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Molecular Ecology Resources Nov 2015With the rise of next-generation sequencing methods, it has become increasingly possible to obtain genomewide sequence data even for nonmodel species. Such data are...
With the rise of next-generation sequencing methods, it has become increasingly possible to obtain genomewide sequence data even for nonmodel species. Such data are often used for the development of single nucleotide polymorphism (SNP) markers, which can subsequently be screened in a larger population sample using a variety of genotyping techniques. Many of these techniques require appropriate locus-specific PCR and genotyping primers. Currently, there is no publicly available software for the automated design of suitable PCR and genotyping primers from next-generation sequence data. Here we present a pipeline called Scrimer that automates multiple steps, including adaptor removal, read mapping, selection of SNPs and multiple primer design from transcriptome data. The designed primers can be used in conjunction with several widely used genotyping methods such as SNaPshot or MALDI-TOF genotyping. Scrimer is composed of several reusable modules and an interactive bash workflow that connects these modules. Even the basic steps are presented, so the workflow can be executed in a step-by-step manner. The use of standard formats throughout the pipeline allows data from various sources to be plugged in, as well as easy inspection of intermediate results with visualization tools of the user's choice.
Topics: Computational Biology; DNA Primers; Genotyping Techniques; Polymerase Chain Reaction; Sequence Analysis, DNA; Transcriptome
PubMed: 25773304
DOI: 10.1111/1755-0998.12403 -
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 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2015Selecting specific primers is crucial for polymerase chain reaction (PCR). Nonspecific primers will bind to unintended genes and result in nonspecific amplicons....
Selecting specific primers is crucial for polymerase chain reaction (PCR). Nonspecific primers will bind to unintended genes and result in nonspecific amplicons. MFEprimer is a program for checking the specificity of PCR primers against the background DNA. In this chapter, we introduce: (1) the factors that affect the specificity of primers; (2) the principle of MFEprimer and its settings; (3) how to use the MFEprimer to examine the specificity of primers.
Topics: Animals; DNA Primers; Humans; Polymerase Chain Reaction
PubMed: 25697662
DOI: 10.1007/978-1-4939-2365-6_15 -
Analytical and Bioanalytical Chemistry Jul 2019Polymerase chain reaction (PCR) is a powerful technique for the detection and quantification of nucleic acids and has enormous applications to research in molecular...
Polymerase chain reaction (PCR) is a powerful technique for the detection and quantification of nucleic acids and has enormous applications to research in molecular biology. Certain inherited diseases, caused by single nucleotide mutations, however, are difficult to identify by PCR, using DNA primers and probes, in a situation where a false diagnosis may lead to incorrect or delayed treatment. With the aim of enhancing the specificity of PCR, we used novel chemically synthesized oligonucleotides containing site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s) as primers and probes. The methyl phosphotriester linkages carry no charge, so the reduction in the electrostatic repulsion of an MPTE-DNA/DNA duplex shows stronger hybridization affinity compared to a DNA/DNA duplex. However, the electrosteric effects introduced by the methyl group may result in instability of the double-stranded DNA (dsDNA) formed. With the use of specific MPTE modification sites and optimization of the number of MPTE modifications, greater delta melting temperature (ΔT) may be obtained, in concert with adjustment of PCR operating conditions, especially with respect to the annealing temperature, to achieve more discriminatory results between the target template and the perfectly matched primer and the mismatched primer. In single nucleotide polymorphism (SNP) genotyping, the results demonstrated that MPTE-modified probes can improve specificity. In summary, MPTE-modified oligonucleotides are a promising DNA analog applied to PCR primers and probes to enhance the specificity and to provide more precise detection results for various applications, such as for genetic diagnosis. In summary, two common DNA polymerases we tested could successfully recognize the MPTE-modified primers and probes. Under the optimal operating conditions, MPTE modification has the ability to improve the discrimination of single nucleotide polymorphism by increasing the ΔT of the perfect match and mismatch sequences and to provide more precise detection results for various applications, such as genetic diagnosis.
Topics: DNA Primers; DNA Probes; DNA-Directed DNA Polymerase; Genotype; Methylation; Molecular Diagnostic Techniques; Phosphates; Polymorphism, Single Nucleotide; Real-Time Polymerase Chain Reaction
PubMed: 31209551
DOI: 10.1007/s00216-019-01865-4 -
Nature May 2022During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to...
During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
Topics: Catalytic Domain; DNA; DNA Primase; DNA Primers; DNA Replication
PubMed: 35508653
DOI: 10.1038/s41586-022-04695-0 -
Methods in Molecular Biology (Clifton,... 2022Overlap extension PCR is one of the routinely used methods to generate mutagenic genes for the functional and structural study of proteins. However, it is time-consuming...
Overlap extension PCR is one of the routinely used methods to generate mutagenic genes for the functional and structural study of proteins. However, it is time-consuming to design the overlapping mutagenic primers and gene primers by manual operation. In this chapter, we present a Python script that is able to search all the possible primer combinations according to the preset definitions and calculate the necessary parameters of each primer for the users, which could facilitate the primer design process. Up to 256 pairs of primers can be provided for selection using this script.
Topics: DNA Primers; Mutagenesis; Mutagenesis, Site-Directed; Polymerase Chain Reaction
PubMed: 35727440
DOI: 10.1007/978-1-0716-2152-3_1 -
Scientific Reports Jun 2016DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading...
DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment.
Topics: Base Sequence; DNA; DNA Primers; DNA-Directed DNA Polymerase; G-Quadruplexes; GC Rich Sequence; Guanine; Polymerase Chain Reaction; Protein Binding; Taq Polymerase
PubMed: 27349576
DOI: 10.1038/srep28769 -
BMC Bioinformatics Jun 2022This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence...
BACKGROUND
This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT‑PCR assay for Sanger sequencing and polymerase-capsid based genotyping.
RESULTS
The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation.
CONCLUSIONS
An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.
Topics: DNA Primers; Norovirus; Real-Time Polymerase Chain Reaction; Sequence Alignment
PubMed: 35717145
DOI: 10.1186/s12859-022-04781-0