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Molecular Ecology Resources Jul 2020Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor...
Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor representation in DNA databases. Here, we searched for a short barcode able to identify marine macrophytes from tissue samples; then, we created a DNA reference library which was used to identify macrophytes in eDNA from coastal sediments. Barcoding of seagrasses, mangroves and marine macroalgae (Chlorophyta, Rhodophyta and Phaeophyceae) was tested using 18 primer pairs from six barcoding genes: the plant barcodes rbcL, matK and trnL, plus the genes ITS2, COI and 18S. The 18S gene showed the highest universality among marine macrophytes, amplifying 95%-100% of samples; amplification performance of the other barcodes was limited. Taxonomy was assigned using a phylogeny-based approach to create an 18S DNA reference library. Macrophyte tissue sequences were accurately identified within their phyla (88%), order (76%), genus (71%) and species (23%). Nevertheless, out of 86 macrophytes tested, only 48% and 15% had a reference sequence at genus and at species level, respectively. Identification at these levels can be improved by more inclusive reference libraries. Using the 18S mini-barcode and the reference library, we recovered eDNA from 21 marine macrophytes in sediments, demonstrating the barcode's ability to trace primary producers that contribute to blue carbon. We expect this barcode to also be useful for other ecological questions, such as tracing macro primary producers in marine food webs.
Topics: Chlorophyta; DNA Barcoding, Taxonomic; DNA Primers; DNA, Plant; Gene Library; Geologic Sediments; Phaeophyceae; Phylogeny; Rhodophyta; Seaweed
PubMed: 32279439
DOI: 10.1111/1755-0998.13164 -
PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.Methods in Molecular Biology (Clifton,... 2023The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of...
The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension gene synthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Ts) are designed so that the 5' half of the DNA is encoded by sense primers and the 3' half of the DNA molecule is encoded by antisense primers. Primer extension is initiated at the center of the DNA and continues bidirectionally to progressively elongate the DNA molecule. Here we provide the protocols necessary for performing TBIO gene synthesis to generate a DNA molecule of interest.
Topics: Polymerase Chain Reaction; DNA Primers; Genetic Engineering; Oligonucleotides; Synthetic Biology
PubMed: 36853457
DOI: 10.1007/978-1-0716-3004-4_6 -
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 -
Biosensors Jul 2022DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect...
DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBR Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5-110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.
Topics: Biosensing Techniques; DNA; DNA Primers; Limit of Detection; Methyltransferases; Nucleic Acid Amplification Techniques
PubMed: 35892466
DOI: 10.3390/bios12080569 -
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 -
Journal of Visualized Experiments : JoVE Aug 2021Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic...
Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.
Topics: Cell-Free System; DNA Primers; Gene Regulatory Networks; Promoter Regions, Genetic; Synthetic Biology
PubMed: 34515672
DOI: 10.3791/62816 -
Journal of Medical Virology Jan 2023Rapid and accurate diagnosis of infections is fundamental to containment of disease. Several monkeypox virus (MPV) real-time diagnostic assays have been recommended by...
Rapid and accurate diagnosis of infections is fundamental to containment of disease. Several monkeypox virus (MPV) real-time diagnostic assays have been recommended by the CDC; however, the specificity of the primers and probes in these assays for the ongoing MPV outbreak has not been investigated. We analyzed the primer and probe sequences present in the CDC recommended MPV generic real-time PCR assay by aligning those sequences against 1730 MPV complete genomes reported in 2022 worldwide. Sequence mismatches were found in 99.08% and 97.46% of genomes for the MPV generic forward and reverse primers, respectively. Mismatch-corrected primers were synthetized and compared to the generic assay for MPV detection. Results showed that the two primer-template mismatches resulted in a ~11-fold underestimation of initial template DNA in the reaction and 4-fold increase in the 95% LOD. We further evaluated the specificity of seven other real-time PCR assays used for MPV and orthopoxvirus (OPV) detection and identified two assays with the highest matching score (>99.6%) to the global MPV genome database in 2022. Genetic variations in the primer-probe regions across MPV genomes could indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real-time generic assay may not be optimal to accurately detect MPV, and the mismatch-corrected assay with full complementarity between primers and current MPV genomes could provide a more sensitive and accurate detection of MPV.
Topics: Humans; Monkeypox virus; Real-Time Polymerase Chain Reaction; DNA Primers; Mpox (monkeypox); Disease Outbreaks; Sensitivity and Specificity
PubMed: 36504122
DOI: 10.1002/jmv.28395 -
Molecular Ecology Resources Aug 2022High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes...
High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.
Topics: DNA Primers; Eukaryota; High-Throughput Nucleotide Sequencing; Metagenomics; Phylogeny; RNA, Ribosomal, 16S; RNA, Ribosomal, 18S; rRNA Operon
PubMed: 35437888
DOI: 10.1111/1755-0998.13623 -
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