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Analytical Chemistry Aug 2023Gene 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...
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.
Topics: Humans; Mutation; Polymerase Chain Reaction; DNA; Lung Neoplasms; DNA Primers
PubMed: 37527514
DOI: 10.1021/acs.analchem.3c01803 -
ACS Nano Feb 2024Developing DNA strand displacement reactions (SDRs) offers crucial technical support for regulating artificial nucleic acid circuits and networks. More recently,...
Developing DNA strand displacement reactions (SDRs) offers crucial technical support for regulating artificial nucleic acid circuits and networks. More recently, enzymatic SDR-based DNA circuits have gained significant attention because of their modular design, high orthogonality signaling, and extremely fast reaction rates. Typical enzymatic SDRs are regulated by relatively long primers (20-30 nucleotides) that hybridize to form stable double-stranded structures, facilitating enzyme-initiated events. Implementing more flexible primer-based enzymatic SDR regulations remains challenging due to the lack of convenient and simple primer control mechanism, which consequently limits the development of enzymatic DNA circuits. In this study, we propose an approach, termed primer switching regulation, that implements programmable and flexible regulations of enzymatic circuits by introducing switchable wires into the enzymatic circuits. We applied this method to generate diverse enzymatic DNA circuits, including cascading, fan-in/fan-out, dual-rail, feed-forward, and feedback functions. Through this method, complex circuit functions can be implemented by just introducing additional switching wires without reconstructing the basic circuit frameworks. The method is experimentally demonstrated to provide flexible and programmable regulations to control enzymatic DNA circuits and has future applications in DNA computing, biosensing, and DNA storage.
Topics: Computers, Molecular; DNA; Nucleic Acids; Nucleotides
PubMed: 38286819
DOI: 10.1021/acsnano.3c12000 -
Journal of Biological Methods 2023Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a...
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
PubMed: 38023773
DOI: 10.14440/jbm.2023.411 -
Journal of Visualized Experiments : JoVE Dec 2023Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal...
Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4 WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.
Topics: Humans; Hepatitis B virus; Real-Time Polymerase Chain Reaction; DNA, Viral; Reproducibility of Results; Hepatitis B; Viral Load; Sensitivity and Specificity
PubMed: 38163269
DOI: 10.3791/66249 -
Mycologia 2023The (formerly ) species complex was previously composed of two morphological varieties: var. and var. . Prior attempts to resolve this morphology-based species complex...
The (formerly ) species complex was previously composed of two morphological varieties: var. and var. . Prior attempts to resolve this morphology-based species complex using molecular techniques have been inconclusive or conflicting. The increased availability of sequenced genomes and isolates identified as var. and var. has allowed us to examine these relationships at a higher resolution and with a broader scope than previously possible. Using comparative genomics, we identified highly variable gene regions and designed primers for four new protein-coding genes for phylogenetics. These were then used alongside three known markers to generate a nuclear multigene genealogy of the species complex. From a collection of 163 isolates belonging to the target taxa, a subset of 29 was chosen to be included in this study (verified with nuclear rDNA internal transcribed spacer 1 [ITS1] and mitochondrial cytochrome oxidase subunit 1 [] sequences). Seventeen isolates of var. were selected to be representative of variations in genotype, morphology, and geographic collection location. The 12 isolates of var. included all available specimens identified either morphologically (in previous studies) or through sequence similarity with ITS1 and . Based on the fulfillment of reciprocal monophyly and observed genealogical concordance under the genealogical concordance phylogenetic species recognition, we determined that the species complex is composed of four genetically distinct species: , and .
Topics: Pythium; Phylogeny; Base Sequence; Genotype; DNA, Ribosomal
PubMed: 37796448
DOI: 10.1080/00275514.2023.2241980 -
Breast Cancer Research and Treatment Nov 2023The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S....
PURPOSE
The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry.
METHODS
We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle.
RESULTS
The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls.
CONCLUSION
We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.
Topics: Cattle; Humans; Female; Animals; Sheep; Breast Neoplasms; Leukemia Virus, Bovine; DNA, Viral; Breast; Mammary Neoplasms, Animal
PubMed: 37517027
DOI: 10.1007/s10549-023-07061-4 -
Food Chemistry. Molecular Sciences Dec 2023Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2...
Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 () gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3'-CCAAACACAACTCCGAAAA-5') and species-specific reverse primers composed of twenty (3'-CCAAACACAACTCCGAAAA-5') and twenty-one (3'-TGGCAAGAATTAGGACGGTTA-5') bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.
PubMed: 37637373
DOI: 10.1016/j.fochms.2023.100181 -
BMC Research Notes Nov 2023DNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the...
OBJECTIVES
DNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the 'Folmer' barcode region at the 5´ start of the cytochrome c oxidase 1 gene (cox1), with universal primers. Previous studies showed failures of standard "universal" primers to amplify this region in psyllids. The aim of the study was the design of a new alternative more reliable primer combination for taxa of the superfamily Psylloidea and its comparison with the performance of the standard "universal" Folmer-primers.
RESULTS
A newly designed degenerate forward primer LCOP-F was developed following comparison of the sequence alignment of the priming site of "universal" primer LCO1490 and the standard insect forward primer LepF1. When combined with the "universal" reverse primer, HCO2198, this new primer pairing was able to generate barcode sequence for all 36 species in 20 genera across the five families of psyllids tested in this study, and these primers were found to be more universally reliable across psyllid taxa than other primer pairs tested.
Topics: Animals; DNA Barcoding, Taxonomic; Hemiptera; Aphids; DNA Primers; Electron Transport Complex IV
PubMed: 37941051
DOI: 10.1186/s13104-023-06585-8 -
Heliyon Jun 2024Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing...
Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing it have yet to be established. The actual status of cross-infection remains to be seen. The purpose of this study was to evaluate the utility of bacterial DNA and serum immunoglobulin G (IgG) antibody titer assays to assess infection of human-pathogenic and dog-pathogenic species in dogs. Four experimental beagles were used for establishing methods. Sixty-six companion dogs at veterinary clinics visiting for treatment and prophylaxis of periodontal disease were used and divided into healthy, gingivitis, and periodontitis groups. Periodontal pathogens such as and were investigated as target bacteria. DNA levels of both bacteria were measured using species-specific primers designed for real-time polymerase chain reaction (PCR). Serum IgG titers of both bacteria were measured by enzyme-linked immunosorbent assay (ELISA). PCR primers were confirmed to have high sensitivity and specificity. However, there was no relationship between the amount of bacterial DNA and the severity of the periodontal disease. In addition, dogs with periodontitis had higher IgG titers against both bacteria compared to dogs in the healthy and gingivitis groups; there was cross-reactivity between the two bacteria. Receiver operating characteristic (ROC) analysis of IgG titers against both bacteria showed high sensitivity (>90 %) and specificity (>75 %). Since both bacteria were distinguished by DNA assays, the combination of these assays may be useful in the evaluation of cross-infection.
PubMed: 38919974
DOI: 10.1016/j.heliyon.2024.e31872 -
Plant Disease Oct 2023The pine pitch canker pathogen is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable...
The pine pitch canker pathogen is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to . Using a globally representative collection of isolates and other closely related species, we have demonstrated that the assay can be used to identify across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.
Topics: Fusarium; Trees; DNA
PubMed: 36867583
DOI: 10.1094/PDIS-04-22-0972-SR