Dr. Torstein Tengs

Characterization of unknown genetic modifications using high throughput sequencing and computational subtraction

Torstein Tengs — Wed, 14 Oct 2009 01:05:03 PDT

BackgroundWhen generating a genetically modified organism (GMO), the primary goal is to give a target organism one or several novel traits by using biotechnology techniques. A GMO will differ from its parental strain in that its pool of transcripts will be altered. Currently, there are no methods that are reliably able to determine if an organism has been genetically altered if the nature of the modification is unknown. ResultsWe show that the concept of computational subtraction can be used to identify transgenic cDNA sequences from genetically modified plants. Our datasets include 454-type sequences from a transgenic line of Arabidopsis thaliana and published EST datasets from commercially relevant species (rice and papaya).ConclusionWe believe that computational subtraction represents a powerful new strategy for determining if an organism has been genetically modified as well as to define the nature of the modification. Fewer assumptions have to be made compared to methods currently in use and this is an advantage particularly when working with unknown GMOs.

Non-prejudiced Detection and Characterization of Genetic Modifications

Torstein Tengs — Wed, 14 Oct 2009 00:58:38 PDT

The application of gene technology is becoming widespread much thanks to the rapid increase in technology, resource, and knowledge availability. Consequently, the diversity and number of genetically modified organisms (GMOs) that may find their way into the food chain or the environment, intended or unintended, is rapidly growing. From a safety point of view the ability to detect and characterize in detail any GMO, independent of publicly available information, is fundamental. Pre-release risk assessments of GMOs are required in most jurisdictions and are usually based on application of technologies with limited ability to detect unexpected rearrangements and insertions. We present an array-based approach to address these problems and show with three examples (GTS 40-3-2 Roundup Ready and event A5547-127 soybean as well as T25 Liberty Link Maize) that the method can detect and characterize GMOs with high accuracy while making very few prior assumptions about the actual genetic modifications or constructs in question. Based on the array results, a simple polymerase chain reaction-scheme is also described that will enable the user to characterize the inserted sequences to DNA sequence level. The method may provide the biotechnology developers and risk regulators with a useful tool to improve pre-market risk assessments as well as seed producers and other food chain and environmental stakeholders with a platform to improve their ability to detect and characterize GMOs.

UniquePrimer - a web utility for design of specific PCR primers and probes

Torstein Tengs — Wed, 14 Oct 2009 00:43:51 PDT

We have developed a web-based tool for design of specific PCR primers and probes. The program allows you to enter primer sequence information as well as an optional probe, and sequence similarity searches (MegaBLAST) will be performed to see if the sequences match the same sequence entry in the specified database. If primers (and probe) match, this will be reported. The program can handle overlapping amplicons, amplification from a single primer, ambiguous bases and other problematic cases.

A quantitative TaqMan MGB real-time polymerase chain reaction based assay for detection of the causative agent of crayfish plague Aphanomyces astaci

Torstein Tengs — Tue, 10 Feb 2009 02:25:53 PST

Here we present the development and first validation of a TaqMan minor groove binder (MGB) real-time polymerase chain reaction (RT-PCR) method for quantitative and highly specific detection of Aphanomyces astaci, the causative agent of crayfish plague. The assay specificity was experimentally assessed by testing against DNA representative of closely related oomycetes, and theoretically assessed by additional sequence similarity analyses comparing the primers and probe sequences to available sequences in EMBL/GenBank. The target of the assay is a 59 bp unique sequence motif of A. astaci found in the internal transcribed spacer 1 of the nuclear ribosomal gene cluster. A standard curve for quantification was established by setting up a four-fold dilution series with genomic A. astaci DNA. The absolute limit of detection (LODabs), defined as the lowest concentration yielding a false negative probability < 5% was found to be approximately 5 PCR forming units (PFU; target template copies) equivalent to less than one A. astaci genome. The absolute limit of quantification (LOQabs) was experimentally established as 10 times the LODabs. Assay performance was also assessed with samples of naturally infected and noninfected susceptible crayfish (Astacus astacus) and carrier crayfish (Pacifastacus leniusculus). The benefits and limitations of the method are discussed, and guidance to practical application and interpretation of analytical results is provided.

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

Torstein Tengs — Fri, 08 Feb 2008 01:28:03 PST

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, ''Single molecule quantification'' (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQabs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQpract) compared to real-time quantitative PCR based on standard curves.

Microarray-based method for detection of unknown genetic modifications

Torstein Tengs — Mon, 04 Feb 2008 00:11:54 PST

BackgroundDue to the increased use of genetic modifications in crop improvement, there is a need to develop effective methods for the detection of both known and unknown transgene constructs in plants. We have developed a strategy for detection and characterization of unknown genetic modifications and we present a proof of concept for this method using Arabidopsis thaliana and Oryza sativa (rice). The approach relies on direct hybridization of total genomic DNA to high density microarrays designed to have probes tiled throughout a set of reference sequences. ResultsWe show that by using arrays with 25 basepair probes covering both strands of a set of 235 vectors (2 million basepairs) we can detect transgene sequences in transformed lines of A. thaliana and rice without prior knowledge about the transformation vectors or the T-DNA constructs used to generate the studied plants.ConclusionsThe approach should allow the user to detect the presence of transgene sequences and get sufficient information for further characterization of unknown genetic constructs in plants. The only requirements are access to a small amount of pure transgene plant material, that the genetic construct in question is above a certain size (here [greater than or equal to] 140 basepairs) and that parts of the construct shows some degree of sequence similarity with published genetic elements.

Francisella philomiragia subsp. noatunensis subsp. nov., isolated from farmed Atlantic cod (Gadus morhua L.)

Torstein Tengs — Mon, 03 Sep 2007 06:31:05 PDT

Seven bacterial isolates from farmed Atlantic cod displaying chronic granulomatous disease were characterized by phenotypic and molecular taxonomic methods. The isolates were Gram-negative, facultatively intracellular, non-motile, strictly aerobic coccobacilli which produced H2S from cysteine-supplemented media and are therefore phenotypically consistent with members of the genus Francisella. Comparison of 16S rRNA gene sequences and six partial housekeeping gene sequences (groEL, shdA, rpoB, rpoA, pgm and atpA) confirmed the organism as a member of the genus Francisella, with Francisella philomiragia as its closest relative (99.3% 16S rRNA gene sequence similarity, 92.2-99.0% housekeeping gene sequence similarity). Despite the close relationship with F. philomiragia, isolates from Atlantic cod could be readily distinguished phenotypically and genetically from F. philomiragia ATCC 25015. DNA-DNA hybridization studies revealed a mean reassociation value of 68 %. Thus, on the basis of phenotypic and molecular genetic evidence, we propose that the strains isolated from Atlantic cod should be recognized as Francisella philomiragia subsp. noatunensis subsp. nov. with the type strain 2005/ 50/F292-6C (5NCIMB 14265T5LMG 23800). Francisella philomiragia ATCC 25015 (5DSM 735) is reclassified as Francisella philomiragia subsp. philomiragia subsp. nov.

Phenotypically different microalgal morphospecies with identical ribosomal RNA: a case of rapid adaptive evolution?

Torstein Tengs — Fri, 25 May 2007 00:02:56 PDT

The agents driving the divergence and speciation of freeliving microbial populations are still largely unknown. We investigated the dinoflagellate morphospecies Scrippsiella hangoei and Peridinium aciculiferum, which abound in the Baltic Sea and in northern temperate lakes, respectively. Electron microscopy analyses showed significant interspecific differences in the external cellular morphology, but a similar plate pattern in the characteristic dinoflagellate armor. Experimentally, S. hangoei grew in a wide range of salinities (0-30), whereas P. aciculiferum only grew in low salinities (0-3). Despite these phenotypic differences and the habitat segregation, molecular analyses showed identical ribosomal DNA sequences (ITS1, ITS2, 5.8S, SSU, and partial LSU) for both morphospecies. Yet, a strong interspecific genetic isolation was indicated by amplified fragment length polymorphism (FST = 0.76) and cytochrome b (cob) sequence divergence (�1.90%). Phylogenetic reconstructions based on ribosomal (SSU, LSU) and mitochondrial (cob) DNA indicated a recent marine ancestor for P. aciculiferum. In conclusion, we suggest that the lacustrine P. aciculiferum and the marine-brackish S. hangoei diverged very recently, after a marine-freshwater transition that exposed the ancestral populations to different selective pressures. This hypothetical scenario agrees with mounting data indicating a significant role of natural selection in the divergence of free-living microbes, despite their virtually unrestricted dispersal capabilities. Finally, our results indicate that identical ITS rDNA sequences do not necessarily imply the same microbial species, as commonly assumed.

Equal performance of TaqMan, MGB, molecular beacon, and SYBR green-based detection assays in detection and quantification of Roundup Ready soybean

Torstein Tengs — Tue, 02 Jan 2007 00:16:56 PST

We have tested and compared the performance of 12 different assays representing four different real-time polymerase chain reaction (PCR) chemistries in the context of genetically modified organism detection. Several different molecular beacon, SYBR Green, TaqMan, and MGB assays were designed for the event specific detection and quantification of the 3' integration junction of GTS 40-3-2 (Roundup Ready) soybean. Sensitivity as well as robustness in the presence of background DNA were tested. None of the PCR-based approaches appeared to be significantly better than any of the other, but the molecular beacon assays had the lowest efficiency and also seemed more sensitive to changes in experimental setup.

Real-time PCR monitoring of estuarine water samples for Pfiesteria piscicida, a dinoflagellate associated with fish kills and human illness

Torstein Tengs — Tue, 19 Dec 2006 05:19:44 PST

No abstract is available for this book chapter.