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DNA extraction

About: DNA extraction is a research topic. Over the lifetime, 6856 publications have been published within this topic receiving 221321 citations. The topic is also known as: DNA extraction.


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Journal ArticleDOI
Julius Marmur1
TL;DR: A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA, and Representative samples have been characterized for their thermal stability and sedimentation behaviour.

11,573 citations

Journal ArticleDOI
TL;DR: A novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates is described, which ought to have wide applicability for clinical detection and other studies.
Abstract: Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.

4,187 citations

Journal ArticleDOI
TL;DR: The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures.
Abstract: Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.

2,918 citations

Journal ArticleDOI
TL;DR: Four methods for purifying crude DNA were evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization and in general, all methods produced DNA pure enough for PCR amplification.
Abstract: A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.

2,826 citations

Journal ArticleDOI
01 Jan 1987-Gene
TL;DR: To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform.

2,483 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023248
2022551
2021219
2020264
2019343
2018309