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Journal ArticleDOI

A procedure for the isolation of deoxyribonucleic acid from micro-organisms

Julius Marmur1
01 Apr 1961-Journal of Molecular Biology (Academic Press)-Vol. 3, Iss: 2, pp 208-218
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.
About: This article is published in Journal of Molecular Biology.The article was published on 1961-04-01. It has received 11573 citations till now. The article focuses on the topics: DNA extraction & DNA.
Citations
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Journal ArticleDOI
TL;DR: This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.

30,291 citations

Journal ArticleDOI
TL;DR: A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described in this paper.
Abstract: A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

10,245 citations

Journal ArticleDOI
TL;DR: The topic of this report is rap,d m,croscale methods for,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI, which is of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s.
Abstract: The topic of this report is rap,d m,croscale methods for ,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI. The D N A produced ,s of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s. In addi t ion to the rapidi ty and convenience of mlmpreps which permit a large number of samples to be processed in just a few hours, the small amount of tissue reqmred (less than 1.0 grams) allows tbr molecular analysis of plants at a very young stage Mm,prep D N A y,elds from leaf tissue of most species tested to date are typ,cally 30-100 big per gram tissue, greater than 50 kb, and remarkably uniform from sample to sample. The first mmlprep procedure we reported fi3r maize D N A isolation (Dellaporta et al , ;'*l,;tze Geneta3 Cr162162 Neu'_~letlrt. 1983) was adapted from a procedure commonly used for }'east D N A preparatmn (Dav,s et al. , 1980) Since th,s report, numerous personal commun,cat ,ons have demonstrated that the mm,prep procedure or a modification thereof, can be apphed to most plant species tested. For example, the method has been successfully used on Ntcottana hlgl~um. N. plumklgmgidtum. N. 3)/t'eJtrt~. L)s~opertcum sp.. Amar,mthm sp . Gl)~me max. Petuma h.~hra&. Several modifications have been apphed by these ,nvestlgators and in our own laboratory m order to extend the appl ,catmn of ram,prep procedures to other plant species. The select,on of a part icular protocol depends to a large degree on the plant spec,es used. However, the procedure reported here was selected to be statable for most situations.

7,263 citations


Cites background from "A procedure for the isolation of de..."

  • ...6 volumes) has been reported tit separate high molecular DNA from polysaccharides (Marmur, 1961) The sodium acetate also yields ,l tight fibrous precipitate th,it is easily washed and dried The DNA will dissolve readily if allowed tit rehydrate ,it 4 ~ for one hour fi~llowed by hght vortexlng...

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  • ...…e t a t e using relatively small amounts of lsopropanol (about O. 6 volumes) has been reported tit separate high molecular DNA from polysaccharides (Marmur, 1961) The sodium acetate also yields ,l tight fibrous precipitate th,it is easily washed and dried The DNA will dissolve readily if allowed…...

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Journal ArticleDOI
J. Marmur1, Paul Doty1
TL;DR: The previously discovered linear relation between the base composition of DNA, expressed in percentage of guanine plus cytosine bases, and the denaturation temperature, T m, has been further investigated and it appears that the measurement of the T m is a satisfactory means of determining base composition in DNA.

4,154 citations

Journal ArticleDOI
TL;DR: It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available and reveal extensive gene diversity within the current concept of "species".
Abstract: DNA-DNA hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.

3,471 citations


Cites methods from "A procedure for the isolation of de..."

  • ...DNA from Gram-negative organisms was prepared as described by Marmur (1961), with reagent volumes adapted to the use of 50 ml centrifuge tubes....

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References
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Journal ArticleDOI
J. Marmur1, Paul Doty1
TL;DR: The previously discovered linear relation between the base composition of DNA, expressed in percentage of guanine plus cytosine bases, and the denaturation temperature, T m, has been further investigated and it appears that the measurement of the T m is a satisfactory means of determining base composition in DNA.

4,154 citations

Journal ArticleDOI

1,885 citations

Journal ArticleDOI
TL;DR: This communication presents a new method for the study of the molecular weight and partial specific volume of macromolecules, with some illustrations based on results with deoxyribonucleic acid (DNA) and several viruses.
Abstract: This communication presents a new method for the study of the molecular weight and partial specific volume of macromolecules, with some illustrations based on results with deoxyribonucleic acid (DNA) and several viruses. The method involves observation of the equilibrium distribution of macromolecular material in a density gradient itself at equilibrium. The density gradient is established by the sedimentation of a low-molecular-weight solute in a solution subject to a constant centrifugal field.

665 citations