Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

Application of cellulase-polyamidoamine dendrimer-modified silica for microwave-assisted chitosan enzymolysis

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with a precise molecular structure, high geometric symmetry, and a large number of terminal groups. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation and characterized by Fourier transform infrared spectroscopy and elemental analysis. A novel immobilized cellulase was developed based on enzyme immobilization onto the prepared PAMAM-grafted silica and applied in microwave-assisted chitosan enzymolysis. The results show that the efficiency of cellulase immobilization increased with increasing generations of PAMAM. A high enzymatic hydrolysis efficiency was obtained for a 7 mg ml^?1 chitosan solution at pH 6.2 and 50 °C with 40 W microwave-assisted enzymolysis (20 min) compared with a conventional enzymolysis protocol (3 h). The experimental results indicate that this rapid and efficient enzymolysis method combines the advantages of both PAMAM and microwave-assisted technology, which can be adapted to high-throughput enzyme assay in biochemical and clinical research.     

Immobilization of cellulase on polyamidoamine dendrimer-grafted silica

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups, and is suitable to carry biomolecules due to its affinity and biocompatibility. In this study, PAMAM was grafted onto the surface of silica by microwave irradiation. A novel media was developed through immobilizing cellulase onto the prepared PAMAM-grafted silica by adsorption and crosslinking methods and applied in hydrolysis of carboxymethyl cellulose. The results demonstrate that the enzyme binding capacity and enzymolysis efficiency increased with generations of PAMAM. The properties of the immobilized cellulase-PAMAM-grafted silica were investigated, which possessed high enzymatic activity and exhibited better stability with respect to pH, temperature compared with free enzyme. The optimal immobilization conditions for adsorption and crosslinking method were respectively obtained at 5 and 4 mg ml⁻¹ cellulase for 2 h of immobilization. A high enzymolysis efficiency was achieved by employing pH 4.8 and 5.8 substrate solution at 60 °C for adsorbed and crosslinked cellulase, respectively. After repeated three run cycles, the retained activities were found to be 75% and 82%. The results indicate that the PAMAM has a good performance as a carrier, and can be potentially adapted to support other biomacromolecules.     

Polyamidoamine dendrimer as a spacer for the immobilization of glucose oxidase in capillary enzyme microreactor

Polyamidoamine dendrimer (PAMAM) is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups. In this study, different generations of PAMAM (G0-G4) were introduced onto the inner wall of fused-silica capillaries by microwave irradiation and a new type of glucose oxidase (GOx) capillary enzyme microreactor was developed based on enzyme immobilization in the prepared PAMAM-grafted fused-silica capillaries. The optimal enzymolysis conditions for β-d-glucose in the microreactor were evaluated by capillary zone electrophoresis. In addition, the enzymolysis efficiencies of different generations of PAMAM-GOx capillary enzyme microreactor were compared. The results indicate that enzymolysis efficiency increased with increasing generations of PAMAM. The experimental results provide the possibility for the development and application of an online immobilized capillary enzyme microreactor.     

Innovative metal oxide-based substrates for DNA microarrays

In the present report, we propose a novel approach to synthesize DNA microarrays that employs immobilization of the nucleic acid molecules onto zinc and iron oxide surfaces through their phosphate backbone. Oxide films were prepared by the sol–gel technique and the resulting surfaces were characterized especially with respect to surface chemistry and morphological features by both X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). ZnO films annealed at T ? 300 °C show the most promising surface features to be employed for DNA microarray preparation, i.e. high density of binding sites (hydroxyl groups), smooth and homogeneous surfaces, high optical transmittance in the visible spectral range suitable for detection using fluorescence, and easy handling during preparation procedures. The analysis of nucleic acid retention on the oxide layers was performed by the scanning of dye-labelled DNA previously printed on the substrate using the DNA microarray robotic arm. Clearly visible spots with regular shape were revealed above the background noise indicating that anchoring of the DNA on the treated surface is efficient and does not interfere with hybridization processes. The use of suitably engineered zinc oxide film makes the immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

The effect of surface probe density on DNA hybridization

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.     

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.     

Gold nano particle decorated graphene core first generation PAMAM dendrimer for label free electrochemical DNA hybridization sensing

A novel first generation (G1) poly(amidoamine) dendrimer (PAMAM) with graphene core (GG1PAMAM) was synthesized for the first time. Single layer of GG1PAMAM was immobilized covalently on mercaptopropionic acid (MPA) monolayer on Au transducer. This allows cost effective and easy deposition of single layer graphene on the Au transducer surface than the advanced vacuum techniques used in the literature. Au nano particles (17.5 nm) then decorated the GG1PAMAM and used for electrochemical DNA hybridization sensing. The sensor discriminates selectively and sensitively the complementary double stranded DNA (dsDNA, hybridized), non-complementary DNA (ssDNA, un-hybridized) and single nucleotide polymorphism (SNP) surfaces. Interactions of the MPA, GG1PAMAM and the Au nano particles were characterized by Ultra Violet (UV), Fourier Transform Infrared (FTIR), Raman spectroscopy (RS), Thermo gravimetric analysis (TGA), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Cyclic Voltmetric (CV), Impedance spectroscopy (IS) and Differntial Pulse Voltammetry (DPV) techniques. The sensor showed linear range 1×10(-6) to 1×10(-12) M with lowest detection limit 1 pM which is 1000 times lower than G1PAMAM without graphene core.     

Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal –NH₂ groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.     

Synthesis and characterization of nanoscale dendritic RGD clusters for potential applications in tissue engineering and drug delivery

Spatial control over the distribution and the aggregation of arginine-glycine-aspartate (RGD) peptides at the nanoscale significantly affects cell responses. For example, nanoscale clustering of RGD peptides can induce integrins to cluster, thus triggering complete cell signaling. Dendrimers have a unique, highly branched, nearly spherical and symmetrical structure with low polydispersity, nanoscale size, and high functionality. Therefore, dendrimers are a class of ideal scaffold for construction of nanoscale dendritic RGD clusters in which RGD loading degree and cluster size can be finely adjusted. This new type of nanoscale dendritic RGD cluster will aid us to better understand the impact of spatial arrangement of RGD on cellular responses and to engineer RGD to trigger more favorable cellular responses. In this study, nanoscale dendritic RGD clusters were synthesized based on Starburst anionic G3.5 and cationic G4.0 polyamidoamine (PAMAM) dendrimers. The multiple terminal functional groups on the outermost layer of the dendrimer were coupled with RGD tripeptides. Biofunctionalized dendrimer structures were found to be highly dependent on the generation and the extent of peptide modification (ie, number of peptides per PAMAM dendrimer). Fluorescein isothiocyanate (FITC)-conjugated PAMAM dendrimers were utilized to monitor cellular internalization of dendrimers by adherent fibroblasts. Anionic G3.5-based dendritic RGD clusters have been shown to have no negative effect on fibroblast viability and a concentration-dependent effect on lowering cell adhesion on tissue culture polystyrene (TCPS) as that of free RGD. A similar concentration-dependent effect in cell viability and adhesion was also observed for cationic G4.0-based dendritic RGD clusters at lower but not at high concentrations. The results imply that the synthesized nanoscale dendritic RGD clusters have great potential for tissue engineering and drug delivery applications.     

In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.     

LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E

Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.     

DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation

DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.     

PAMAM-Lys,a Novel Vaccine Delivery Vector,Enhances the Protective Effects of the SjC23 DNA Vaccine against Schistosoma japonicum Infection

Background

Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection.

Methodology/Principal Findings

Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ.

Conclusion/Significance

Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection.     

Osteogenic Surface Modification Based on Functionalized Poly-P-Xylylene Coating

The biotechnology to immobilize biomolecules on material surfaces has been developed vigorously due to its high potentials in medical applications. In this study, a simple and effective method was designed to immobilize biomolecules via amine-N-hydroxysuccinimide (NHS) ester conjugation reaction using functionalized poly-p-xylylene coating on material surfaces. The NHS ester functionalized coating is synthesized via chemical vapor deposition, a facile and solvent-less method, creating a surface which is ready to perform a one-step conjugation reaction. Bone morphogenetic protein 2 (BMP-2) is immobilized onto material surfaces by this coating method, forming an osteogenic environment. The immobilization process is controlled at a low temperature which does not damage proteins. This modified surface induces differentiation of preosteoblast into osteoblast, manifested by alkaline phosphatase (ALP) activity assay, Alizarin Red S (ARS) staining and the expression of osteogenic gene markers, Alpl and Bglap3. With this coating technology, immobilization of growth factors onto material surface can be achieved more simply and more effectively.     

High quality SNPs/Indels mining and characterization in ginger from ESTs data base

Ginger (Zingiber officinale Rosc.) is an important herb of the family Zingiberaceae. It is accepted as a universal cure for a multitude of diseases in Indian systems of medicine and its rhizomes are equally popular as a spice ingredient throughout Asia. SNPs, the definitive genetic markers, representing the finest resolution of a DNA sequence, are abundantly found in populations having a lower rate of mutation and are used for genomic analysis. The public ESTs sequences mostly lack quality files, making high quality SNPs detection more difficult since it is exclusively based on sequence comparisons. In the present study, current dbESTs of NCBI was mined and 38115 ginger ESTs sequences were obtained and assembled into contigs using CAP3 program. In this analysis, recent software tool QualitySNP was used to detect 11523 potential SNPs sites, 8810 high quality SNPs and 1008 indels polymorphisms with a frequency of 1.61 SNPs / 10 kbp. Of ESTs libraries generated from three ginger tissues together, rhizomes had a frequency of 0.32 SNPs and 0.03 indels per 10 kbp whereas the leaves had a frequency of 2.51 SNPs and 0.23 indels per 10 kbp and root is showing relative frequency of 0.76/10 kbp SNPs and 0.02/10 kbp indels. The present analysis provides additional information about the tissue wise presence of haplotypes (222), distribution of high quality exonic (2355) and intronic (6455) SNPs and information about singletons (7538) in addition to contigs transitions and transversions ratio (0.57). Among all tissue detected SNPs, transversions number is higher in comparison to the number of transitions. Quality SNPs detected in this work can be used as markers for further ginger genetic experiments.     

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.     

Eye color prediction using single nucleotide polymorphisms in Saudi population

BackgroundDNA prediction of eye color represent one application of the externally visible characteristics (EVC), which attained growing interest in the field of DNA forensic phenotyping. This is mainly due to its ability to narrow the pool of suspects without the need to compare any retrieved DNA material from the crime scene to a reference DNA. Several methods and multiplex genetic panel were proposed with variable prediction accuracy between different populations. However, such panel was not previously tested in the Saudi population, nor any populations of the Middle East and North Africa origin.MethodA panel of eleven single nucleotide polymorphisms (SNPs) was tested for their association with three eye colors (brown, hazel, and intermediate) in 80 volunteer Saudi individuals. SNPs and haplotype association test with eye colors were performed to identify the top significant SNPs with the three eye colors. Also, multinomial logistic regression was used to construct the prediction model using a training set of 60 subjects, and a validation set of 20 subjects. The goodness of fit parameter of the model to correctly predicts each eye color as compared to the other was performed.ResultsEye color was significantly associated with rs12913832, rs7170852, and rs916977 that are located within HERC2. SNP rs12913832 was the top significant SNP (p-value = 1.78E?15) that accounted for the association in this region, as the other SNPs were not significant after adjusting for rs12913832. A prediction model containing five SNPs showed high prediction accuracy with Area Under the receiver operating characteristic Curves (AUC) equals to 0.95 and 0.83 for brown and intermediate eye colors, respectively. However, the model’s performance was very low for predicting the hazel eye color with AUC equals 0.75.DiscussionDespite the small sample size of our study, we reported very significant SNP associations with eye color. Our model to predict eye colors based on DNA material showed high accuracy for brown and intermediate eye colors. The eye color prediction-model underperformed for the hazel eye colors, suggesting that larger sample size, as well as more comprehensive set of SNPs, could improve the model-prediction accuracy.     

Comprehensive SNP Scan of DNA Repair and DNA Damage Response Genes Reveal Multiple Susceptibility Loci Conferring Risk to Tobacco Associated Leukoplakia and Oral Cancer

Polymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants affecting OSCC development in Indian population, the “maximally informative” method of SNP selection from HapMap data to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253 leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in predisposition to OSCC and leukoplakia.     

Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.