Ethidium Bromide Agarose Gel Electrophoresis Research Articles

Abstract A196: Characterization of stability and biological activity of the cancer gene therapy biologic SNS01 following storage at ambient and freezing temperatures

Abstract Introduction: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post-translational addition of a hypusine residue. Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identified as markers of neoplastic growth and metastasis. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modified eIF5A. In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and overexpression of a non-hypusinable mutant of eIF5A potently induces apoptosis in multiple cancer cell types. SNS01 is a nanoparticle specifically designed for the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B-cell-specific (B29) promoter driving expression of a pro-apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI). Methods: Ethidium-bromide agarose gel electrophoresis was used to detect heparin-mediated release of nucleic acids from nanoparticles. Dynamic light scattering (DLS) was used to assess the short-term stability of SNS01 by monitoring changes in size distribution, polydispersity, and zeta potential. In vitro biological activity of SNS01 was assessed using RT-qPCR to measure eIF5A and eIF5AK50R transgene expression as well as FACS detection of AnnexinV-FITC labeled apoptotic cells. Anti-tumoral activity of SNS01 was evaluated using a subcutaneous human myeloma model (KAS-6/1 cells) in SCID mice with twice-weekly intra-venous injections of SNS01. Results: SNS01 nanoparticles were found to be relatively stable for as long as 48 hours at room temperature with no significant loss in nucleic acid integrity. The nanoparticles had an average diameter of 146.2 nm, a polydispersity of 0.287 and an average zeta potential of 35.3+0.7 mV. These parameters were quite stable over 48 hours. SNS01 could also be frozen at − 80°C for at least one month with no observable loss in biological activity. Twice-weekly intra-venous injections into myeloma tumor-bearing mice of a frozen SNS01 preparation was found to have anti-tumoral activity comparable to freshly prepared SNS01 (frozen SNS01: 89 % tumor inhibition, p = 0.00158; SNS01: 84 % tumor inhibition, p = 0.00003). Conclusions: SNS01 is relatively stable at room temperature and was also found to be stable and biologically active following freezing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A196.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

AbstractA monolithic stationary phase was prepared via free radical co‐polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized with 2‐chloro‐N,N‐diethylethylamine hydrochloride (DEAE‐Cl) for anion‐exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α‐pUC19 culture in a ribonuclease/protease‐free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single‐stage 5 min anion‐exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr‐AGE), enzyme restriction analysis and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. This non‐toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level. Copyright © 2007 Society of Chemical Industry

Detection of Bartonella henselae and Afipia felis DNA by polymerase chain reaction in specimens from patients with cat scratch disease.

Polymerase chain reaction (PCR) amplification and colorimetric identification of amplicons were performed to detect Bartonella henselae and Afipia felis DNA in specimens from patients who were clinically and histologically suspected of having cat scratch disease. PCR products were revealed using 2% ethidium bromide agarose-gel electrophoresis and identified with specific probes in a commercial colorimetric hybridization assay (DEIA) (GEN-ETI-K; DiaSorin, Italy). Six paraffin-embedded lymph node biopsies from 18 patients as well as 18 samples of peripheral whole blood and 18 sera were investigated. Bartonella henselae DNA was recovered from the whole blood of four patients, and Bartonella henselae and Afipia felis DNA were detected in one patient's lymph node biopsy. This study suggests that PCR-DEIA is sufficiently sensitive to be considered feasible for the molecular diagnosis of cat scratch disease.

Detection of Mycobacterium paratuberculosis in stool samples of patients with inflammatory bowel disease by IS900-based PCR and colorimetric detection of amplified DNA

The hypothesis that Crohn's disease (CD) and ulcerative colitis (UC) may result from mycobacterial infection has been proposed. Among the atypical mycobacteria, Mycobacterium paratuberculosis has been often involved in the pathogenesis of CD and UC. The polymerase chain reaction (PCR) with a single primer pair from the nucleotide sequence of the `Insertion Sequence' IS900 of M. paratuberculosis followed by a non-isotopic ELISA-like detection method of amplification products for the specific detection of M. paratuberculosis in human feces was developed. Fifteen (46.8%) of the 32 stool samples from patients with histologically confirmed CD, and nine (33.3%) of the 27 stool samples from patients with UC had a 229-bp fragment of M. paratuberculosis DNA detected by ethidium bromide agarose gel electrophoresis. Of the 41 stool samples used as negative control, 30 were from healthy subjects, nine from patients with other non-specific gastrointestinal diseases, and two from patients with colon cancer. Only one of these samples, namely from one of the patients with colon cancer, was positive by PCR. With regard to cultural technique, eight stool samples from patients with CD, five samples from patients with UC and one sample from a patient with colon cancer allowed the mycobacterial growth. The amplified PCR products were identified by using a colorimetric detection procedure designed DNA Enzyme ImmunoAssay (DEIA), based on the hybridization of the denatured DNA with a non-radioactively labelled inter-primer specific oligonucleotide probe. Severe precautions were taken to exclude either the possible contamination among the samples or false-positive results. Our findings confirm other works in which M. paratuberculosis has been considered the putative etiologic agent responsible for CD and UC. In addition, the newly developed PCR-DEIA technique, revealing a higher sensitivity than cultural technique and being much more rapid, represents a useful tool for both epidemiological and therapeutic purposes.

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens.

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10(2) bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans. The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.

Polymerase Chain Reaction Amplification for Direct Detection of Chicken Anemia Virus DNA in Tissues and Sera

Direct detection of chicken anemia virus (CAV) DNA in tissues and sera was investigated by a polymerase chain reaction (PCR) assay. Using a pair of primers constructed to amplify the coding sequence of the CAV DNA genome, the PCR assay was shown to be extremely sensitive, being able to detect 1 fg of CAV replicative form DNA. The oligonucleotide primers used for the PCR yielded 583 base-pair (bp) amplified product, which was sized by ethidium bromide-agarose gel electrophoresis. Tissue samples from seven cases of suspected chicken infectious anemia were obtained for CAV isolation. DNA extracted from the homogenized suspension of pooled tissues of each case was analyzed by the PCR assay. Results showed that five of the seven cases were positive for CAV DNA by PCR, whereas CAV was isolated from four cases only. The PCR assay also detected CAV DNA in two of 37 serum samples from disease-free chickens. The specificity of PCR was confirmed by chemiluminescence dot-blot analysis of the amplified products.

Unusual DNA structures at the integration site of an HIV provirus

Supercoiled pHXBc2 DNA (containing the genome of the human immunodeficiency virus type 1 and human sequences) migrated more slowly than linear DNA in native and ethidium bromide agarose gel electrophoresis at 4.5 volts/cm, suggesting the presence of unusual DNA structures. S1 nuclease analysis of pHXBc2 revealed two S1 hypersensitive sites. Site I was located within a 25 bp direct repeat in host DNA 0.6 kB upstream from the 5′ LTR. Site II was mapped 0.2 kB upstream from the vif gene start site. Sequence analysis showed that Site I sequences could assume different unusual DNA structures, whereas sequences at Site II could assume either slipped or H-DNA forms. Unusual DNA structures in host DNA may be associated with active chromatin regions and may favor proviral integration.