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Chembiochem : a European Journal of... Apr 2024Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an... (Review)
Review
Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an aqueous, protecting group free system, without the need for expensive enzymes. However, these primer extension reactions suffer from a lack of fidelity, low reaction rates, low overall yields, and short primer extension lengths. This review outlines a detailed mechanistic pathway for non-enzymatic template-directed primer extension and presents a review of the thermodynamic driving forces involved in entropic templating. Through the lens of entropic templating, the rate and fidelity of a reaction are shown to be intrinsically linked to the reactivity of the activating agent used. Thus, a strategy is discussed for the optimization of non-enzymatic template-directed primer extension, providing a path towards cost-effective in vitro synthesis of RNA and DNA.
Topics: DNA Primers; Nucleic Acids; DNA; RNA; Thermodynamics; Templates, Genetic
PubMed: 38282207
DOI: 10.1002/cbic.202300859 -
Scientific Reports Oct 2020The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity...
The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.
Topics: Animals; Biodiversity; DNA Barcoding, Taxonomic; DNA Primers; DNA, Ribosomal; Databases, Genetic; Fishes; Gene Library; Mitochondria; Species Specificity
PubMed: 33087755
DOI: 10.1038/s41598-020-74902-3 -
Proceedings of the National Academy of... Nov 2020Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single...
Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
Topics: DNA; DNA Primers; Emulsions; Gene Library; Genome; High-Throughput Nucleotide Sequencing; Humans; Models, Theoretical; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Templates, Genetic
PubMed: 33087570
DOI: 10.1073/pnas.2017138117 -
Science China. Life Sciences May 2022Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely. The past two decades have witnessed the increasing application of...
Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely. The past two decades have witnessed the increasing application of next-generation 16S rRNA gene sequencing in the investigation of bacterial communities. However, deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial 16S. Here, we designed polymerase chain reaction (PCR) primer sets, including 799F/1107R, 322F/796R, and 322F-Dr/796Rs (primer pair 322F/796R with a penultimate-base substitution in 322F), that can specifically amplify bacterial 16S from plant total DNAs. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets. Both 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial communities. The primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome, which revealed 10-10 and 10-10 bacteria per gram fresh weight, respectively. These 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome, which will significantly advance studies on the plant-related microbiome.
Topics: Bacteria; DNA Primers; DNA, Bacterial; DNA, Plant; Plants; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 34309738
DOI: 10.1007/s11427-021-1953-5 -
Scientific Reports Jun 2020Even though ladybirds are well known as economically important biological control agents, an integrative framework of DNA barcoding research was not available for the...
Even though ladybirds are well known as economically important biological control agents, an integrative framework of DNA barcoding research was not available for the family so far. We designed and present a set of efficient mini-barcoding primers to recover full DNA barcoding sequences for Coccinellidae, even for specimens collected 40 years ago. Based on these mini-barcoding primers, we obtained 104 full DNA barcode sequences for 104 species of Coccinellidae, in which 101 barcodes were newly reported for the first time. We also downloaded 870 COI barcode sequences (658 bp) from GenBank and BOLD database, belonging to 108 species within 46 genera, to assess the optimum genetic distance threshold and compare four methods of species delimitation (GMYC, bPTP, BIN and ABGD) to determine the most accurate approach for the family. The results suggested the existence of a 'barcode gap' and that 3% is likely an appropriate genetic distance threshold to delimit species of Coccinellidae using DNA barcodes. Species delimitation analyses confirm ABGD as an accurate and efficient approach, more suitable than the other three methods. Our research provides an integrative framework for DNA barcoding and descriptions of new taxa in Coccinellidae. Our results enrich DNA barcoding public reference libraries, including data for Chinese coccinellids. This will facilitate taxonomic identification and biodiversity monitoring of ladybirds using metabarcoding.
Topics: Animals; Coleoptera; DNA Barcoding, Taxonomic; DNA Primers; Databases, Genetic; Phylogeny; Polymerase Chain Reaction
PubMed: 32572078
DOI: 10.1038/s41598-020-66874-1 -
Methods in Molecular Biology (Clifton,... 2023Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However,...
Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.
Topics: Spices; Real-Time Polymerase Chain Reaction; DNA Primers; Biological Assay; Carica
PubMed: 37608099
DOI: 10.1007/978-1-0716-3358-8_2 -
STAR Protocols Mar 2023Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the...
Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022)..
Topics: Base Sequence; Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA Primers; DNA
PubMed: 36853735
DOI: 10.1016/j.xpro.2022.102037 -
Methods in Molecular Biology (Clifton,... 2023Quantitative PCR is one of the fundamental steps performed when processing routine casework in a forensic laboratory. Quantitative PCR of Alu repeats using a SYBR Green...
Quantitative PCR is one of the fundamental steps performed when processing routine casework in a forensic laboratory. Quantitative PCR of Alu repeats using a SYBR Green master mix can produce calculated estimates of how much DNA was extracted from a sample. This process offers more efficiency, human specificity, and can be performed faster than other outdated quantification methods, such as slot blot or yield gel. A qPCR master mix is prepared and consists of Alu-F primers, Alu-R primers, water, and SYBR Green master mix. The Alu-F and Alu-R primers target Alu sequences that are present hundreds of thousands of times throughout the human genome and are effective markers for human DNA quantification. During qPCR, the 7500 system facilitates the amplification of target Alu repeats. The SYBR Green I fluorescent dye intercalates between the amplified dsDNA targets. During each amplification cycle, the 7500 system agitates the SYBR Green I dye, resulting in a fluorescence signal that is recorded when it passes a specified Ct value. After qPCR amplification is complete, a standard curve is created and used to determine how much DNA a sample contains. This chapter provides instructions on how to accurately prepare a 96-well plate for qPCR, use the 7500 system and associated software to set up the qPCR amplification, and interpret the corresponding results produced.
Topics: Humans; Polymerase Chain Reaction; DNA; Nucleic Acid Amplification Techniques; DNA Primers; Fluorescent Dyes; Benzothiazoles; Real-Time Polymerase Chain Reaction
PubMed: 37439981
DOI: 10.1007/978-1-0716-3295-6_10 -
Angewandte Chemie (International Ed. in... Oct 2021The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA...
The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.
Topics: Arabinose; Crystallography, X-Ray; DNA Primers; Deuterium Oxide; Imidazoles; Kinetics; Nucleic Acid Conformation; Nucleotides; RNA; Structure-Activity Relationship; Templates, Genetic; Water
PubMed: 34428345
DOI: 10.1002/anie.202109714 -
Journal of Microbiological Methods May 2021Unlike fungi, which have a universally accepted barcode marker, universal primers still lack in myxomycetes. Typically, DNA barcode primers were designed based on...
Unlike fungi, which have a universally accepted barcode marker, universal primers still lack in myxomycetes. Typically, DNA barcode primers were designed based on comparing existing myxomycetes sequences and targeting the conserved regions. However, the extreme genetic diversity within major myxomycetes groups and the frequent occurrence of group I introns have made the development of universal DNA barcode a severe challenge. The emergence of next-generation sequencing provides an opportunity to address this problem. We sequenced the mixed genomic DNA of 81 myxomycetes and extracted the SSU gene's reads using next-generation sequencing. After alignment and assembly, we designed a set of SSU primers that matched all potential SNPs, avoided all known group I intron insertion sites, and were highly conserved between major myxomycetes orders. This set of SSU primers has the potential to become one of the universal primer combinations. Due to the high genetic divergence caused by long and complicated evolutionary histories, the lack of universal barcode primers is common in protists. Our research provides a new method to solve this problem.
Topics: DNA Primers; Fungal Proteins; Genetic Variation; High-Throughput Nucleotide Sequencing; Myxomycetes; Phylogeny
PubMed: 33722637
DOI: 10.1016/j.mimet.2021.106203