Efficient DNA Extraction Method Research Articles

A rapid and efficient method for DNA extraction from paraffin wax embedded tissue for PCR amplification

DNA from archival, formaldehyde fixed, paraffin wax embedded human tissue, suitable for amplification by the polymerase chain reaction (PCR), was obtained using a microwave method based on the capture of DNA by magnetic beads. Fragments of the alpha-1-antitrypsin gene (AAT) and the apolipoprotein E gene (APOE) were amplified successfully from human liver and brain tissue, respectively. This procedure provides a more rapid, simple and efficient method for reproducibly obtaining DNA from preserved tissue that has been kept in storage for up to 30 years.

A modified CTAB DNA extraction procedure for plants belonging to the family proteaceae

This paper describes rapid and efficient DNA extraction methods for mature leaves, resting buds and seedling leaves of genera in the family Proteaceae. The procedures combine and modify previously published techniques. The DNA can be digested by restriction endonucleases and is suitable for subsequent PCR amplification.

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.

Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems

A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.

A simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection

Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer, and detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. We developed a simple, easy and efficient non-organic method of DNA extraction from cervical scrapes for reliable detection of HPV DNA sequences. The method involves incubation of cell nuclei in higher concentration of proteinase K at 65°C for 2.5 h. Following prolonged incubation at higher temperature, the enzyme is autoinactivated and the DNA isolated can be used directly for analysis without further purification. The recovery of DNA is more than 95% and it can be easily cleaved by restriction enzymes and is suitable for amplification by the polymerase chain reaction (PCR). The whole procedure is carried out in a single Eppendorf tube and a large number of specimens can be processed at a time without any error of handling. DNA extracted from a single smear sample is sufficient to conduct as many as four different molecular biology tests. This provides an opportunity for verification of sensitivity, specificity and reliability of each test for diagnosis of HPV infection without resorting to biopsy.

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.

Tracking microorganisms and genes in the environment.

Proposed intentional releases of GEMs into the environment necessitate the development of appropriate methodologies for tracing organisms and their genes in various environmental samples. Studies have been conducted to determine the sensitivities and limitations of various methods for determining the fate of GEMs and their genes in the environment. Selective viable plate count procedures can be designed to detect the introduced organisms with high sensitivity; but they are restricted by potential mutations affecting the expression of the selective characteristic in the introduced organism, the occurrence of the particular selective characteristic in the indigenous organisms, and the need to culture the organism. The accuracy of this approach is greatly improved by colony hybridization procedures that use a specific gene probe to detect the introduced genes, but this approach is still only as sensitive as the plating procedure. Direct extraction of DNA from environmental samples, coupled with dot blot hybridization with radiolabeled probe DNA or solution hybridization, gives a high degree of both sensitivity and precision. This approach does not require culturing of the organism; and even if an introduced gene moves into a new organism or if the introduced organism is viable but nonculturable, the gene probe methods will detect the persistence of the introduced genes in the environment. Efficient direct DNA extraction methods have been developed and tested following in vitro experimental additions of GEMs to sediment and water samples.

Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica)

Abstract. Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica). Nusantara Bioscience 3: 105-111. A simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels has been described here. In the present study, three kinds of plant DNA extraction protocols were studied and the target was to establish the water-saturated ether (WSE) with 1.25 M NaCl method as the most efficient protocol for removing the highly concentrated polysaccharides from genomic DNA of woody fruit crops. This method involves the modified CTAB or SDS procedure employing a purification step to remove polysaccharides using the WSE method. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet became easily dissolved in TE buffer. Using these three methods, DNA was extracted from samples of 60 mango genotypes, including young, mature, old, frosted old and withered old leaves. Compared with the three studied DNA extraction protocols of mango, it was found that the WSE method with NaCl had the highest value of average percentage (85.44%) in DNA content of the mango genotypes. The average yield of DNA ranged from 5.05 µg/µL to11.28 µg/µL. DNA was suitable for PCR and RAPD analyses and long-term storage for further use.