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The Analyst Jan 2020Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of...
Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 10 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.
Topics: DNA; DNA Primers; Fluorescence Resonance Energy Transfer; Nucleic Acid Amplification Techniques; Polymorphism, Single Nucleotide; Recombinases; Thermus thermophilus
PubMed: 31793929
DOI: 10.1039/c9an01701a -
Genetics and Molecular Research : GMR Apr 2017The objective of this article was to develop TRAP (target region amplification polymorphism) primers for castor bean, with the goal of making functional markers...
The objective of this article was to develop TRAP (target region amplification polymorphism) primers for castor bean, with the goal of making functional markers available for genetic studies about the species. To do this, oligonucleotides were designed based on ESTs, obtained from the NCBI (National Center for Biotechnology Information) databank, which code enzymes involved in metabolic routes of fatty acid synthesis, ricin synthesis, and resistance to castor bean pathogens. The forward primers were designed with the help of the Primer3 software and, for the reverse, six arbitrary primers were used. To standardize the amplification reactions, the following criteria were used to select the primers: sizes between 18 and 20 bp, guanine/cytosine (GC) in the range of 40 to 60%, and average annealing temperature between 55° and 62°C. The design quality of the primers was verified using the Net Primer application. Fifty-six primers were designed, which had an average GC percentage of 53.2%. A total of 336 combinations were obtained using the 56 fixed and 6 arbitrary primers. Based on polymerase chain reaction, 330 combinations (89%) presented good amplification patterns for the genomic DNA of castor bean. The size of the fragments amplified varied between 50 and 2072 bp. The TRAP primers designed and validated in this study are the first for castor bean and represent a significant increase in the molecular markers for this species.
Topics: Amplified Fragment Length Polymorphism Analysis; Base Pairing; DNA Primers; Expressed Sequence Tags; Polymorphism, Genetic; Ricinus
PubMed: 28407195
DOI: 10.4238/gmr16029647 -
International Journal of Medicinal... 2022This study aimed to obtain a set of specific inter simple sequence repeat (ISSR) primers and establish a stable and accurate intraspecific identification method for...
This study aimed to obtain a set of specific inter simple sequence repeat (ISSR) primers and establish a stable and accurate intraspecific identification method for Ganoderma lingzhi. A total of 117 G. lingzhi strains were identified using internal transcribed spacer sequences from 147 strains determined as G. lingzhi via simple morphological identification. Based on the sequences obtained, specific ISSR primers for G. lingzhi were screened and validated, and 15 specific ISSR primers showed polymorphic banding pattern with clear band resolution. Subsequently, ISSR PCRs of the 15 specific primers were performed for the 117 G. lingzhi strains. As expected, DNA analysis of the ISSR markers could distinguish G. lingzhi strains, with similarity coefficients ranging from 0.11 to 0.89. Thus, the 15 specific ISSR primers can be used for intraspecific identification and polymorphism analysis of G. lingzhi.
Topics: Agaricales; DNA Primers; Ganoderma; Genetic Variation; Microsatellite Repeats; Reishi
PubMed: 35695595
DOI: 10.1615/IntJMedMushrooms.2022043141 -
Analytical and Bioanalytical Chemistry Nov 2022A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for...
A simple, sensitive, specific and fast method based on the loop-mediated isothermal amplification (LAMP) technique and cleavable molecular beacon (CMB) was developed for chicken authentication detection. LAMP and CMB were used for DNA amplification and amplicon analysis, respectively. Targeting the mitochondrial cytochrome b gene of chickens, five primers and one CMB probe were designed, and their specificity was validated against nine other animal species. The structure of CMB and concentrations of dNTPs, MgSO, betaine, RNase H2, primers and CMB were optimized. The CMB-LAMP assay was completed within 17 min, and its limit of detection for chicken DNA was 1.5 pg μL. Chicken adulteration as low as 0.5% was detected in beef, and no cross-reactivity was observed. Finally, this assay was successfully applied to 20 commercial meat products. When combined with our developed DNA extraction method (the extraction time was 1 min: lysis for 10 s, washing for 20 s and elution for 30 s), the entire process (from DNA extraction to results analysis) was able to be completed within 20 min, which is at least 10 min shorter than other LAMP-based methods. Our method showed great potential for the on-site detection of chicken adulteration in meat.
Topics: Cattle; Animals; Chickens; Nucleic Acid Amplification Techniques; Meat; DNA Primers; DNA; Sensitivity and Specificity
PubMed: 36152037
DOI: 10.1007/s00216-022-04342-7 -
Methods in Molecular Biology (Clifton,... 2022Fungal and oomycete plant pathogens are responsible for the devastation of various ecosystems such as forest and crop species worldwide. In an effort to protect such...
Fungal and oomycete plant pathogens are responsible for the devastation of various ecosystems such as forest and crop species worldwide. In an effort to protect such natural resources for food, lumber, etc., early detection of non-indigenous phytopathogenic fungi in new areas is a key approach in managing threats at their source of introduction. A workflow was developed using high-throughput sequencing (HTS), more specifically metabarcoding, a method for rapid and higher throughput species screening near high-risk areas, and over larger geographical spaces. Biomonitoring of fungal and oomycete entities of plant pathogens (e.g., airborne spores) regained from environmental samples and their processing by metabarcoding is thoroughly described here. The amplicon-based approach goes from DNA and sequencing library preparation using custom-designed polymerase chain reaction (PCR) fusion primers that target the internal transcribed spacer 1 (ITS1) from fungi and oomycetes and extends to multiplex HTS with the Ion Torrent platform. In addition, a brief and simplified overview of the bioinformatics analysis pipeline and other available tools required to process amplicon sequences is also included. The raw data obtained and processed enable users to select a bioinformatics pipeline in order to directly perform biodiversity, presence/absence, geographical distribution, and abundance analyses through the tools suggested, which allows for accelerated identification of phytopathogens of interest.
Topics: Biological Monitoring; DNA Primers; Ecosystem; Oomycetes; Plants
PubMed: 35819612
DOI: 10.1007/978-1-0716-2517-0_18 -
Molecular Ecology Resources May 2022Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of... (Review)
Review
Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.
Topics: Biodiversity; DNA Barcoding, Taxonomic; DNA Primers; Gene Library; Polymerase Chain Reaction
PubMed: 34551203
DOI: 10.1111/1755-0998.13512 -
Molecular Ecology Resources Jan 2023Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially... (Review)
Review
Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed "the predator problem". We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.
Topics: Animals; Food Chain; Predatory Behavior; DNA Primers; Polymerase Chain Reaction; DNA; Diet
PubMed: 36017818
DOI: 10.1111/1755-0998.13705 -
Methods in Molecular Biology (Clifton,... 2022The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides...
The FastPCR software is an integrated tool environment for PCR primer and probe design and for prediction of oligonucleotide properties. The software provides comprehensive tools for designing primers for most PCR and perspective applications, including standard, multiplex, long-distance, inverse, real-time with TaqMan probe, Xtreme Chain Reaction (XCR), group-specific, overlap extension PCR for multifragment assembling cloning, and isothermal amplification (Loop-mediated Isothermal Amplification). A program is available to design specific oligonucleotide sets for long sequence assembly by ligase chain reaction and to design multiplexed of overlapping and nonoverlapping DNA amplicons that tile across a region(s) of interest for targeted next-generation sequencing, competitive allele-specific PCR (KASP)-based genotyping assay for single-nucleotide polymorphisms and insertions and deletions at specific loci, among other features. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. FastPCR includes various bioinformatics tools for analysis and searching of sequences, restriction I-II-III-type enzyme endonuclease analysis, and pattern searching. The program also supports the assembly of a set of contiguous sequences, consensus sequence generation, and sequence similarity and conservancy analysis. FastPCR performs efficient and complete detection of various repeat types with visual display. FastPCR allows for sequence file batch processing that is essential for automation. The software is available for download at https://primerdigital.com/fastpcr.html and online version at https://primerdigital.com/tools/pcr.html .
Topics: Computer Simulation; DNA Primers; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Oligonucleotides; Polymerase Chain Reaction; Software
PubMed: 34773626
DOI: 10.1007/978-1-0716-1799-1_16 -
Nucleosides, Nucleotides & Nucleic Acids 2020Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of...
Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3'-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3'-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.
Topics: DNA; DNA Primers; Dimerization; Polymerase Chain Reaction; Thermodynamics
PubMed: 32799617
DOI: 10.1080/15257770.2020.1803354 -
Chembiochem : a European Journal of... Mar 2018Swap and extend: The autonomous synthesis of single-stranded DNA molecules of arbitrary size and sequence composition can easily be achieved by primer exchange reaction...
Swap and extend: The autonomous synthesis of single-stranded DNA molecules of arbitrary size and sequence composition can easily be achieved by primer exchange reaction (PER) cascades, in which the sequential polymerase-mediated extension of DNA primers is guided by catalytic hairpins. This highlight illustrates the potential of this method for applications in DNA nanotechnology.
Topics: DNA Primers; DNA, Single-Stranded; Nanostructures; Nanotechnology; Nucleic Acid Conformation
PubMed: 29239531
DOI: 10.1002/cbic.201700639