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Chembiochem : a European Journal of... Sep 2020DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional...
DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high-throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA-encoded information safer, if not unhackable. Mixing information-carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA-encrypted information, heating will cause reshuffling and generate endonuclease-resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)-like function for detecting PCR by an interceptor and a self-destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high-throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys.
Topics: Computer Security; DNA; DNA Primers; Humans; Polymerase Chain Reaction
PubMed: 32270906
DOI: 10.1002/cbic.202000149 -
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 -
Molecular Ecology Resources May 2020Telomere length has been used as a proxy of fitness, aging and lifespan in vertebrates. In the last decade, dozens of articles reporting on telomere dynamics in the... (Review)
Review
Telomere length has been used as a proxy of fitness, aging and lifespan in vertebrates. In the last decade, dozens of articles reporting on telomere dynamics in the fields of ecology and evolution have been published for a wide range of taxa. With this growing interest, it is necessary to ensure the accuracy and reproducibility of telomere length measurement techniques. Real-time quantitative PCR (qPCR) is routinely applied to measure relative telomere length. However, this technique is highly sensitive to several methodological variables and the optimization of qPCR telomere assays remains highly variable between studies. Therefore, standardized guidelines are required to enable the optimization of robust protocols, and to help in judging the validity of the presented results. This review provides an overview of preanalytical and analytical factors that can lead to qPCR inconsistencies and biases, including: (a) sample type, collection and storage; (b) DNA extraction, storage and quality; (c) qPCR primers, laboratory reagents, and assay conditions; and (d) data analysis. We propose a minimum level of information for publication of qPCR telomere assays in evolutionary ecology considering the methodological pitfalls and sources of error. This review highlights the complexity of the optimization and validation of qPCR for telomere measurement per se, demonstrating the importance of transparency and clarity of reporting methodological details required for reliable, reproducible and comparable qPCR telomere assays. We encourage efforts to implement standardized protocols that ensure the rigour and quality of telomere dynamics studies.
Topics: Animals; DNA; DNA Primers; Ecology; Humans; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Telomere
PubMed: 32133733
DOI: 10.1111/1755-0998.13152 -
Scientific Reports Apr 2021Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into...
Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.
Topics: DNA Primers; DNA Probes; Genome, Viral; Mutation; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33903676
DOI: 10.1038/s41598-021-88532-w -
Analytica Chimica Acta Mar 2021Rolling circle amplification (RCA) is an efficient enzymatic isothermal reaction that using circular probe as a template to generate long tandem single-stranded DNA or... (Review)
Review
Rolling circle amplification (RCA) is an efficient enzymatic isothermal reaction that using circular probe as a template to generate long tandem single-stranded DNA or RNA products under the initiation of short DNA or RNA primers. As a simplified derivative of natural rolling circle replication which synthesizes copies of circular nucleic acids molecules such as plasmids, RCA amplifies the circular template rapidly without thermal cycling and finds various applications in molecular biology. Compared with other amplification strategies, RCA has many obvious advantages. Firstly, because of the strict complementarity required in ligation of a padlock probe, it endows the RCA reaction with high specificity and can even be utilized to distinguish single base mismatches. Secondly, through the introduction of multiple primers, exponential amplification can be achieved easily and leads to a good sensitivity. Thirdly, RCA products can be customized by manipulating circular templates to generate functional nucleic acids such as aptamer, DNAzymes and restriction enzyme sites. Moreover, the RCA has good biocompatibility and is especially suitable for in situ detection. Therefore, RCA has attracted considerable attention as an efficient and potential tool for highly sensitive detection of biomarkers. Herein, we comprehensively introduce the fundamental principles of RCA technology, summarize it from three aspects including initiation mode, amplification mode and signal output mode, and discuss the recent application of RCA-based biosensor in this review.
Topics: Biosensing Techniques; DNA; DNA Primers; DNA, Catalytic; Nucleic Acid Amplification Techniques
PubMed: 33516384
DOI: 10.1016/j.aca.2020.12.062 -
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 -
Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.Journal of Microbiological Methods Dec 2020The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic... (Review)
Review
The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed.
Topics: Animals; Bacteria; DNA Primers; Environmental Monitoring; Harmful Algal Bloom; Nucleic Acid Amplification Techniques; Nucleic Acids; Real-Time Polymerase Chain Reaction; Schistosoma; Wastewater; Water Microbiology
PubMed: 33159993
DOI: 10.1016/j.mimet.2020.106099 -
PloS One 2023Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other...
Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease.
Topics: Infant, Newborn; Humans; Cell-Free Nucleic Acids; DNA; Nucleic Acids; Persistent Infection; Chagas Disease; DNA Primers; Extracellular Vesicles; Neglected Diseases
PubMed: 37682970
DOI: 10.1371/journal.pone.0282814 -
BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2 -
Molecules (Basel, Switzerland) Oct 2019Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics... (Review)
Review
Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.
Topics: Aptamers, Nucleotide; DNA Primers; DNA, Single-Stranded; Gene Library; High-Throughput Nucleotide Sequencing; Nucleic Acid Amplification Techniques; Nucleic Acids; Polymerase Chain Reaction; SELEX Aptamer Technique
PubMed: 31591283
DOI: 10.3390/molecules24193598