Autoradiography and Phosphorimaging.

Many of the commonly used techniques in molecular cloning depend on methods to map accurately the distribution of radioactive atoms on two-dimensional (2D) surfaces. Without this ability, methods such as Southern blotting, northern hybridizations, radiolabeled DNA sequencing, and library screening would not have been possible. In the 1970s and 1980s-the pioneering days of molecular cloning-imaging of 2D surfaces was obtained using autoradiography. In this technique, ß-particles emitted by radioactive specimens were recorded on X-ray film, producing a latent image that can be converted to a true image by developing and fixing the film. Autoradiography was a lot of fun, but it was also messy. In the impatient excitement of wanting to see how an experiment had turned out, people used to hold the newly developed X-ray films in their metal frames up to the darkroom light. Drips of the final wash would run down their arms, clothes would be stained, and shoes ruined. It is hardly surprising that autoradiography was quickly abandoned when sensitive phosphorimagers came onto the market at the end of the 1990s.

Cloning in Plasmid Vectors: Directional Cloning.

This protocol describes the standard, old-fashioned but reliable procedure for cloning linear DNA fragments whose ends are incompatible with each other but are compatible with those of the linearized vector.

Observing protein interaction dynamics to chemically defined chromatin fibers by colocalization single-molecule fluorescence microscopy.

In eukaryotic cells, the genome is packaged into chromatin and exists in different states, ranging from open euchromatic regions to highly condensed heterochromatic regions. Chromatin states are highly dynamic and are organized by an interplay of histone post-translational modifications and effector proteins, both of which are central in the regulation of gene expression. For this, chromatin effector proteins must first search the nucleus for their targets, before binding and performing their role. A key question is how chromatin effector proteins search, interact with and alter the different chromatin environments. Here we present a modular fluorescence based in vitro workflow to directly observe dynamic interactions of effector proteins with defined chromatin fibres, replicating different chromatin states. We discuss the design and creation of chromatin assemblies, the synthesis of modified histones, the fabrication of microchannels and the approach to data acquisition and analysis. All of this with the aim to better understand the complex in vivo relationship between chromatin structure and gene expression.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Clinical implication and viral mutation in basal core promoter/pre-core of hepatitis B virus C/D recombinant.

BACKGROUND AND AIM: Hepatitis B virus (HBV) C/D recombinant is predominant in Tibet in Western China. Although the geographical and ethnic distributions of the C/D recombinant have been described, the clinical implication and the characteristics of viral mutation in the basal core promoter (BCP)/pre-core (PC) region remain unclear. METHODS: A total of 174 chronic HBV carriers, including 115 with chronic hepatitis B, 45 with liver cirrhosis, and 14 with hepatocellular carcinoma, were enrolled. Using next-generation sequencing, the S and BCP/PC genes were determined and analyzed. RESULTS: Genotypes B, C2, D, and C/D recombinant were detected in 1.1% (2/174), 19.5% (34/174), 0.6% (1/174) and 78.7% (137/174) of the patients, respectively. The clinical parameters and viral mutation frequency in the BCP/PC region were compared between C2- and C/D recombinant-infected patients. The distribution of C2 and C/D did not differ by disease status or liver function. Significantly higher levels of HBV DNA (6.7 ± 1.6 vs. 5.9 ± 1.5, p = 0.014), HBeAg (263.5 vs. 20.0, p = 0.013) and A1762T/G1764A double-mutations (81.0 vs. 61.8%, p = 0.018), but a lower frequency of G1896A stop mutation (33.6 vs. 76.5%, p < 0.001) was observed in patients with the C/D recombinant than in patients with genotype C2. The clonal frequencies of A1762T, G1764A, G1896A and A1846T were lower in patients with C/D than C2. CONCLUSION: The C/D recombinant has different mutation pattern in the BCP/PC region compared with genotype C2. The lower clonal frequencies of BCP/PC mutations may explain the higher levels of HBV DNA and HBeAg in C/D-infected patients.

A novel GMO biosensor for rapid ultrasensitive and simultaneous detection of multiple DNA components in GMO products.

Since the introduction of genetically modified organisms (GMOs), there has been on-going and continuous concern and debates on the commercialization of products derived from GMOs. There is an urgent need for development of highly efficient analytical methods for rapid and high throughput screening of GMOs components, as required for appropriate labeling of GMO-derived foods, as well as for on-site inspection and import/export quarantine. In this study, we describe, for the first time, a multi-labeling based electrochemical biosensor for simultaneous detection of multiple DNA components of GMO products on the same sensing interface. Two-round signal amplification was applied by using both an exonuclease enzyme catalytic reaction and gold nanoparticle-based bio-barcode related strategies, respectively. Simultaneous multiple detections of different DNA components of GMOs were successfully achieved with satisfied sensitivity using this electrochemical biosensor. Furthermore, the robustness and effectiveness of the proposed approach was successfully demonstrated by application to various GMO products, including locally obtained and confirmed commercial GMO seeds and transgenetic plants. The proposed electrochemical biosensor demonstrated unique merits that promise to gain more interest in its use for rapid and on-site simultaneous multiple screening of different components of GMO products.

Development of event-specific PCR detection methods for genetically modified tomato Huafan No. 1.

BACKGROUND: The genetically modified (GM) tomato Huafan No. 1 is a commercial GM event created in China, and the development of specific methods for its identification and quantification is necessary under labeling regulations for genetically modified organisms (GMOs). The event-specific polymerase chain reaction (PCR) method is the most used method for identification of GMOs in routine analysis. RESULTS: The 3' junction sequence of transgene integration in GM Huafan No. 1 tomato was revealed by thermal asymmetric interlaced PCR (TAIL-PCR) and sequencing analysis. Based on the revealed 3' integration junction sequence, both conventional and real-time PCR assays were developed and validated for GM Huafan No. 1 tomato identification. In the conventional PCR assay, the limit of detection (LOD) was 20 haploid genome copies. In the real-time PCR assay, the LOD and limit of quantification (LOQ) were estimated to be 5 and 10 tomato haploid genome copies, respectively. Furthermore, the developed PCR methods were well validated by in-house validation. CONCLUSION: Our results suggest that the developed event-specific PCR methods can be routinely used for identification and quantification of GM Huafan No. 1 tomato.

The fate of recombinant plasmids during composting of organic wastes.

Composting was investigated as a means for safe disposal of organic waste containing bacteria that carry transgenes in recombinant plasmids. To generate model recombinant plasmids, a mobile IncQ plasmid, RSF1010, and a non-mobile plasmid, pGFP, were genetically modified to carry a DNA segment encoding both green fluorescent protein and kanamycin resistance and were designated as RSF1010-GFPK and pGFPK. Escherichia coli (E. coli) C600 harboring these plasmids were inoculated into chicken manure specimens that were placed in compost at 20 and 60 cm from the bottom of a 1.0-m high compost bin. Control specimens were held at ambient temperature. By day 10, compost temperatures at the lower and upper levels of the bin had reached 45.3 and 61.5 degrees C, respectively, and at both levels the target E. coli had been inactivated and the plasmids had lost their capacity to be transformed or mobilized. Furthermore, based on real time Polymerase chain reaction (PCR), the transgene fragments along with the host chromosomal DNA fragment from specimens at the upper level had been degraded beyond the detection limit. However, at the lower level where temperatures remained below 48 degrees C these fragment persisted to day 21. At ambient temperatures (0-8 degrees C), the E. coli, plasmids and the transgene fragments persisted in manure specimens throughout the 21 day test period. The study showed the potential for composting as a safe procedure for disposal of bacteria carrying transgenes in recombinant plasmids.

Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhanced surface plasmon resonance imaging.

Technologies today available for the DNA detection rely on a combination of labeled probes hybridized to target sequences which are amplified by polymerase chain reaction (PCR). Direct detection methods that eliminate the requirement for both PCR and labeling steps could afford faster, cheaper and simpler devices for the analysis of small amounts of unamplified DNA. In this work we describe the results obtained in the ultrasensitive detection of non-amplified genomic DNA. We analyzed certified reference materials containing different amounts of genetically modified DNA by using a detection method which combines the nanoparticle-enhanced surface plasmon resonance imaging (SPRI) biosensing to the peptide nucleic acids (PNAs) improved selectivity and sensitivity in targeting complementary DNA sequences. The method allowed us to obtain a 41 zM sensitivity in targeting genomic DNA even in the presence of a large excess of non-complementary DNA.

Analysis of DNA ligation by microchip electrophoresis.

We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 bp with good quantification (DNA concentration, <8 ng/l) within 5 min, for the analysis of DNA ligation. On analysis of an electropherogram of a ligation mixture of the pTAC1-T vector and a 789 bp PCR-amplified DNA fragment, the presence of recombinant DNA was easily detected by comparison with an electropherogram obtained without ligase. On analysis of a ligation mixture of pUC19/Eco RI without alkaline phosphatase treatment and a 667 bp Eco RI-digested fragment of foreign DNA, several peaks observed in the electropherogram corresponded to the formation of monomeric and polymeric insert DNAs, self-ligated vector DNA, and recombinant DNA. On the other hand, several peaks were also observed in the electropherogram of the ligation mixture of pUC19/Eco RI with alkaline phosphatase treatment and the 667 bp Eco RI-digested fragment of foreign DNA, the fluorescence intensity corresponding to recombinant DNA apparently being increased. These results indicate the potential of microchip electrophoresis for the analysis of DNA ligation, it offering high resolution in a short time.

Toxoplasma gondii: expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages.

In this study, effects of GRA1 organelle-targeted expression on macrophage functions were investigated. The recombinant plasmid pCMV/myc/ER-GRA1 was constructed and then was transfected into murine macrophage RAW264.7 by Lipofectamine, selected by resistance of G418. The selected mono-clone cell line was named ER-GRA1-RAW264.7. The expression of GRA1 was localized in ER of ER-GRA1-RAW264.7 cells by indirect immunofluorescence detection. GRA1 mRNA expression level in ER-GRA1-RAW264.7 cell was significantly enhanced with a concomitant increase in its growth and adherence activity. Fluorescence intensity of intracellular calcium in ER-GRA1-RAW264.7, ER-ctrl-RAW264.7 and RAW264.7 cells in the presence of 1 mmol/l arachidonic acid (AA) were assayed by confocal microscopy using calcium-sensitive dye, Fluo-3 AM. Cytoplasm [Ca2+]i peaked at about 18 s after AA treatment, and cytoplasm [Ca2+]i of RAW264.7 cell almost instantly stepped up after AA was added, and peaked in 3 s, with a minor cytoplasm [Ca2+]i vibration subsequently. These results demonstrated that the expression of GRA1 in ER of macrophages promotes both growth and adherence of macrophages and modulates the intracellular calcium release stimulated by AA.

Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples.

Real time PCR assays were developed to detect and quantify the transgene DNA of a commercially released Bacillus thuringiensis (Bt) corn (Zea mays L.) hybrid (DKC42-23), which was derived from the event MON863 and also carried a neomycin phosphotransferase gene (the nptII gene). We applied the real time PCR assays to investigate the persistence of the transgene DNA in a field trial grown with DKC42-23 over 3 years, in combination with bacterial natural transformation. The results showed that under continuous cultivation of DKC42-23, its transgene DNA was detectable in the field plots all year around. Meanwhile, when soil DNA extracts from DKC42-23 plots were used as donor in bacterial natural transformation, successful recovery of kanamycin resistant (Km(R)) transformants indicated that the nptII gene carried by DKC42-23 could be taken up and integrated into naturally competent Pseudomonas stutzeri pMR7 cells, leading to the restoration of the antibiotic resistance of P. stutzeri pMR7. However, after the cultivation of a soybean line in the same plots for the subsequent growing season, the presence of transgene DNA of DKC42-23 was reduced to undetectable levels at the end of that growing season. Therefore, existing corn-soybean crop rotation practices reduce the availability of transgene DNA in soil and thus minimize the risks that might be attributable to horizontal gene transfer. The real time PCR assays are useful for investigating the persistence of transgene DNA derived from the MON863 event in soil environments.

Cellular barcoding tool for clonal analysis in the hematopoietic system.

Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.

A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco.

A novel Potato virus Y (PVY) isolate, L26, recovered from a Frontier potato line was initially typed as a PVY(NTN) strain using multiplex RT-PCR and serological assays. However, L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of the PVY (NTN) strain. The whole genome sequence was determined for L26 and two other PVY(NTN) isolates, HR1 and N4, from Idaho that did induce vein necrosis in tobacco. The sequence of all three isolates was similar to typical European PVY(NTN) isolates that contain three recombination junctions in their genome. The sequence of the L26 genome was nearly identical to the genomes HR1, N4, and to a previously characterized PVY(NTN) isolate, 423-3, differing by only five nucleotides in the entire ca. 9.7-kb genome, only one resulting in a corresponding amino acid change, D-205 to G-205 in the central region of HC-Pro. Two "signature" amino acid residues, thought involved in induction of the vein necrosis syndrome in tobacco, K-400 and E-419, were present in the C-terminal region of HC-Pro of all three isolates. Multiple alignment of the whole genome sequences of L26 and other PVY(NTN) isolates whose phenotype in tobacco has been reported, suggests that a single nucleotide change (A-1,627 to G-1,627) resulting in the single amino acid change (D-205 to G-205) in the HC-Pro cistron of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Secondary structure modeling of the HC-Pro protein predicts the G-205 residue, and the previously identified residues K-400 and E-419, would all be located on the exposed surface of the protein. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and includes other element(s), in addition to the C-terminal fragment of HC-Pro.

Waste recombinant DNA: effectiveness of thermo-treatment to manage potential gene pollution.

Heating at 100 degrees C for 5-10 min is a common method for treating wastewater containing recombinant DNA in many bio-laboratories in China. In this experiment, plasmid pET-28b was used to investigate decay efficiency of waste recombinant DNA during thermo-treatment. The results showed that the decay half-life of the plasmid was 2.7-4.0 min during the thermo-treatment, and even heating for 30 min the plasmids still retained some transforming activity. Low pH promoted the decay of recombinant DNA, but NaCl, bovine serum albumin and EDTA, which existed in the most wastewater from bio-laboratories, protected DNA from degradation. Thus, the decay half-life of plasmid DNA may be longer than 2.7-4.0 min practically. These results suggest that the effectiveness of heating at 100 degrees C for treating waste recombinant DNA is low and a gene pollution risk remains when those thermo-treated recombinant DNAs are discharged into the environment. Therefore other simple and effective methods should be developed.

Validity assessment of the detection method of maize event Bt10 through investigation of its molecular structure.

In March 2005, U.S. authorities informed the European Commission of the inadvertent release of unauthorized maize GM event Bt10 in their market and subsequently the grain channel. In the United States measures were taken to eliminate Bt10 from seed and grain supplies; in the European Union an embargo for maize gluten and brewer's grain import was implemented unless certified of Bt10 absence with a Bt10-specific PCR detection method. With the aim of assessing the validity of the Bt10 detection method, an in-depth analysis of the molecular organization of the genetic modification of this event was carried out by both the company Syngenta, who produced the event, and the European Commission Joint Research Centre, who validated the detection method. Using a variety of molecular analytical tools, both organizations found the genetic modification of event Bt10 to be very complex in structure, with rearrangements, inversions, and multiple copies of the structural elements (cry1Ab, pat, and the amp gene), interspersed with small genomic maize fragments. Southern blot analyses demonstrated that all Bt10 elements were found tightly linked on one large fragment, including the region that would generate the event-specific PCR amplicon of the Bt10 detection method. This study proposes a hypothetical map of the insert of event Bt10 and concludes that the validated detection method for event Bt10 is fit for its purpose.

Simultaneous detection of recombinant DNA segments introduced into genetically modified crops with multiplex ligase chain reaction coupled with multiplex polymerase chain reaction.

We developed a multiplex polymerase chain reaction (PCR)-multiplex ligase chain reaction (LCR) (MPCR-MLCR) technique as a novel approach for the simultaneous detection of recombinant DNA segments (e.g., promoters, trait genes, and terminators) of genetically modified (GM) crops. With this technique, target DNA regions were amplified by multiplex PCR, the PCR products were then subjected to multiplex LCR as template DNAs, and the LCR products were then analyzed by polyacrylamide gel electrophoresis and subsequent fluorescent scanning. Seven recombinant DNA segments commonly introduced into some GM crop lines were selected as target DNA regions. In addition, another MPCR-MLCR system for the simultaneous detection of three endogenous DNA segments was designed as a positive control test. The specificity and sensitivity of the method were examined. The method allowed us to detect GM crops comprehensively and is expected to be utilized for efficient screening of GM crops into which any one of the seven recombinant DNA segments have been introduced, and for profiling the segments.

The antitumor and immunoadjuvant effects of IFN-alpha in combination with recombinant poxvirus vaccines.

PURPOSE: IFN-alpha is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-alpha with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen. EXPERIMENTAL DESIGN: The phenotypic and functional effects of IFN-alpha were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-alpha administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-alpha and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice. RESULTS: We identified a dose and schedule of IFN-alpha that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8(+)) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-alpha distal to the site of vaccination. The combination of IFN-alpha and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA(+) adenocarcinomas. In mice with pancreatic tumors, IFN-alpha slowed tumor growth, induced CTL activity, and increased CD8(+) tumor-infiltrating lymphocytes. CONCLUSIONS: These data suggest that IFN-alpha can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.

One-step DNA-programmed growth of luminescent and biofunctionalized nanocrystals.

Colloidal semiconductor nanocrystals are widely used as lumiphores in biological imaging because their luminescence is both strong and stable, and because they can be biofunctionalized. During synthesis, nanocrystals are typically passivated with hydrophobic organic ligands, so it is then necessary either to replace these ligands or encapsulate the nanocrystals with hydrophilic moieties to make the lumiphores soluble in water. Finally, biological labels must be added to allow the detection of nucleic acids, proteins and specific cell types. This multistep process is time- and labour-intensive and thus out of reach of many researchers who want to use luminescent nanocrystals as customized lumiphores. Here, we show that a single designer ligand--a chimeric DNA molecule--can controllably program both the growth and the biofunctionalization of the nanocrystals. One part of the DNA sequence controls the nanocrystal passivation and serves as a ligand, while another part controls the biorecognition. The synthetic protocol reported here is straightforward and produces a homogeneous dispersion of nanocrystal lumiphores functionalized with a single biomolecular receptor. The nanocrystals exhibit strong optical emission in the visible region, minimal toxicity and have hydrodynamic diameters of approximately 6 nm, which makes them suitable for bioimaging. We show that the nanocrystals can specifically bind DNA, proteins or cells that have unique surface recognition markers.