChianca, Fendeler, Junior
et alFront Oral Health (2025) 6, 1520945
Abstract: Microorganisms have an important role in the pathogenesis of endodontic disease. Significant advances have been made to increase the sensitivity of microbial detection, identification and enumeration in endodontic samples. The aim of the present study is to compare culture and whole-genome amplification (WGA) followed by PCR assays in the detection of bacteria before and after chemical mechanical preparation (CMP) of root canals.Ten uniradicular teeth with primary endodontic infections were analyzed. Microbiological samples were collected before and after CMP using paper points, which were separated into two groups: (i) culture assay samples were plated onto Brucella agar with 5% defibrinated sheep's blood, menadione and hemin and incubated anaerobically for 14 days at 36°C; (ii) DNA was extracted from molecular assay samples and subject to WGA by isothermal strand displacement with Phi29 DNA polymerase followed by PCR to determine the presence of bacteria.In both assays, samples before CMP showed the presence of bacteria in all 10 teeth. After CMP, however, bacterial detection differed in the assays performed (p = 0.0198). The presence of bacteria was detected in 70% (7 of 10) of the samples by WGA followed by PCR, where only 10% (1 of 10) had demonstrated bacterial growth in the culture method.The combination of WGA followed by PCR increased the detection of microorganisms from root canal samples after endodontic treatment using NaOCl as a CMP irrigant. So this combination of techniques can represent an important tool to improve the detection of microorganisms in endodontic research.© 2025 Chianca, Fendeler, Junior, Pereira, Póvoa, Antunes, Antunes and Iorio.
Dual miRNAs Imaging Platform Based on HRCA-Cas12a by Replacing PAM with Bubble to Reduce False PositiveWang, Zhao, Yin
et alAnal Chem (2025) 97 (5), 3053-3062
Abstract: Detection and imaging of dual miRNAs based on AND logic gates can improve the accuracy of the early diagnosis of disease. However, a single target may lead to false positive. Hence, this work rationally integrates hyperbranched rolling circle amplification (HRCA) with Cas12a by replacing the PAM sequence with a bubble to sensitively detect and image miRNA-10b and miRNA-21 based on the AND logic gate. When miRNA-10b and miRNA-21 are both present, the two padlocks are linked into circular DNA as a template for RCA. Long ssDNA products are generated under the catalysis of phi29 DNA polymerase, which are cis-cleaved by Cas12a and activated the trans-cleavage of Cas12a to generate fluorescent signals. Subsequently, the primer hybridizes with the products of cis-cleavage and is extended as the dsDNA substrate of Cas12a to produce more fluorescent signals. However, a single target produces significant fluorescent signals leading to false positive due to the presence of protospacer adjacent motif (PAM) on the padlock. After PAM is removed from the padlock, the primer and RCA products form bubbles to replace PAM, which activate Cas12a without affecting sensitivity and reduce false positive. The introduction of a primer enables the second utilization of phi29 and Cas12a, increasing the signal-to-noise ratio. HRCA and Cas12a exhibit optimal activity in the T4 ligase buffer, achieving one-pot detection of dual miRNAs. In addition, the HRCA-Cas12a method enables the intracellular visualization of dual miRNAs. It exhibits the ability to distinguish different types of cancer cells based on the expression level of miRNAs.
膜杰作
Star Staining
