scholarly journals DNA base flipping analytical pipeline

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Peng Zhang ◽  
Florian D. Hastert ◽  
Anne K. Ludwig ◽  
Kai Breitwieser ◽  
Maria Hofstätter ◽  
...  
Keyword(s):  
High Resolution ◽  
Melting Temperature ◽  
High Resolution Melting ◽  
Dna Bases ◽  
Sequence Specificity ◽  
Temperature Analysis ◽  
Base Flipping ◽  
Mobility Shift ◽  
Dna Sequence Specificity ◽  
Dna Base

Abstract DNA base modifications and mutations are observed in all genomes throughout the kingdoms of life. Proteins involved in their establishment and removal were shown to use a base flipping mechanism to access their substrates. To better understand how proteins flip DNA bases to modify or remove them, we optimized and developed a pipeline of methods to step-by-step detect the process starting with protein–DNA interaction, base flipping itself and the ensuing DNA base modification or excision. As methylcytosine is the best-studied DNA modification, here we focus on the process of writing, modifying and reading this DNA base. Using multicolor electrophoretic mobility shift assays, we show that the methylcytosine modifier Tet1 exhibits little DNA sequence specificity with only a slight preference for methylated CpG containing DNA. A combination of chloroacetaldehyde treatment and high-resolution melting temperature analysis allowed us to detect base flipping induced by the methylcytosine modifier Tet1 as well as the methylcytosine writer M.HpaII. Finally, we show that high-resolution melting temperature analysis can be used to detect the activity of glycosylases, methyltransferases and dioxigenases on DNA substrates. Taken together, this DNA base flipping analytical pipeline (BaFAP) provide a complete toolbox for the fast and sensitive analysis of proteins that bind, flip and modify or excise DNA bases.

scholarly journals Novel DNA Bis-intercalation by MLN944, a Potent Clinical Bisphenazine Anticancer Drug

2004 ◽  
Vol 279 (44) ◽  
pp. 46096-46103 ◽  
Author(s):  
Jixun Dai ◽  
Chandanamalie Punchihewa ◽  
Prakash Mistry ◽  
Aik Teong Ooi ◽  
Danzhou Yang
Keyword(s):  
Dna Binding ◽  
Anticancer Drug ◽  
Topoisomerase I ◽  
Specific Binding ◽  
Binding Mode ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Phase I Clinical Trials ◽  
Mobility Shift ◽  
Dna Sequence Specificity

The new bisphenazine anticancer drug MLN944 is a novel cytotoxic agent with exceptional anti-tumor activity against a range of human and murine tumor models both invitroand invivo. MLN944 has recently entered Phase I clinical trials. Despite the structural similarity with its parent monophenazine carboxamide and acridine carboxamide anticancer compounds, MLN944 appears to work by a distinct mechanism of inhibiting DNA transcription rather than the expected mechanism of topoisomerase I and II inhibition. Here we present the first NMR structure of MLN944 complexed with d(ATGCAT)2DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5′-TpG site, with the carboxamide amino linker lying in the major groove of DNA. The MLN944 molecule adopts a significantly unexpected conformation and side chain orientation in the DNA complex, with the N10 on the phenazine ring protonated at pH 7. The phenazine chromophore of MLN944 is very well stacked with the flanking DNA base pairs using the parallel base-stacking intercalation binding mode. The DNA sequence specificity and the groove recognition of MLN944 binding is determined by several site-specific hydrogen bond interactions with the central G:C base pair as well as the favorable stacking interactions with the 5′-flanking thymine. The specific binding site of MLN944 is known to be recognized by a number of important transcription factors. Our electrophoretic gel mobility shift assay results demonstrated that the c-Jun DNA binding to the AP-1 site is significantly inhibited by MLN944 in a dose-dependent manner. Thus, the exceptional biological activity of MLN944 may be due to its novel DNA binding mode leading to a unique mechanism of action.

scholarly journals Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling

BioMetals ◽  
2020 ◽  
Vol 33 (4-5) ◽  
pp. 169-185
Author(s):  
Kristel Berg ◽  
Hege Lynum Pedersen ◽  
Ingar Leiros
Keyword(s):  
Biochemical Characterization ◽  
Ferric Uptake Regulator ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Mobility Shift ◽  
Dna Oligomers ◽  
Dna Sequence Specificity ◽  
Aliivibrio Salmonicida ◽  
Target Dna ◽  
Mobility Shift Assay

Abstract Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models. The protein interacts specifically with DNA at binding sites designated as ‘Fur boxes’. In the present study, we have investigated the interaction between Fur from the fish pathogen Aliivibrio salmonicida (AsFur) and its target DNA using a combination of biochemical and in silico methods. A series of target DNA oligomers were designed based on analyses of Fur boxes from other species, and affinities assessed using electrophoretic mobility shift assay. Binding strengths were interpreted in the context of homology models of AsFur to gain molecular-level insight into binding specificity.

scholarly journals Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260087
Author(s):  
Beatriz Iandra da Silva Ferreira ◽  
Natália Lins da Silva-Gomes ◽  
Wagner Luis da Costa Nunes Pimentel Coelho ◽  
Vanessa Duarte da Costa ◽  
Vanessa Cristine de Souza Carneiro ◽  
...  
Keyword(s):  
High Resolution ◽  
Melting Temperature ◽  
Molecular Diagnosis ◽  
Molecular Diagnostics ◽  
High Resolution Melting ◽  
Diagnostic Testing ◽  
Molecular Diagnostic ◽  
Clinical Specimens ◽  
P Gene ◽  
Molecular Diagnostic Testing

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

scholarly journals Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM)

2021 ◽  
pp. 1-8
Author(s):  
Xiaochun Zhang ◽  
Huan Yu ◽  
Ziwei Wang ◽  
Qi Yang ◽  
Ruocheng Xia ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

2021 ◽  
Vol 90 ◽  
pp. 104741
Author(s):  
Maëllys Kevin ◽  
Guillaume Girault ◽  
Yvan Caspar ◽  
Moulay Ali Cherfa ◽  
Christiane Mendy ◽  
...  
Keyword(s):  
High Resolution ◽  
Western Europe ◽  
High Resolution Melting ◽  
Epidemiological Surveillance ◽  
Pcr Assay

scholarly journals Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chayapol Tungphatthong ◽  
Santhosh Kumar J. Urumarudappa ◽  
Supita Awachai ◽  
Thongchai Sooksawate ◽  
Suchada Sukrong
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Dna Barcode ◽  
Herbal Medicines ◽  
Efficient Tool ◽  
Hrm Analysis ◽  
Mitragyna Speciosa ◽  
Leaf Powder

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

scholarly journals A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 199
Author(s):  
Xiaochun Zhang ◽  
Huan Yu ◽  
Qi Yang ◽  
Ziwei Wang ◽  
Ruocheng Xia ◽  
...  
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Its Region ◽  
Its Sequencing ◽  
Melting Temperatures ◽  
Hrm Analysis ◽  
National Drug ◽  
Species Specific ◽  
Its1 And Its2

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

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