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The Journal of Molecular Diagnostics :... Nov 2022Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the...
Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.
Topics: Humans; Taq Polymerase; Alleles; DNA; DNA Primers; Polymerase Chain Reaction
PubMed: 36058471
DOI: 10.1016/j.jmoldx.2022.08.002 -
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 -
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 -
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 -
Cold Spring Harbor Protocols Jul 2017Capillary sequencing of DNA remains a mainstay in the toolkit of molecular biologists. Whether the technique is used to verify that a clone is constructed properly or to...
Capillary sequencing of DNA remains a mainstay in the toolkit of molecular biologists. Whether the technique is used to verify that a clone is constructed properly or to validate an interesting variant found by next-generation whole-genome sequencing, capillary sequencing is an extremely versatile tool. This method for cycle sequencing relies on the use of the ABI3730xl capillary sequencer. The template can be prepared from plasmid minipreps for direct sequencing of clones to identify a mutation or structure of interest, or amplified products can be sequenced individually with each of the two primers used in LongAmp amplification reactions. Both template preparations use primers designed specifically for "targets"-either for direct sequencing or for validation.
Topics: DNA; DNA Primers; Plasmids; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 27803269
DOI: 10.1101/pdb.prot094607 -
BMC Bioinformatics Feb 2017Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction...
BACKGROUND
Molecular genetic markers are one of the most informative and widely used genome features in clinical and environmental diagnostic studies. A polymerase chain reaction (PCR)-based molecular marker is very attractive because it is suitable to high throughput automation and confers high specificity. However, the design of taxon-specific primers may be difficult and time consuming due to the need to identify appropriate genomic regions for annealing primers and to evaluate primer specificity.
RESULTS
Here, we report the development of a Tool for Identification of Primers for Multiple Taxa (TipMT), which is a web application to search and design primers for genotyping based on genomic data. The tool identifies and targets single sequence repeats (SSR) or orthologous/taxa-specific genes for genotyping using Multiplex PCR. This pipeline was applied to the genomes of four species of Leishmania (L. amazonensis, L. braziliensis, L. infantum and L. major) and validated by PCR using artificial genomic DNA mixtures of the Leishmania species as templates. This experimental validation demonstrates the reliability of TipMT because amplification profiles showed discrimination of genomic DNA samples from Leishmania species.
CONCLUSIONS
The TipMT web tool allows for large-scale identification and design of taxon-specific primers and is freely available to the scientific community at http://200.131.37.155/tipMT/ .
Topics: DNA Primers; Genetic Markers; Genome, Protozoan; Internet; Leishmania; Polymerase Chain Reaction; User-Computer Interface
PubMed: 28187714
DOI: 10.1186/s12859-017-1485-3 -
Methods in Molecular Biology (Clifton,... 2023High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or...
High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.
Topics: Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Polymerase Chain Reaction; DNA Primers
PubMed: 36781650
DOI: 10.1007/978-1-0716-3024-2_20 -
Methods in Molecular Biology (Clifton,... 2022Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in...
Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.
Topics: DNA Primers; DNA, Complementary; RNA; RNA, Viral; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Viroids
PubMed: 34845693
DOI: 10.1007/978-1-0716-1464-8_14