Salmon Genomic DNA Research Articles

Electrochemical detection of Piscirickettsia salmonis genomic DNA from salmon samples using solid-phase recombinase polymerase amplification.

Electrochemical detection of solid-phase isothermal recombinase polymerase amplification (RPA) of Piscirickettsia salmonis in salmon genomic DNA is reported. The electrochemical biosensor was constructed by surface functionalization of gold electrodes with a thiolated forward primer specific to the genomic region of interest. Solid-phase RPA and primer elongation were achieved in the presence of the specific target sequence and biotinylated reverse primers. The formation of the subsequent surface-tethered duplex amplicons was electrochemically monitored via addition of streptavidin-linked HRP upon completion of solid-phase RPA. Successful quantitative amplification and detection were achieved in less than 1h at 37°C, calibrating with PCR-amplified genomic DNA standards and achieving a limit of detection of 5 · 10-8μgml-1 (3 · 103 copies in 10μl). The presented system was applied to the analysis of eight real salmon samples, and the method was also compared to qPCR analysis, observing an excellent degree of correlation. Graphical abstract Schematic of use of electrochemical RPA for detection of Psiricketessia salmonis in salmon liver.

Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid‐based veterinary vaccine

Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar) leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

The effect of single wall carbon nanotube metallicity on genomic DNA-mediated chirality enrichment

Achieving highly enriched single wall carbon nanotubes (SWNTs) is one of the major hurdles today because their chirality-dependent properties must be uniform and predictable for use in nanoscale electronics. Due to the unique wrapping and groove-binding mechanism, DNA has been demonstrated as a highly specific SWNT dispersion and fractionation agent, with its enrichment capabilities depending on the DNA sequence and length as well as the nanotube properties. Salmon genomic DNA (SaDNA) offers an inexpensive and scalable alternative to synthetic DNA. In this study, SaDNA enrichment capabilities were tested on SWNT separation with varying degrees of metallicity that were formulated from mixtures of commercial metallic (met-) and semiconducting (sem-) abundant SWNTs. The results herein demonstrate that the degree of metallicity of the SWNT sample has a significant effect on the SaDNA enrichment capabilities, and this effect is modeled based on deconvolution of the near-infrared (NIR) absorption spectra and verified with photoluminescence emission (PLE) measurements. Using molecular dynamics and circular dichroism, the preferential SaDNA mediated separation of the (6, 5) sem-tube is shown to be largely influenced by the presence of met-SWNTs.

On modeling biomolecular–surface nonbonded interactions: application to nucleobase adsorption on single-wall carbon nanotube surfaces

In this work we explored the selectivity of single nucleobases towards adsorption on chiral single-wall carbon nanotubes (SWCNTs) by density functional theory calculations. Specifically, the adsorption of molecular models of guanine (G), adenine (A), thymine (T), and cytosine (C), as well as of AT and GC Watson–Crick (WC) base pairs on chiral SWCNT C(6, 5), C(9, 1) and C(8, 3) model structures, was analyzed in detail. The importance of correcting the exchange–correlation functional for London dispersion was clearly demonstrated, yet limitations in modeling such interactions by considering the SWCNT as a molecular model may mask subtle effects in a molecular–macroscopic material system. The trend in the calculated adsorption energies of the nucleobases on same diameter C(6, 5) and C(9, 1) SWCNT surfaces, i.e. G > A > T > C, was consistent with related computations and experimental work on graphitic surfaces, however contradicting experimental data on the adsorption of single-strand short homo-oligonucleotides on SWCNTs that demonstrated a trend of G > C > A > T (Albertorio et al 2009 Nanotechnology 20 395101). A possible role of electrostatic interactions in this case was partially captured by applying the effective fragment potential method, emphasizing that the interplay of the various contributions in modeling nonbonded interactions is complicated by theoretical limitations. Finally, because the calculated adsorption energies for Watson–Crick base pairs have shown little effect upon adsorption of the base pair farther from the surface, the results on SWCNT sorting by salmon genomic DNA could be indicative of partial unfolding of the double helix upon adsorption on the SWCNT surface.

Enrichment of (6,5) Single Wall Carbon Nanotubes Using Genomic DNA

Single wall carbon nanotubes (SWNTs) have attracted attention because of their potential in a vast range of applications, including transistors and sensors. However, immense technological importance lies in enhancing the purity and homogeneity of SWNTs with respect to their chirality for real-world electronic applications. In order to achieve optimal performance of SWNTs, the diameter, type, and chirality have to be effectively sorted. Any employed strategy for sorting SWNTs has to be scalable, nondestructible, and economical. In this paper, we present a solubilization and chirality enrichment study of commercially available SWNTs using genomic DNA. On the basis of the comparison of the photoluminescence (PL) and near-infrared absorption measurements from the SWNTs dispersed with salmon genomic DNA (SaDNA) and d(GT)20, we show that genomic DNA specifically enriches (6,5) tubes. Circular dichroism and classical all-atom molecular dynamics simulations reveal that the genomic double-stranded SaDNA prefers to interact with (6,5) SWNTs as compared to (10,3) tubes, meanwhile single-stranded d(GT)20 shows no or minimal chirality preference. Our enrichment process demonstrates enrichment of >86% of (6,5) SWNTs from CoMoCat nanotubes using SaDNA.

Mixed DNA/oligo (ethylene glycol) functionalized gold surfaces improve DNA hybridization in complex media

Reliable, direct "sample-to-answer" capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s-->pi(*) transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein adsorption, facilitating detection of small DNA target sequences from these undiluted, unpurified complex biological mixtures unachievable with previously reported, analogous ssDNA/11-mercapto-1-undecanol monolayer surfaces [P. Gong, C.-Y. Lee, L. J. Gamble, D. G. Castner, and D. W. Grainger, Anal. Chem. 78, 3326 (2006)].

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

BackgroundWe have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested.ResultsWe have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA.ConclusionWe validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.

Gene transfer: potential to enhance the genome of Atlantic salmon for aquaculture

Over the past 20 years we have generated stable lines of transgenic Atlantic salmon possessing either antifreeze protein (AFP) genes or a salmon growth hormone (GH) gene construct. The AFP gene transfer studies were initiated in 1982. The AFP transgene integrated into salmon genomic DNA and AFP has been found in the blood of all 5 generations to date. However, AFP levels are low and a means to raise these levels needs to be developed. Our GH gene transfer studies were initiated in 1989. Evidence to date indicates that a single copy of the GH transgene integrated into chromosomal DNA and has been passed down in Mendelian fashion, along with its rapid growth phenotype, over 6 generations. Laboratory studies indicate that our GH transgene enhances growth rates with Atlantic salmon reaching market size (4–6 kg) a year earlier than non-transgenics cultured commercially in Atlantic Canada. This GH gene transfer technology was patented and licensed to Aqua Bounty Farms Inc., and the transgenic salmon are currently under review by various government regulatory authorities in the USA and Canada for use in commercial aquaculture ventures. Our experience with the regulatory authorities, the industry and the press indicates that the successful introduction of transgenic salmon into the aquaculture industry involves issues concerning not only science but also food safety, environmental safety, animal welfare and consumer acceptance. This communication centres on our experience with Atlantic salmon and outlines our plans and progress towards demonstrating the safety of transgenic fish to the consumer and to the environment.

Identification of a Genetic Marker That Discriminates Ocean-Type and Stream-Type Chinook Salmon in the Columbia River Basin

Abstract A marker based on randomly amplified polymorphic DNA (RAPD), OT-38, was discovered that nonlethally discriminates between stream-type and ocean-type populations of chinook salmon Oncorhynchus tshawytscha in the Columbia River basin, including the threatened fall-run (ocean-type) and spring-run (stream-type) Snake River populations. This marker was developed by amplifying chinook salmon genomic DNA with a single RAPD primer, sequencing the termini of the polymorphic products, and designing primer pairs for allele-specific amplification. It was used to assay 18−80 individuals from several wild and hatchery populations differing in year-class, freshwater life history, and location along the Columbia River. OT-38 unambiguously distinguished ocean-type from stream-type populations in 93.1% of the chinook salmon sampled.

Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA.

A new technique is described for the rapid detection of specific nucleic acid sequences in unamplified DNA samples. The method consists of using two nucleic acid probes complementary to different sites on a target DNA sequence. The two probes are each labeled with different fluorescent dyes. When mixed with a sample containing the target DNA, the two probes hybridize to their respective binding sites on the same target DNA molecule. The sample is then analyzed by a laser-based ultrasensitive fluorescence system capable of detecting single fluorescent molecules at two different wavelength channels simultaneously. Since the probes are bound to the same target DNA molecule, their signals appear simultaneously. Thus, coincident detection of both dyes provides the necessary specificity to detect an unamplified, single-copy target DNA molecule in a homogeneous assay. If the target is not present, only uncorrelated events originating from free probes will be observed at either channel. Phage lambda DNA in a background of salmon genomic DNA was detected as a two-dye coincident signal at a relative concentration of one lambda molecule per salmon genome. In a control sample, cleavage of the lambda DNA between the two probe binding sites eliminated the coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applications to genetic analysis are discussed.

A growth hormone pseudogene in the salmon genome.

Representatives of the fish family Salmonidae were reported to possess two nonallelic growth hormone (GH)-encoding genes. In addition to those, we found a third GH-like sequence in a chum salmon genomic DNA library. A number of point mutations and large deletions abolished the possibility of expressing this sequence, showing that the chum salmon genomic DNA contains a GH pseudogene besides functional GH genes.

Gene Structure Of Chum Salmon Somatolactin, A Presumed Pituitary Hormone Of The Growth Hormone/Prolactin Family

From a chum salmon genomic DNA library, clones coding for somatolactin (SL), a presumed pituitary hormone belonging to the GH/PRL family, were isolated and analyzed. The salmon SL gene was 16 kilobases (kb) in length, this being the largest among mammalian GH (approximately 2 kb) and PRL (approximately 10 kb) and fish GH (approximately 4 kb) genes. As in the case of mammalian GH/PRL genes, the salmon SL gene was comprised of five exons in contrast to rainbow trout and Atlantic salmon GH genes, which each have six exons. Upstream from the transcriptional initiation site were found one TATA box identical to those of mammalian PRL genes and four consensus sequences corresponding to the Pit-1/GHF-1 binding element which is essential to the expression of mammalian GH and/or PRL genes. The similarity in amino acid sequences and organization of the SL gene with mammalian GH/PRL genes indicates that the SL gene is quite likely produced from an ancestral gene common to GH/PRL genes by gene duplication. Based on these considerations, an evolutionary model for the GH/PRL/SL gene family was made and shows the common ancestral gene to originate not from duplication of a primordial gene but from shuffling of independent exons and a regulatory sequence.