-
BioTechniques Oct 2019Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available... (Review)
Review
Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available qPCR-based methods for miRNA expression analysis and discuss their advantages and disadvantages. Existing techniques involve the use of stem-loop reverse transcriptase-PCR, polyadenylation of RNAs, ligation of adapters or RT with complex primers, using universal or miRNA-specific qPCR primers and/or probes. Many of these methods are oriented towards the expression analysis of mature miRNAs and few are designed for the study of pre-miRNAs and pri-miRNAs. We also discuss findings from articles that compare results from existing methods. Finally, we suggest key points for the improvement of available techniques and for the future development of additional methods.
Topics: Computer Simulation; DNA Primers; Gene Expression; High-Throughput Nucleotide Sequencing; MicroRNAs; Polyadenylation; Real-Time Polymerase Chain Reaction; Software
PubMed: 31560239
DOI: 10.2144/btn-2019-0065 -
Nature Protocols Dec 2021Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of... (Review)
Review
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cell Line, Tumor; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Primers; Deoxyribonucleases, Type II Site-Specific; Electroporation; Gene Editing; Genome, Human; Humans; Osteoblasts; Plasmids; Polymerase Chain Reaction; Primary Cell Culture; RNA, Guide, CRISPR-Cas Systems; T-Lymphocytes
PubMed: 34773119
DOI: 10.1038/s41596-021-00626-x -
MSphere Feb 2021Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in...
Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols. Short amplicons targeting different variable regions (V-regions) or ranges thereof (V1-V2, V1-V3, V3-V4, V4, V4-V5, V6-V8, and V7-V9) were investigated for differences in the composition outcome due to primer choices. Next, the influence of clustering (operational taxonomic units [OTUs], zero-radius OTUs [zOTUs], and amplicon sequence variants [ASVs]), different databases (GreenGenes, the Ribosomal Database Project, Silva, the genomic-based 16S rRNA Database, and The All-Species Living Tree), and bioinformatic settings on taxonomic assignment were also investigated. We present a systematic comparison across all typically used V-regions using well-established primers. While it is known that the primer choice has a significant influence on the resulting microbial composition, we show that microbial profiles generated using different primer pairs need independent validation of performance. Further, comparing data sets across V-regions using different databases might be misleading due to differences in nomenclature (e.g., versus ) and varying precisions in classification down to genus level. Overall, specific but important taxa are not picked up by certain primer pairs (e.g., is missed using primers 515F-944R) or due to the database used (e.g., in GreenGenes and the genomic-based 16S rRNA Database). We found that appropriate truncation of amplicons is essential and different truncated-length combinations should be tested for each study. Finally, specific mock communities of sufficient and adequate complexity are highly recommended. In 16S rRNA gene sequencing, certain bacterial genera were found to be underrepresented or even missing in taxonomic profiles when using unsuitable primer combinations, outdated reference databases, or inadequate pipeline settings. Concerning the last, quality thresholds as well as bioinformatic settings (i.e., clustering approach, analysis pipeline, and specific adjustments such as truncation) are responsible for a number of observed differences between studies. Conclusions drawn by comparing one data set to another (e.g., between publications) appear to be problematic and require independent cross-validation using matching V-regions and uniform data processing. Therefore, we highlight the importance of a thought-out study design including sufficiently complex mock standards and appropriate V-region choice for the sample of interest. The use of processing pipelines and parameters must be tested beforehand.
Topics: Computational Biology; DNA Primers; DNA, Bacterial; Feces; Gastrointestinal Microbiome; Genetic Variation; High-Throughput Nucleotide Sequencing; Humans; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 33627512
DOI: 10.1128/mSphere.01202-20 -
Brazilian Journal of Microbiology :... Jul 2019Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be...
Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be effectively addressed, if the conventional detection methods are replaced with DNA-based molecular methods. Accordingly, it was aimed to develop a robust PCR protocol for specific detection of Salmonella spp. Out of the different primers screened, one pair of primers developed in this study targeting invA gene demonstrated 100% inclusivity for a wide range of Salmonella serotypes and 100% exclusivity for wide range of non-target species. The in silico analysis of the nucleotide sequence obtained from the PCR product suggests its potential as a hybridization probe for genus specific detection of Salmonella spp. contamination. The PCR protocol was sensitive enough to detect 15 cells per reaction using crude DNA prepared within a short time directly from artificially contaminated shrimp tissue. The study demonstrated that the result of PCR reaction can come out on the same day of sample arrival. Incorporation of this pair of primers in a multiplex PCR designed for simultaneous detection of four common seafood-borne human pathogens yielded 147 bp, 302 bp, 403 bp, and 450 bp distinct DNA bands specifically targeting E. coli, toxigenic Vibrio cholerae, Salmonella spp., and V. parahaemolyticus, respectively in a single PCR tube. The PCR methods developed in this study has the potential to be used in the seafood processing plants for effective monitoring of CCPs required for implementation of HACCP-based quality assurance system.
Topics: Animals; DNA Primers; Food Contamination; Multiplex Polymerase Chain Reaction; Palaemonidae; Salmonella; Shellfish
PubMed: 31006836
DOI: 10.1007/s42770-019-00072-8 -
Nature Communications Apr 2022One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of...
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
Topics: Algorithms; DNA Primers; Likelihood Functions; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35410464
DOI: 10.1038/s41467-022-29500-4 -
PloS One 2023qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However...
AIMS
qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6.
METHODS
The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer.
RESULTS
Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition.
CONCLUSIONS
Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.
Topics: Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA; DNA Primers; Genomics
PubMed: 37083935
DOI: 10.1371/journal.pone.0284538 -
Molecular Ecology Dec 2023Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different... (Review)
Review
Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different trophic levels, and to evaluate impacts on interspecific interactions. Molecular approaches such as DNA metabarcoding are boosting our ability to perform biodiversity inventories. Nevertheless, even though a few studies have recently attempted exhaustive reconstructions of communities, holistic assessments remain rare. The majority of metabarcoding studies published in the last years used just one or two markers and analysed a limited number of taxonomic groups. Here, we provide an overview of emerging approaches that can allow all-taxa biological inventories. Exhaustive biodiversity assessments can be attempted by combining a large number of specific primers, by exploiting the power of universal primers, or by combining specific and universal primers to obtain good information on key taxa while limiting the overlooked biodiversity. Multiplexes of primers, shotgun sequencing and capture enrichment may provide a better coverage of biodiversity compared to standard metabarcoding, but still require major methodological advances. Here, we identify the strengths and limitations of different approaches, and suggest new development lines that might improve broad scale biodiversity analyses in the near future. More holistic reconstructions of ecological communities can greatly increase the value of metabarcoding studies, improving understanding of the consequences of ongoing environmental changes on the multiple components of biodiversity.
Topics: Biodiversity; DNA; DNA Barcoding, Taxonomic; DNA Primers; Ecology
PubMed: 36762839
DOI: 10.1111/mec.16881 -
Nature Structural & Molecular Biology May 2023The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural...
The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.
Topics: Humans; DNA Primase; DNA; DNA Replication; DNA Primers; RNA
PubMed: 37069376
DOI: 10.1038/s41594-023-00971-3 -
BMC Microbiology Jun 2020Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been...
BACKGROUND
Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been primarily used for the discrimination of bacterial strains. Reproducible DNA fingerprint patterns for various phage types were generated using either of the two primers.
RESULTS
The similarity index of replicates ranged from 89.4-100% for BOXA2R-PCR, and from 90 to 100% for BOXA1R-PCR. The method of DNA isolation (p = 0.08) and the phage propagation conditions at two different temperatures (p = 0.527) had no significant influence on generated patterns. Rep-PCR amplification products were generated from different templates including purified phage DNA, phage lysates and phage plaques. The use of this method enabled comparisons of phage genetic profiles to establish their similarity to related or unrelated phages and their bacterial hosts.
CONCLUSION
The findings suggest that repetitive-PCR could be used as a rapid and inexpensive method to preliminary screen phage isolates prior to their selection for more comprehensive studies. The adoption of this rapid, simple and reproducible technique could facilitate preliminary characterisation of a large number of phage isolates and the investigation of genetic relationship between phage genotypes.
Topics: Bacteriophages; DNA Primers; DNA, Viral; Genotyping Techniques; Phylogeny; Polymerase Chain Reaction; Temperature
PubMed: 32527227
DOI: 10.1186/s12866-020-01770-2 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507