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Yi Chuan = Hereditas Apr 2022Flanking genomic sequences refer to the DNA sequences flanking specific sites of known sequences in chromosome, which contain information such as candidate genes,... (Review)
Review
Flanking genomic sequences refer to the DNA sequences flanking specific sites of known sequences in chromosome, which contain information such as candidate genes, transcriptional regulation, chromosome structure, and biosafety, and play an important role in genomics research. Flanking sequence acquisition technologies are mainly used in the cloning of regulatory sequences such as promoters and enhancers, identification of T-DNA or transposon insertion sites, chromosome walking, genome-wide gap filling, etc. It is an important means of structural genomics research and functional genomics research. It is applied in the identification of transgenic plants and animals and their safety management. With the development of molecular biology, many methods for obtaining flanking sequences have been established, including plasmid rescue, inverse PCR, ligation-mediated PCR, semi-random primer PCR, whole-genome resequencing etc. In this review, we summarize and compared different methods for acquiring flanking genomic sequence. The principles and research progress of each approach are discussed.
Topics: Animals; Chromosome Walking; DNA Primers; Genomics; Plants, Genetically Modified; Polymerase Chain Reaction
PubMed: 35437239
DOI: 10.16288/j.yczz.21-415 -
Proceedings of the National Academy of... Apr 2022Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers...
Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers generated by primase and provides a springboard for loading other replication factors. Here we provide the structural and functional analysis of the human Polα interaction with a mismatched template:primer. The structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template:primer containing a T-C mismatch at the growing primer terminus was solved at a 2.9 Å resolution. It revealed the absence of significant distortions in the active site and in the conformation of the substrates, except the primer 3′-end. The T-C mismatch acquired a planar geometry where both nucleotides moved toward each other by 0.4 Å and 0.7 Å, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polα has a low affinity to DNA and is not able to discriminate against a mispaired template:primer in the absence of deoxynucleotide triphosphate (dNTP). Strikingly, in the presence of cognate dNTP, Polα showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polα extends the T-C mismatch with a 249-fold lower efficiency due to reduction of the polymerization rate constant by 38-fold and reduced affinity to the incoming nucleotide by 6.6-fold. Thus, a mismatch at the postinsertion site affects all factors important for primer extension: affinity to both substrates and the rate of DNA polymerization.
Topics: Catalytic Domain; DNA Polymerase I; DNA Primers; DNA Replication; Humans; Kinetics
PubMed: 35467978
DOI: 10.1073/pnas.2111744119 -
Methods in Molecular Biology (Clifton,... 2023Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of...
Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R ≥ 0.9999 and efficiency (E) = 100 ± 5% for the best primer pair of each gene, which serves as the prerequisite for using the 2 method for data analysis.
Topics: Reverse Transcriptase Polymerase Chain Reaction; DNA, Complementary; DNA Primers; Reproducibility of Results; Real-Time Polymerase Chain Reaction
PubMed: 36995635
DOI: 10.1007/978-1-0716-3131-7_20 -
Toxins Nov 2022is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate...
is one of the most poisonous plants, and its accidental ingestion has frequently occurred in humans and livestock. It is vital to develop a rapid and accurate identification method for the timely rescue of oleander-poisoned patients and the investigation of poisoning cases. In this study, a specific and highly sensitive quantitative real-time PCR (qPCR)-based method was developed to identify oleander in mixture systems and simulated forensic specimens (SFS). First, a new pair of oleander-specific primers, JZT-BF/BR, was designed and validated. Then, a qPCR method was developed using the primers, and its detective sensitivity was examined. The results showed that JZT-BF/BR could specifically identify oleander in forage and food mixtures, and qPCR was capable of accurate authentication even at a low DNA concentration of 0.001 ng/μL. This method was further applied to the analysis of SFS containing different ratios of . The method was confirmed to be applicable to digested samples, and the detection limit reached 0.1% (/) oleander in mixture systems. Thus, this study undoubtedly provides strong support for the detection of highly toxic oleander and the diagnosis of food poisoning in humans and animals.
Topics: Animals; Humans; Nerium; Real-Time Polymerase Chain Reaction; Poisons; Plants, Toxic; DNA Primers
PubMed: 36356026
DOI: 10.3390/toxins14110776 -
Nature Communications Jul 2023The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is...
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
Topics: Bacteriophage T4; DNA Primase; DNA Helicases; DNA Replication; DNA Primers; DNA, Viral
PubMed: 37474605
DOI: 10.1038/s41467-023-40106-2 -
Journal of Bioscience and Bioengineering Feb 2021Recombinase polymerase amplification (RPA) is a technique that is used to specifically amplify a target nucleic acid sequence. Unlike the polymerase chain reaction...
Recombinase polymerase amplification (RPA) is a technique that is used to specifically amplify a target nucleic acid sequence. Unlike the polymerase chain reaction (PCR), RPA is performed at a constant temperature between 37 and 42°C. Therefore, it can be potentially used for the onsite detection of various pathogens when combined with DNA extraction and amplicon detection techniques. In this study, we prepared recombinant recombinase and single-stranded DNA-binding protein from T4 phage and used them to examine the effects of reaction conditions and additives on the efficiency of RPA. The results revealed that the optimal pH was 7.5-8.0, optimal potassium acetate concentration was 40-80 mM, and optimal reaction temperature was 37-45°C although dimethyl sulfoxide at 5% v/v and formamide at 5% v/v inhibited the reaction. Our results suggest that RPA could be conducted using a wider range of optimal reaction conditions than those required for PCR and that RPA is highly suitable for point-of-care use.
Topics: DNA Primers; Genetic Engineering; Nucleic Acid Amplification Techniques; Recombinases; Solvents; Temperature
PubMed: 33177003
DOI: 10.1016/j.jbiosc.2020.10.001 -
Biophysical Journal Apr 2023The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent...
The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization and therefore require effective suppression of hybridization errors. A simple and effective method to achieve that is to add blocker strands, also called clamps, to the PCR mixture. These strands bind to the unwanted target sequence, thereby blocking the primer mishybridization. Because of its simplicity, this method is applicable to a broad nucleic-acid-based biotechnology. However, the precise mechanism by which blocker strands suppress PCR errors remains to be understood, limiting the applicability of this technique. Here, we combine experiments and theoretical modeling to reveal this mechanism. We find that the blocker strands both energetically destabilize the mishybridized complex and sculpt a kinetic barrier to suppress mishybridization. This combination of energetic and kinetic biasing extends the viable range of annealing temperatures, which reduces design constraint of the primer sequence and extends the applicability of PCR.
Topics: DNA Primers; Polymerase Chain Reaction; Nucleic Acid Hybridization; Nucleic Acids; Temperature
PubMed: 36823986
DOI: 10.1016/j.bpj.2023.02.028 -
The Science of the Total Environment Feb 2021Fish environmental DNA (eDNA) studies have made substantial progress during the past decade, and significant advances in monitoring fishes have been gained by taking... (Review)
Review
Fish environmental DNA (eDNA) studies have made substantial progress during the past decade, and significant advances in monitoring fishes have been gained by taking advantage of this technology. Although a number of reviews concerning eDNA are available and some recent fish eDNA reviews focused on fisheries or standard method have been published, a systematic review of methodology of fish eDNA and its applications in ecology and environment has not yet been published. To our knowledge, this is the first review of fish eDNA for solving ecological and environmental issues. First, the most comprehensive literature analysis of fish eDNA was presented and analyzed. Then, we systematically discuss the relevant experiments and analyses of fish eDNA, and infers that standard workflow is on the way to consensus. We additionally provide reference sequence databases and the primers used to amplify the reference sequences or detecting fish eDNA. The abiotic and biotic conditions affecting fish eDNA persistence are also summarized in a schematic diagram. Subsequently, we focus on the major achievements of fish eDNA in ecology and environment. We additionally highlight the exciting new tools, including in situ autonomous monitoring devices, CRISPR nucleic acid detection technology, and meta-omics technology for fish eDNA detection in future. Ultimately, methodology of fish eDNA will provide a wholly new paradigm for conservation actions of fishes, ecological and environmental management at a global scale.
Topics: Animals; Biodiversity; DNA Primers; Environmental Monitoring; Fisheries; Fishes
PubMed: 33059148
DOI: 10.1016/j.scitotenv.2020.142622 -
Scientific Reports Jan 2024mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP)...
mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.
Topics: RNA, Messenger; RNA; Biological Assay; DNA Primers; DNA, Complementary
PubMed: 38200031
DOI: 10.1038/s41598-023-49651-8 -
Scientific Reports Jun 2022DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and...
DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolε extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3' end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3' end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolε has low affinity to DNA (K of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolε/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2'OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.
Topics: DNA; DNA Polymerase II; DNA Primers; DNA Replication; Humans; Kinetics; Magnesium; Nucleotides; Templates, Genetic
PubMed: 35715491
DOI: 10.1038/s41598-022-14602-2