Chloroform-isoamyl Alcohol Extraction Research Articles

Preparing Bacterial Genomic DNA.

We describe two alternative procedures for purifying bacterial chromosomal DNA. The first procedure incorporates the use of a commercial kit based on silica membrane technology. This approach relies on the selective binding of DNA to a silica-based column in the presence of chaotropic salts (guanidine salts). Polysaccharides and proteins do not bind well to the column and flow through. Their residual traces, along with the guanidine salts, are removed during alcohol-based wash steps. The DNA is then selectively eluted under low-salt conditions. This method is quick and easy and yields genomic DNA suitable for most downstream applications. The second procedure, implemented for many years in our laboratory before the appearance of commercial kits, is based on the ability of the cationic detergent cetyl trimethyl ammonium bromide (CTAB) to complex with polysaccharides and proteins, producing an emulsion that can be removed by chloroform-isoamyl alcohol extraction. This procedure is therefore especially suited to working with Gram-negative bacteria, which typically produce large amounts of polysaccharides. Its main advantage, besides cost-effectiveness, is the high yield of the DNA obtained; its main disadvantage is that the workflow is relatively cumbersome.

Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method

The analysis of environmental DNA (eDNA) using metabarcoding has increased in use as a method for tracking biodiversity of ecosystems. Little is known about eDNA in marine human-modified environments, such as commercial ports, which are key sites to monitor for anthropogenic impacts on coastal ecosystems. To optimise an eDNA metabarcoding protocol in these environments, seawater samples were collected in a commercial port and methodologies for concentrating and purifying eDNA were tested for their effect on eukaryotic DNA yield and subsequent richness of Operational Taxonomic Units (OTUs). Different filter materials [Cellulose Nitrate (CN) and Glass Fibre (GF)], with different pore sizes (0.5 µm, 0.7 µm and 1.2 µm) and three previously published liquid phase extraction methods were tested. The number of eukaryotic OTUs detected differed by a factor of three amongst the method combinations. The combination of CN filters with phenol-chloroform-isoamyl alcohol extractions recovered a higher amount of eukaryotic DNA and OTUs compared to GF filters and the chloroform-isoamyl alcohol extraction method. Pore size was not independent of filter material but did affect the yield of eukaryotic DNA. For the OTUs assigned to a highly successful non-indigenous species, Styelaclava, the two extraction methods with phenol significantly outperformed the extraction method without phenol; other experimental treatments did not contribute significantly to detection. These results highlight that careful consideration of methods is warranted because choice of filter material and extraction method create false negative detections of marine eukaryotic OTUs and underestimate taxonomic richness from environmental samples.

Evaluation of three DNA extraction methods from fungal cultures

Polymerase chain reaction (PCR) based assays have been developed to amplify DNA of fungal pathogens as culture-based detection methods show low sensitivity. In order to perform a sensitive, specific, and reliable PCR based assay, the availability of pure DNA as well as an easy-to-perform DNA extraction protocol is essential. The existing protocols for DNA extraction used for bacteria or viruses show poor release of fungal DNA. In this study, we evaluated three different methods of DNA extraction and compared their efficacy in the extraction of DNA from filamentous fungi, yeasts, and dermatophytes commonly isolated in our laboratory. It was found that the Fungi/Yeast Genomic DNA Isolation Kit (Norgen Biotek Corp, Ontario, Canada) demonstrated satisfactory extraction of DNA from all the fungi analyzed as compared to that of the Qiamp DNA extraction kit (Qiagen GmbH, Dusseldorf, Germany) or the Phenol Chloroform Isoamyl alcohol extraction method which failed to extract amplifiable DNA from many of the fungal species. Thus, we recommend the use of Fungi/Yeast Genomic DNA Isolation Kit (Norgen) with modifications for the extraction of DNA from fungal cultures.

Extraction of Jute Genomic DNA: Difficulties and Solutions

The plant parts of jute are composed of highly viscous substances and phenolic compounds which make nuisance to extract good quality genomic DNA (gDNA). Here different methods viz., phenol chloroform isoamylalcohol extraction and ethanol precipitation method; potassium acetate method and Cetyl trimethylammonium bromide (CTAB) method were applied for isolating the jute gDNA from leaves of 14 and 40 day’s plants. All of these methods were unable to extract good quality gDNA from jute leaves. That is why, in this experiment, the concentration and chemical compositions of CTAB method were modified for obtaining good quality jute gDNA. Polyvinylpyrrolidone (PVP) was added in the CTAB extraction buffer and β mercaptoethanol was used while grinding the leaf tissues with CTAB extraction buffer. It was found that good quality gDNA was obtained using modified CTAB method from 14 day’s plant’s leaves but low quality gDNA was obtained from 40 day’s plant’s leaves. These were confirmed by 0.8% agarose gel electrophoresis. Considering the visual quality of the banding patterns and their reproducibly MHR24 (5/-TTCCCTCCCA-3/) and MHR21 (5-/CCCGAAGCGA-3/) primers were selected out of 8 Random Amplified Polymorphic DNA (RAPD) primers for PCR. A total number of 11 loci were identified by these RAPD primers. Popgen32 software was used for analyzing the RAPD data. The number of polymorphic loci was one and the percentage was 9.09 which stated that a low level of genetic variations was existed among the jute accessions. In the dendrogram, jute accession CC875 (C. capsularis) was grouped in one cluster while CC894 (C. capsularis) and CC896 (C. capsularis) accessions were grouped in another cluster. DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11320Int J Appl Sci Biotechnol, Vol. 2(4): 516-520

Physico-chemical growth requirements and molecular characterization of indigenous Spirulina

The present study was designed to explore the indigenous Spirulina and its mass cultivation by optimizing the physico-chemical growth requirements. One hundred and twenty samples were collected from different soils and water from three districts (Sargodha, Lahore and Faisalabad) of Punjab. Collected samples were shifted to laboratory immediately under sterile conditions to avoid contamination and were kept under dim light at 30°C. Then Spirulina was isolated from collected samples and cultivated under different nutrient, temperature and light regimens to get its maximum bio-mass in our laboratory. Our results showed that maximum growth of indigenous Spirulina was obtained at 30°C and at 1500 lux light intensity. Nitrogen concentrations of 0.625, 1.25 and 1.875 g/L had no effect on the growth, while phosphate concentrations of 0.5, 1.0 and 1.5 g/L had a minimal and gradual effect on growth as the concentrations were increased. For the confirmation and molecular characterization of indigenous Spirulina, DNA was isolated by chloroform-isoamyl alcohol extraction method and its polymerase chain reaction (PCR) was carried out by using specific primer of 16s rDNA gene and PCR products were run on gel giving an amplicon size of 700 bp. Our study shows that Spirulina can be grown in lab conditions by optimizing the physico-chemical growth requirements. Key words: Amplicon, biomass, physico-chemical, polymerase chain reaction (PCR), Spirulina.

Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens

Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol–chloroform–isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol–chloroform–isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol–chloroform–isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate ‘universal’ nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol–chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a ‘universal’ nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.

Model for the molecular genetic diagnosis of endometrial cancer using K-ras mutation analysis.

Model for the molecular genetic diagnosis of endometrial cancer using K-ras mutation analysis.

An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents

Routine methods of extraction of DNA from blood involve the enrichment of cells by Ficoll-Hypaque gradient centrifugation followed by lysis of the cells with extraction buffer, proteinase K digestion of the lysate, and phenol: chloroform-isoamyl alcohol extraction. These methods generally require large amounts of blood, which poses a problem with pediatric patients. To overcome this, we developed a new method of extracting DNA directly from whole blood. This method involves the treatment of whole blood with an equal volume of NaI (3 M final concentration) followed by chloroform:isoamyl alcohol extraction to clear hemoglobin and cell debris. The clear aqueous layer is then mixed with isopropanol to obtain DNA. A large number of samples can easily be handled by this extraction procedure, as it can be carried out in 30 min and requires only a microcentrifuge.

The kinetoplast DNA structure ofTrypanosoma cf. carassii

The structure of the kinetoplast DNA (kDNA) network of fish trypanosomes is described for the first time. The kDNA was isolated from lysed cells of trypanosomes grown in culture by differential centrifugation combined with chloroform-isoamyl alcohol extraction. Electron microscopy of spread DNA networks revealed the existence of two kinds of mutually interlocked circular molecules. The maxicircle size was 27.5 ± 2.5 kbp (kilobase pairs) and that of the predominant minicircles 1.9 ± 0.1 kbp.

The use of agarose gel electrophoresis to measure the size of DNA molecules in crude cell lysates

We have used agarose gel electrophoresis to measure the size of DNA molecules taken directly from crude cell lysates. Pulse-labeled DNA from CV-1 cells that was denatured migrated as small molecules the size of Okazaki pieces when it was subjected to electrophoresis on 0.7% agarose gels under alkaline and neutral conditions. The gels also displayed the incorporation of the Okazaki pieces into bulk DNA when the DNA was labeled by pulse-chase techniques. The migration of the DNA from crude cell lysates was identical to DNA purified by chloroform-isoamyl alcohol extraction, and estimates of the size of the pulse-labeled molecules were similar by electrophoresis and by alkaline sucrose gradient centrifugation. These results indicate that, under these experimental conditions, agarose gell electrophoresis can be used in the same manner as alkaline sucrose gradient centrifugation to analyze replicating DNA from crude cell lysates.