Short DNA Sequences Research Articles

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria.

Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.

Rarely acquired type II-A CRISPR-Cas spacers mediate anti-viral immunity through the targeting of a non-canonical PAM sequence.

The Streptococcus pyogenes type II-A CRISPR-Cas systems provides adaptive immunity through the acquisition of short DNA sequences from invading viral genomes, called spacers. Spacers are transcribed into short RNA guides that match regions of the viral genome followed by a conserved NGG DNA motif, known as the PAM. These RNA guides, in turn, are used by the Cas9 nuclease to find and destroy complementary DNA targets within the viral genome. While most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by NGG sequences, there is a small fraction that target non-canonical PAMs. Whether these spacers originate through accidental acquisition of phage sequences and/or provide efficient defense is unknown. Here we found that many of them match phage target regions flanked by an NAGG PAM. Despite being scarcely present in bacterial populations, NAGG spacers provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro; with both activities comparable to spacers that target sequences followed by the canonical AGG PAM. In contrast, acquisition experiments showed that NAGG spacers are acquired at very low frequencies. We therefore conclude that discrimination against these sequences occurs during immunization of the host. Our results reveal unexpected differences in PAM recognition during the spacer acquisition and targeting stages of the type II-A CRISPR-Cas immune response.

DNA barcoding Clithon sp. (Gastropoda: Neritidae) from Badur Beach, Madura, Indonesia, based on COI gene molecular marker

Abstract. Djoemharsjah IS, Ambarwati R, Rahayu DA. 2023. DNA barcoding Clithonsp. (Gastropoda: Neritidae) from Badur Beach, Madura, Indonesia, based on COI gene molecular marker. Biodiversitas 24: xxxx. Neritidae is polymorphic with various shell colors and patterns; therefore, they were doubts about the identifying species from the genus Clithon, namely Clithon sp. from Badur Beach of Madura Island, Sumenep District, East Java Province, Indonesia. In addition, DNA barcoding could provide genetic information using short DNA sequences to quickly and precisely identify species. This study aimed to identify the genus Clithon sp. from Badur Beach, based on Cytochrome Oxidase subunit I(COI) genes and analysis of phylogenetic relationships. The research methods include sampling, sample preservation, morphological identification of species, DNA isolation, amplification, electrophoresis, and sequencing using the Sanger method with genetic analysis using bioinformatics software. The results of COI barcode identification obtained a DNA sequence length of 490bp with a similarity value of the three Clithon sp. samples between 96.75 to 98.97%. The identification with the Barcode of Life Data System consisted of three variations of nucleotide bases, and the average value of the genetic distance with the in-group was 1.74% as Clithon sp. The Phylogenetic tree Clithon sp. from Badur Beach, was in the same clade as Clithon sowerbianum Récluz 1843 and Clithon mertonianum Récluz 1843 with the Neighbor-Joining Tree and Maximum Likelihood methods with bootstrap values between 96-100. Therefore, the COI barcode DNA markers analysis successfully identified Clithon sp. from Badur Beach, Madura, Indonesia as C. sowerbianum.

Peran Mini-Barcode Internal Transcribed Spacer 2 (ITS2) untuk Identifikasi Molekuler Spesies Anggrek

AbstractMolecular identification using short orthological DNA sequences (DNA barcoding) has been applied for the classification of orchid species which is a major step in biodiversity management, conservation, breeding, authenticating components of herbal products, and tracking the adulteration of orchid species. One of the most widely used loci for phylogenetic inference at the generic and infrageneric levels in plants was ITS located between 18S rDNA, 5.8S rDNA, and 26S rDNA. The ITS or ITS2 region has been suggested as a plant barcode in some previous studies. However, DNA barcoding using the entire ITS genome is considered less effective and efficient in the process of PCR amplification and sequencing. Besides, complete DNA barcodes are difficult to obtain from herbarium samples and herbal products because some DNA sequences have been degraded. DNA mini-barcodes were developed over the past ten years to overcome issues related to DNA barcoding. DNA mini-barcodes use shorter DNA segments for PCR amplification, so they can identify species effectively and efficiently compared to the regular DNA barcoding. Specific primers that encode the ITS1 and ITS2 regions need to be designed for the PCR amplification. DNA mini-barcoding ITS has proven useful in species identification, classification studies, and authentication of specific orchid species. Therefore, a new rapid identification method based on the ITS mini-barcode is expected to be established, especially for orchid species. Keywords : DNA mini-barcode, Internal Transcribed Spacer, Orchid, Primer Design

Determining MSI status by a one-step PCR melting curve analysis of Chinese-specific mononucleotide repeats.

e15532 Background: Microsatellite instability (MSI) is a hypermutated phenotype primarily caused by the deficiency of DNA mismatch repair activity. Conventional MSI detection platforms are time consuming and need extra normal tissue. Due to the short DNA sequences of microsatellite loci in MSI samples, the method can be simplified to a one-step approach to differentiate MSI status by observing the melting curve. Herein a novel feasible MSI status detection method based on one step-PCR in single tumor tissue was developed, and validated in colorectal cancer samples. Methods: Specific microsatellite sites shorter than 12 bp in length (feasibility of fluorescence probes) were selected from public databases for analysis. In tumor tissues from 98 CRC patients with known MSI status, microsatellite sites with the highest receiver operating characteristic (ROC)-area under the ROC curve (AUC) were eventually selected for the final PCR panel design. The cut-off value of this assay was defined by the number of melting peaks in a specific Tm value range. An independent set of 199 tumor tissues from CRC patients with known MSI by Sanger sequencing was recruited to validate the performance of this novel PCR MSI kit. Results: The ROC-AUCs of the pre-selected eight mononucleotide repeats were 0.82, 0.93, 0.79, 0.78, 0.98, 0.95, 0.92, and 0.79. When 0 or 1 marker had a melting peak within the specified Tm value range, the sample was identified as "MSS/MSI-low (MSI-L)"; when 2 or more markers had melting peaks within the specified Tm value range, the sample was determined to be "MSI-high (MSI-H)". In the clinical sample set, compared with the results given by conventional MSI detection method, the sensitivity of the 8-gene panel for MSI-H was 99.4% (166/167), the specificity of the 8-gene panel was 100%, and the overall concordance reached to 99.5%. Conclusions: The novel PCR-MSI detection method using 8-gene panel developed in this study was feasible and accessible in clinical practice, and a large-scale clinical validation is in progress. [Table: see text]

Detection of progression and regression of genitourinary cancers by circulating tumor DNA (ctDNA).

e16508 Background: Circulating tumor DNA (ctDNA) are short DNA sequences shed by tumor cells into systemic circulation. The sensitivity and specificity of single ctDNA test to predict relapse or recurrence in solid tumor vary widely and the utility of monitoring serial ctDNA trends has not been well-described in genitourinary cancers. Methods: We conducted a retrospective study including adult patients with genitourinary cancers at the hematology-oncology clinic at William Beaumont - Royal Oak and Troy Hospitals, Michigan from, August 2021 to May 2022, who had ctDNA testing done with imaging available for comparison. We evaluated the correlation between ctDNA test, both single test as well as dynamic trends in value over time, with imaging findings. We also calculated lead time for uptrending ctDNA to detect disease progression compared to imaging. Results: 21 total ctDNA test results from eight patient were included in the study; five had renal cell cancer, two had urothelial cancer and one had prostate cancer. The median age at diagnosis was 62 years (Range: 45-82); 90% were Caucasian and 50% were female. Majority (87.5%) had Stage III or IV disease. Out of the 21 total ctDNA samples, all 16 positive ctDNA results had evidence of disease on corresponding imaging: 13 showed progression while 3 showed regression with residual disease compared to prior imaging. Of the 5 negative ctDNA test results, 2 showed no evidence of disease and 3 showed regression with residual disease on corresponding imaging. Sensitivity and specificity of a single positive ctDNA test to detect evidence of disease on imaging was 84.2% and 100%, respectively. Six patients had multiple ctDNA test results with 12 pairs of serial ctDNA values, of which 9 were up-trending, and 3 were down-trending. Both sensitivity and specificity of up-trending ctDNA values to detect progression and down-trending ctDNA values to detect regression on imaging were 100%. Uptrending ctDNA values predicted disease progression with a median lead time of 55.5 days compared to imaging. Conclusions: Given the high sensitivity and specificity of serial ctDNA monitoring to detect disease progression and regression in our study, we conclude that this may be a valid way to reliably monitor for changes in disease status in genitourinary cancers before they become evident on imaging studies. Further clinical studies are needed to prove its utility in detecting immediate changes in disease status and guide therapeutic intervention in genitourinary cancers. [Table: see text] [Table: see text]

Aptamer-Based Point-of-Care Devices: Emerging Technologies and Integration of Computational Methods.

Recent innovations in point-of-care (POC) diagnostic technologies have paved a critical road for the improved application of biomedicine through the deployment of accurate and affordable programs into resource-scarce settings. The utilization of antibodies as a bio-recognition element in POC devices is currently limited due to obstacles associated with cost and production, impeding its widespread adoption. One promising alternative, on the other hand, is aptamer integration, i.e., short sequences of single-stranded DNA and RNA structures. The advantageous properties of these molecules are as follows: small molecular size, amenability to chemical modification, low- or nonimmunogenic characteristics, and their reproducibility within a short generation time. The utilization of these aforementioned features is critical in developing sensitive and portable POC systems. Furthermore, the deficiencies related to past experimental efforts to improve biosensor schematics, including the design of biorecognition elements, can be tackled with the integration of computational tools. These complementary tools enable the prediction of the reliability and functionality of the molecular structure of aptamers. In this review, we have overviewed the usage of aptamers in the development of novel and portable POC devices, in addition to highlighting the insights that simulations and other computational methods can provide into the use of aptamer modeling for POC integration.

OnTarget: in silico design of MiniPromoters for targeted delivery of expression.

MiniPromoters, or compact promoters, are short DNA sequences that can drive expression in specific cells and tissues. While broadly useful, they are of high relevance to gene therapy due to their role in enabling precise control of where a therapeutic gene will be expressed. Here, we present OnTarget (http://ontarget.cmmt.ubc.ca), a webserver that streamlines the MiniPromoter design process. Users only need to specify a gene of interest or custom genomic coordinates on which to focus the identification of promoters and enhancers, and can also provide relevant cell-type-specific genomic evidence (e.g. accessible chromatin regions, histone modifications, etc.). OnTarget combines the provided data with internal data to identify candidate promoters and enhancers and design MiniPromoters. To illustrate the utility of OnTarget, we designed and characterized two MiniPromoters targeting different cell populations relevant toParkinson Disease.

Taxonomic identification accuracy from BOLD and GenBank databases using over a thousand insect DNA barcodes from Colombia.

Recent declines of insect populations at high rates have resulted in the need to develop a quick method to determine their diversity and to process massive data for the identification of species of highly diverse groups. A short sequence of DNA from COI is widely used for insect identification by comparing it against sequences of known species. Repositories of sequences are available online with tools that facilitate matching of the sequences of interest to a known individual. However, the performance of these tools can differ. Here we aim to assess the accuracy in identification of insect taxonomic categories from two repositories, BOLD Systems and GenBank. This was done by comparing the sequence matches between the taxonomist identification and the suggested identification from the platforms. We used 1,160 COI sequences representing eight orders of insects from Colombia. After the comparison, we reanalyzed the results from a representative subset of the data from the subfamily Scarabaeinae (Coleoptera). Overall, BOLD systems outperformed GenBank, and the performance of both engines differed by orders and other taxonomic categories (species, genus and family). Higher rates of accurate identification were obtained at family and genus levels. The accuracy was higher in BOLD for the order Coleoptera at family level, for Coleoptera and Lepidoptera at genus and species level. Other orders performed similarly in both repositories. Moreover, the Scarabaeinae subset showed that species were correctly identified only when BOLD match percentage was above 93.4% and a total of 85% of the samples were correctly assigned to a taxonomic category. These results accentuate the great potential of the identification engines to place insects accurately into their respective taxonomic categories based on DNA barcodes and highlight the reliability of BOLD Systems for insect identification in the absence of a large reference database for a highly diverse country.

Abstract 991: Leveraging sequences missing from the human genome to detect cancer

Abstract Introduction: Cancer diagnosis using cell-free DNA (cfDNA) has the potential to improve treatment and survival but has several technical limitations. Methods: Here, we used neomers, short DNA sequences (13-17bp in length) that are largely absent from the healthy human genome, but appear in the tumor genome due to somatic mutations, to detect cancer at early stages from cfDNA. cfDNA was extracted from 1mL of plasma, and utilized from Whole Genome Sequencing at 5X coverage. Results: First, we analyzed over 2,500 cancer whole-genome sequences (WGS), to show that a neomer-based classifier can distinguish twenty-one different tumor types with higher accuracy than state-of-the-art methods. Refinement of this classifier via manually selected neomer sets, identified additional cancer features with greater precision. We next generated cfDNA WGS data from >600 patients and non-cancerous controls across four different types of cancers (prostate, lung, ovarian, colorectal), and demonstrated that neomers can precisely identify cancer from a limited amount of plasma for all four cancer types, with exampled sensitivities of 93.73% and 73.47% for lung and ovarian cancers, respectively. Additionally, using both luciferase and massively parallel reporter assays (MPRAs), we show that our neomer-driven approach can detect cancer-associated non-coding mutations that affect gene regulatory activity. Conclusions: Our results show that neomers provide a novel, sensitive, specific and potentially broadly applicable cancer detection tool. This approach has several advantages over existing strategies for liquid biopsies: 1) it is based on standard next-generation sequencing which is already widely available in the clinic; 2) it requires low amounts of starting material and is completely tumor agnostic; 3) it searches the entire genome and is not restricted to known driver mutations. 4) it allows the detection of early-stage disease. Citation Format: Ofer Yizhar Barnea, Ilias Georgakopoulos-Soares, Nadav Ahituv, Jocelyn Chapman, Martin Hemberg, Ioannis Mouratidis, Konstantinos Syrigos, Nikolaos Syrigos, Ioannis Vathiotis, Mayank Mahajan, Emmanouil Panagiotou, Andriani Charpidou, Mark Kvale, Candace S. Chan, Ryder Easterlin. Leveraging sequences missing from the human genome to detect cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 991.

Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategies.

Oligonucleotide-based therapies are a promising approach for treating a wide range of hard-to-treat diseases, particularly genetic and rare diseases. These therapies involve the use of short synthetic sequences of DNA or RNA that can modulate gene expression or inhibit proteins through various mechanisms. Despite the potential of these therapies, a significant barrier to their widespread use is the difficulty in ensuring their uptake by target cells/tissues. Strategies to overcome this challenge include cell-penetrating peptide conjugation, chemical modification, nanoparticle formulation, and the use of endogenous vesicles, spherical nucleic acids, and smart material-based delivery vehicles. This article provides an overview of these strategies and their potential for the efficient delivery of oligonucleotide drugs, as well as the safety and toxicity considerations, regulatory requirements, and challenges in translating these therapies from the laboratory to the clinic.

Filling reference libraries with diatom environmental sequences: strengths and weaknesses

Diatom species identification with DNA metabarcoding is an economical, fast and reliable alternative to identification via light microscopy for river quality monitoring. Using a short DNA sequence of the rbcL gene and ‘Diat.barcode’, a reference barcode library, enables the identification of more than 90% of the environmental sequences to species level in French rivers. But the completeness of this library is much lower in other regions, such as the tropical French overseas departments. A barcode library completion method using high-throughput sequencing data combined with microscopy count data from natural samples (Rimet et al. 2018) was applied and tested in rivers of Martinique and Guadeloupe (West Indies), for which only 45% of the environmental sequences could be identified to species level using Diat.barcode v9. Assigning barcodes to the most abundant species in the islands by this method is illustrated with Ulnaria goulardii and two new species belonging to Nupela and Epithemia, which are also described in this paper. The more complex situation of morphologically similar species is illustrated by reference to Gomphonema designatum and G. bourbonense. Using a combination of molecular and morphological data, their conspecificity, as G. bourbonense, is demonstrated with their reference barcodes. However, when several morphologically similar species and several environmental sequences belonging to the same clade are present, it is not possible to relate the barcodes to corresponding morphological species. Applying this method enabled the Diat.barcode library (v.10) to be updated, with 84% of the environmental sequences from the West Indies now identifiable at the species level. However, many morphological species still lack barcodes. In these cases, more classical methods, such as cell isolation, Sanger sequencing and morphological observations of cultures, must be applied.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

LNA blockers for improved amplification selectivity

LNA-containing oligonucleotides bind DNA more tightly than standard DNA, so they can interact with targeted sequences and affect multiple processes. When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs. Using a short rAAV and synthetic DNA sequence as a model, we studied the length, number, and positioning of LNA bases to improve blocker effectiveness. Oligonucleotides 18–24 bases long with LNAs at every other position were most effective. Highly degenerate targets were used to characterize the impact of mismatches on blocking. Mismatches at LNA ends had little impact on blocking activity. Single and double mismatches were tolerated with longer blockers, especially if the mismatches were near LNA ends. Shorter LNAs were more selective, with > 1 mismatch preventing effective blocking. Neither the strand to which a blocker bound nor the distance between the blocker and priming sites greatly impacted blocking efficiency. We used these findings to design blockers of wild-type DNA versus the single-base A1AT PiZ allele. Blockers are most specific when the mismatch is located away from the LNA 5′ end. Pairs of partially overlapping blockers on opposite strands with a centrally-located mismatch have maximal activity and specificity.

The evolution of early-life telomere length, pace-of-life and telomere-chromosome length dynamics in birds.

Telomeres, the short DNA sequences that protect chromosome ends, are an ancient molecular structure, which is highly conserved across most eukaryotes. Species differ in their telomere lengths, but the causes of this variation are not well understood. Here, we demonstrate that mean early-life telomere length is an evolutionary labile trait across 57 bird species (representing 35 families in 12 orders) with the greatest trait diversity found among passerines. Among these species, telomeres are significantly shorter in fast-lived than in slow-lived species, suggesting that telomere length may have evolved to mediate trade-offs between physiological requirements underlying the diversity of pace-of-life strategies in birds. This association was attenuated when excluding studies that may include interstitial telomeres in the estimation of mean telomere length. Curiously, within some species, larger individual chromosome size predicts longer telomere lengths on that chromosome, leading to the hypothesis that telomere length also covaries with chromosome length across species. We show that longer mean chromosome length or genome size tends to be associated with longer mean early-life telomere length (measured across all chromosomes) within a phylogenetic framework constituting up to 31 bird species. These associations were strengthened when excluding highly influential outliers. However, sensitivity analyses suggested that they were susceptible to sample size effects and not robust to the exclusion of studies that may include interstitial telomeres. Combined, our analyses generalize patterns previously found within a few species and provide potential adaptive explanations for the 10-fold variation in telomere lengths observed among birds.

T-Toxin Virulence Genes: Unconnected Dots in a Sea of Repeats.

In 1970, the Southern Corn Leaf Blight epidemic ravaged U.S. fields to great economic loss. The outbreak was caused by never-before-seen, supervirulent, Race T of the fungus Cochliobolus heterostrophus. The functional difference between Race T and O, the previously known, far less aggressive strain, is production of T-toxin, a host-selective polyketide. Supervirulence is associated with ~1 Mb of Race T-specific DNA; only a fraction encodes T-toxin biosynthetic genes (Tox1). Tox1 is genetically and physically complex, with unlinked loci (Tox1A, Tox1B) genetically inseparable from breakpoints of a Race O reciprocal translocation that generated hybrid Race T chromosomes. Previously, we identified 10 genes for T-toxin biosynthesis. Unfortunately, high-depth, short-read sequencing placed these genes on four small, unconnected scaffolds surrounded by repeated A+T rich sequence, concealing context. To sort out Tox1 topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealed Tox1 gene arrangement and the breakpoints. Six Tox1A genes are arranged as three small islands in a Race T-specific sea (~634 kb) of repeats. Four Tox1B genes are linked, on a large loop of Race T-specific DNA (~210 kb). The race O breakpoints are short sequences of race O-specific DNA; corresponding positions in race T are large insertions of race T-specific, A+T rich DNA, often with similarity to transposable (predominantly Gypsy) elements. Nearby, are 'Voyager Starship' elements and DUF proteins. These elements may have facilitated Tox1 integration into progenitor Race O and promoted large scale recombination resulting in race T. IMPORTANCE In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism. Long read DNA sequencing technology allowed in depth structural comparisons between the sole, previously known, much less aggressive, version of the pathogen and the supervirulent version and revealed, in meticulous detail, the structure of the unique virulence-causing DNA. These data are foundational for future analysis of mechanisms of DNA acquisition from a foreign source.

Design of Specific Primer for Methallothionein Gene of Tor Fish (Tor tambra)

The use of heavy metal biomarkers is needed to detect and integrate heavy metals at the molecular level. Metallothionein gene is a gene that is expressed in fish organs that have been heavily polluted. The aim of the study was to find specific primers for methallothionein of tor fish. Detection of the metallothionein gene expression in PCR instruments requires a primer in the form of a short chain DNA sequence as a specific target DNA identifier. The primer design was performed in silico using the NCBI site and multiple-aligned using Geneious Prime bioinformatic software. Primers were designed according to the conserved region of these genes. The primers specificity was checked using Primer BLAST tools in NCBI. The results showed that the forward primer 1 (5'- GAT TGC GCC AAG ACT GGA ACT –3') and reverse primer 1 (5' – ATC ACG TTG ACC TCC TCA CTG -3') qualified as good primers with an amplicon size 186 bp.

Recent developments in application of nucleic acid aptamer in food safety

Food, a basic necessity of life, is often contaminated with harmful agents like pathogenic microorganisms, biotoxins, allergens, antibiotic residues, pesticides, heavy metals etc. Application of efficient, sensitive, reliable and cost-effective analytical technologies to detect the level of contaminants is one of the first steps in ensuring food safety. Oligonucleotide aptamers comprising of short sequences of DNA or RNA are emerging as an alternative to antibodies in development of novel biosensors or bioassays for analysis of various food contaminants. In the pursuit of developing a perfect analytical method, several design strategies involving nanomaterials, biological reagents, microfluidics etc. have resulted in several disruptive innovations. Currently, aptamers are utilised in affinity columns and various sorbent surfaces for selective extraction of analyte from food matrix. Taking advantages of target responsive characteristic and cross-linking ability of aptamers, hydrogel based sensing materials have been developed. In this review, we present an overview on the recent developments in application of aptamers in evaluating food safety and the influence of food matrices on the performance of aptamer based analytical methods.