Quantitation of plasmid DNA in aqueous two-phase systems by fluorescence analysis

The development of aqueous two-phase systems for plasmid purification from Escherichia coli cell lysates requires a reliable DNA quantitation method. Plasmid DNA was quantified by fluorescence using PicoGreen nucleic acid stain. Linearity was obtained up to 40 ng plasmid ml^–1. Two polyethyleneglycol (PEG)/salt systems were studied, PEG 600/K₂HPO₄ and PEG 300/K₂HPO₄. The average plasmid recovery was 41% in the bottom phase of the first system and 35% in the top phase of the second system. This method has proved to be simple and reproducible.     

Fluorometric determination of DNA in mammalian cell cultures by PicoGreen

An intercalating fluorochrome, PicoGreen, was assessed for its ability to determine the concentration of DNA in clarified mammalian cell culture broths containing monoclonal antibodies. Fluorescent signal suppression was ameliorated by sample dilution or by performing the assay above the pI of secreted IgG. The source of fluorescence in clarified culture broth was validated by incubation with RNase A and DNase I. At least 91.8% of fluorescence was attributable to nucleic acid and pre-digestion with RNase A was shown to be a requirement for successful quantification of DNA in such samples.     

Assay for the quantification of intact/fragmented genomic DNA

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.     

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml^–1) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.     

Validity of DNA analysis to determine cell numbers in tissue engineering scaffolds

The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell lines and primary cells to λDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 × 10⁵ cells ml⁻¹. From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate standard to use for the assay was from the cell type being examined.     

A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair

Neutral filter elution assay is one of the methods used for detection of DNA double strand breaks (DSBs). However, it is laborious, expensive, and hazardous (radiolabeled precursors for DSB detection and scintillation counter for quantification), making it a less preferred method for DSB detection. In the present study, an attempt was made to improve the existing neutral filter elution assay by making use of fluorescent dye (PicoGreen) and microfiltration assembly for eluting the fragmented DNA, thereby reducing the cost and time required for the assay. We studied the effect of dye dilution, pH conditions, and cell number as a part of method standardization. X-ray dose–response and repair kinetics in lymphocytes as well as cell lines were studied for validating the sensitivity of the assay. A linear dose–response relationship for DSBs was observed at a cell number of 4 × 10⁵ cells, a dye dilution of 500-fold, and at pH 10. Repair kinetics revealed a time-dependent repair of DSBs up to 360 min of posttreatment, indicating its usefulness in DSB repair studies. In conclusion, the present modified method is more efficient (in terms of cell number), cost effective, less time-consuming, and less hazardous compared to the existing method.     

Comparison of three common DNA concentration measurement methods

Accurate measurement of DNA concentration is important for DNA-based biological applications. DNA concentration is usually determined by the ultraviolet (UV) absorption, fluorescence staining, and diphenylamine reaction methods. However, the best method for quality assurance of measurements is unknown. Here, we comprehensively compared these methods using different types of samples. We found that all three methods accurately determined the concentrations of high-purity DNA solutions. After digestion of DNA samples, concentration measurements revealed that the PicoGreen dye method was very sensitive to the degradation of DNA. The three methods displayed different anti-jamming ability when contaminants such as transfer RNA (tRNA), protein, and organic chemicals were included in DNA solutions. The diphenylamine reaction method gave the highest accuracy, with an average error of approximately 10% between measured and true values. The PicoGreen dye method was influenced by tRNA and protein, and the UV absorption method was susceptible to all kinds of impurities. Overall, the diphenylamine reaction method gave the most accurate results when DNA was mixed with contaminants, the PicoGreen dye method was most suitable for degraded DNA samples or DNA extracted from processed products, and the UV absorbance method was best for evaluating the impurities in DNA solutions.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

一种新型可用于DNA分子量标准的质粒构建

将首尾带有EcoR Ⅰ酶切位点的2．0kb、1．5kb、1．0kb、0．75kb、0．5kb、0．25kb六个长度的片段逐步连接到pGEM-3zf( )质粒上的B．am H I位点中,构建的质粒用EcoR Ⅰ进行单酶切,经电泳可以获得七条DNA带与设计结果完全一致,可用于DNA电泳试验中分子量标准。     

萘降解质粒ND1.860和NAH7的DNA同源性研究

通过DNA:DNA杂交技术,用NAH7质粒的全部EcoR Ⅰ片段或ECOR Ⅰ A片段作为~(32)P标记的DNA探针,研究了萘降解质粒ND1.860和NAH7之间的DNA同源性。在ND1.860的9个HindⅢ片段中,5个与NAH7有同源性,其中3个与NAH7的编码萘降解途径的EcoR Ⅰ A片段有同源性。     

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.     

外源质粒DNA对小鼠脾脏物质能量代谢的影响

研究外源质粒DNA经胃肠道途径对免疫激活状态下小鼠脾脏代谢的影响。给Balb/c小鼠灌喂质粒pcDNA3 200μg,分别在灌喂后4h和18h分离脾脏,提取脾脏总RNA。利用Affymetrix基因表达谱芯片对灌喂质粒pcDNA3后的Balb/c小鼠脾脏进行基因表达谱研究。采用Genmmapp和MAPPFinder软件进行功能聚类分析。对上调倍数两倍及两倍以上的基因进行功能聚类发现,灌喂后4h,外源质粒刺激小鼠脾脏产生免疫应答的同时,脾脏中嘌呤代谢及蛋白质合成,胆固醇合成、脂肪酸合成、糖酵解、三羧酸循环及线粒体氧化磷酸化途径等大量代谢过程受到诱导。在灌喂后18h也得到类似的结果。结果表明外源质粒DNA可通过胃肠道途径吸收影响小鼠脾脏物质能量代谢过程。结果有助于在分子水平上深入了解外源质粒DNA经胃肠道吸收后的作用机制。     

Immunogold for detection of antigen on nitrocellulose paper

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.     

Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures

A simple and quick method has been developed to isolate plasmid DNA from Lactococcus lactis using overnight or stationary-phase cultures which therefore eliminates the need for subculturing for generating log-phase cultures that are necessary with existing methods. The new method was effective in isolating plasmids, from 1.4 to 64 kb, from the three subspecies of Lactococcus lactis. The resultant DNA was of high yield and purity and therefore no additional purification steps were required for down-stream molecular procedures.