DNA recovery from agarose gels with a simple centrifuge-driven sephadex filtration

Conventional methods of DNA recovery from agarose gel generally require expensive equipment, extended elution times, or considerable handling of the sample after elution. We developed a simple protocol for a quick and effective recovery of DNA from agarose gels with good yield and quality. Using a Sephadex resin filled spin column, DNA fragments of 500 bp to 6 kb in an agarose gel slice were easily recovered by a 2 min centrifugation. The recovery efficiencies were over 40% -50% and the eluted DNA can be used directly for downstream application, such as polymerase chain reactions （PCR） and restriction enzyme digestion. This method could also be used to recover large DNA fragment （48 kb） without degradation. The use of Sephadex helps to remove small molecular impurities from agarose and it also reduces the chance of clogging the column filter caused by direct contact with agarose.

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus （WSSV） in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based mieroarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the mieroarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62μg/mL, with a woven long shelf life for 6 months at 4℃ or 8 months at -20℃. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.

DNA Binding and Cleavage Property of Pyrenyl-macrocyclic Polyamine Conjugate

We reported the synthesis of a simple bifunctional molecule and the interaction between this molecule and DNA were studied by UV and the DNA cleavage behavoior was determined by agarose gel electrophoresis.

Investigation of an inflammatory viral disease HBV in cardiac patients through polymerase chain reaction

In the present study 100 cardiac patients were randomly selected from the cardiology ward, Allied Hospital Faisalabad, Pakistan. All the selected cases were analysed for different parameters like Hepatitis B surface Antigen (HbsAg), Bilirubin, Alkaline phosphatase, serum glutamic pyruvic transaminase, and serum glutamic-oxaloacetic transaminase. Out of total 16% patients were lying in the age of 21-30 year, 25% in the age of 31-40 year, 35% in the age of 41-50 year, 19% in the age of 51-60 years and 5% patients in the age of 61-70 years. No subject was found positive in age group 21-30 years patients. 35% patients have higher value of SGPT while, other 26% were with higher value of SGOT. Rest of the 32 and 24% have higher ALP and Bilirubin levels, respectively. Assay profile revealed that ALP level was increased with increasing age, body mass index, Creactive protein, diabetes, smoking, sex, serum uric acid, lead, cadmium, hypercholesterolemia, lesion of liver and cardiovascular disease. The serum of the eight HbsAg positive cases were tested for the presence of HBV through PCR and no sample was found positive. At the end of the study, PCR amplified samples were run on 1.5% agarose gel to confirm the case.

Sorting Semiconducting Single-Walled Carbon Nanotubes by Water-Soluble Polyfluorene Assisted Electrophoresis and Its Application in Field-effect Transistors

We report a considerably promising method based on agarose gel electrophoresis （AGE） to separate single-walled carbon nanotubes by adding a water-soluble polyfluorene （w-PFO） as surfactant into the agarose gel. In this effective method, the AGE/w-PFO gel network will trap more semiconducting single-walled carbon nanotubes （SWNTs） with the assistance ofw-PFO, for the strong interaction between w-PFO and semiconducting species. The optical absorbance, photoluminescence emission and resonant Raman scattering characterization were used to ver- ify the separation effect. The purity of separated semiconducting species is as high as （98±1）%. The demonstrated field effect transistors give the on/off ratio and mobility about 27000 and 10.2 cm^2·V^-1·s^-1, respectively.

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

Exploiting biased reptation for continuous flow preparative DNA fractionation in a versatile microfluidic platform

A new approach is presented for preparative,continuous flow fractionation of sub-10-kbp DNA fragments,which exploits the variation in the field-dependent mobility of the DNA molecules based on their length.Orthogonally pulsed electric fields of significantly different magnitudes are applied to a microchip filled with a sieving matrix of 1.2% agarose gel.Using this method,we demonstrate a high-resolution separation of 0.5,1,2,5,and 10 kbp DNA fragments within 2 min.During the separation,DNA fragments are also purified from other ionic species.Preparative fractionation of sub-10-kbp DNA molecules plays an important role in second-generation sequencing.The presented device performs rapid high-resolution fractionation and it can be reliably manufactured with simple microfabrication procedures.