Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washings

1. Sialic acid has been found to interfere with three colorimetric reactions used for the estimation of DNA: a modified diphenylamine reaction at 100° (Dische, 1930), the nitrophenylhydrazine method (Webb & Levy, 1955) and the diphenylamine reaction at 30° (Burton, 1956). 2. Evidence is presented that sialic acid is present in hydrolysates obtained from gastric wash-out material. 3. A mathematical method for correcting for interference from sialic acid in the diphenylamine reaction at 30° is described. 4. The diphenylamine reaction has been modified to make it suitable for the estimation of DNA in the presence of sialic acid. The modifications are to increase the concentration of diphenylamine to 2% and to perform the reaction at 6–13° for 48hr. These modifications increase the sensitivity 25% above Burton's (1956) modification of the diphenylamine reaction. 5. The precipitation, extraction and recovery of DNA from gastric wash-out material have been investigated.     

A new colorimetric method for the determination of 6-aminopenicillanic acid.

A new, sensitive colorimetric method for the estimation of 6-aminopenicillanic acid is described. The procedure is based upon formation of a 2,4-pentanedione derivative of 6-aminopenicillanic acid followed by a second reaction with p-dimethylaminobenzaldehyde (Ehrlich's reagent), resulting in a red product which absorbs at 538 nm. The absorbance response is linear from 0 to 350 μg of 6-aminopenicillanic acid. Penicillins do not interfere with the assay, but 6-aminopenicilloic acid does.     

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P_i is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.     

Microbial metabolism of aryl sulphonates A re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with thep-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

An improved method for DNA isolation from polyphenols rich leaf samples of Annona squamosa L.

A reliable and efficient method for isolating Annona squamosa L. genomic DNA, free from polyphenols and polysaccharides has been developed. Different methods involving use of CTAB and SDS with or without modifications were used. A CTAB based extraction method which uses diatomite to remove polyphenols and polysaccharides proved to be the best. This method allowed recovery of good quality DNA in sufficient quantity suitable for complete digestion by restriction endonucleases and amplifiable in polymerase chain reaction as compared to other methods.     

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.     

Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation

Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples.     

One-step molybdate method for rapid determination of inorganic phosphate in the presence of protein

A colorimetric method for the determination of γ-carboxyglutamic acid (Gla) is presented. The procedure is based on the following results. (a) Gla is quantitatively converted into a proline derivative by reaction with acetaldehyde. (b) This derivative is spectrophotometrically detected by the secondary amine reagent: nitroprusside and acetaldehyde in alkaline medium. Under the reported conditions, Beer's law is obeyed for concentrations of Gla varying from 0.5 to 5 × 10^?4m. The method has been used to determine the Gla content in urine samples.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/μl or 25 ng of T4 gene 32 protein/μl to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.     

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.     

On-line integration of PCR and cycle sequencing in capillaries: from human genomic DNA directly to called bases

A fully integrated system has been developed for genetic analysis based on direct sequencing of polymerase chain reaction (PCR) products. The instrument is based on a serially connected fused-silica capillary assembly. The technique involves the use of microreactors for small-volume PCR and for dye-terminator cycle-sequencing reaction, purification of the sequencing fragments, and separation of the purified DNA ladder. Four modifications to the normal PCR protocol allow the elimination of post-reaction purification. The use of capillaries as reaction vessels significantly reduced the required reaction time. True reduction in reagent cost is achieved by a novel sample preparation procedure where nanoliter volumes of templates and sequencing reaction reagent are mixed using a micro- syringe pump. The remaining stock solution of sequencing reaction reagent can be reused without contamination. The performance of the whole system is demonstrated by one-step sequencing of a specific 257-bp region in human chromosome DNA. Base calling for the smaller fragments is limited only by the resolving power of the gel. The system is simple, reliable and fast. The entire process from PCR to DNA separation is completed in ~4 h. Feasibilities for development of a fully automated sequencing system in the high-throughput format and future adaptation of this concept to a microchip are discussed.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °Cin the dark. These conditions are essential for the stability of the LiCHI₂ reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by ¹H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by ¹H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.     

A colorimetric procedure for measuring the enzymatic hydrolysis of terminal galactose from GM ganglioside

The enzymatic hydrolysis of the terminal galactose from GM₁-ganglioside is monitored by a colorimetric procedure. The NADH generated from the oxidation of released galactose with NAD and galactose dehydrogenase is employed to reduce p-iodonitrotetrazolium and the absorbance of the product, p-iodonitrotetrazolium formazan, is measured. The method can detect as little as 0.5 nmol of galactose. Hydrolysis of GM₁-ganglioside is accomplished using β-galactosidase from the marine gastropod Turbo cornutus. The enzymatic release of galactose is maximal at pH 3.5, and the reaction rate is linearly proportional to incubation time for 30 min, under the conditions employed. The presence of GM₂-ganglioside in the reaction mixture, after hydrolysis has occurred, is demonstrated by thin-layer chromatography.     

Colorimetric screening assay for cystine plus cysteine in legume seed meals

A colorimetric assay for cystine plus cysteine in pure proteins has been adapted to legume seed meals. The procedure involves incubation of legume seed meals for 1 hr at 38°C with sodium borohydride in 8 m urea, destruction of the sodium borohydride, and colorimetric determination of thiols produced with Ellman's reagent. A comparison of values from this procedure and from performic acid oxidation of 33 legume seed meals is presented and shows good correlation for peas (Pisum sativum) and lentils (Lens culinaris), with somewhat equivocal results for beans (Phaseolus vulgaris) and fava beans (Vicia faba).     

Colorimetric method for the determination of ketoses using phenol-acetone-boric acid reagent (PABR)

A purplish pink color developed when ketose solution (100 μl) was mixed with phenolacetone-boric acid reagent (0.5 ml 5% phenol, 2% acetone, 4% boric acid) and then treated with 96% sulfuric acid (1.4 ml). The absorbance of the reaction mixture was measured at 568 nm after 60 min at 37°C. This method allowed the simple determination of 3–50 nmol of d-fructose with coefficient of variation 7.8% for 3 nmol and 2.8% for 50 nmol. Carbohydrates other then ketoses did not interfere with this reaction. The influence of various chemicals on the colorimetric reaction and applicability of the method for determination of ketoses in natural products are presented.     

Sequence-specific spin labeling of DNA

Several DNA fragments deriving from plasmid pBR322 were used to determine the modification sites caused by the reaction with alkylating spin-labeling probes. At a high spin-label concentration, all guanines became alkylated, causing the cleavage of the phosphodiester bonds upon the treatment with piperidine. The lengths of the breakage products of 5'-end labeled DNA treated with spin labels were compared with the length of DNA scission products generated by Maxam-Gilbert procedure for DNA sequence analysis. The distribution of the guanine modifications is dependent on the amount of the reagent used for the alkylation and the ionic conditions of the reaction. The frequency of alkylation by spin labels was greatly enhanced within continuous runs of guanines in DNA. The stabilization of the DNA structure by magnesium or spermine directs the spin-label binding specifically to the most exposed region of DNA fragment containing GGTGG sequence. The sequence-dependent interaction of spin labels with DNA enables the development of the method for the selective spin labeling of DNA molecule.