its protocol for dna isolation

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Veterinary Reseurch Comm unications, 14 (D90) 447-451Copyright 0 Khwer Academ ic Publishers bv - Printed in the Netherlands

Short CommunicationA RAPID AND SIMPLIFIED PROTOCOL FOR DNA ISOLATIONFROM BACTERIAD.L. TROYER, A. REED2, D. OBERS+ AND M.M. CHENGAPPA2Department of Anatomy and Physiology, Department of Laboratory Medicine andDepartment of Pathology, 228 Veterinary Medical Science Building, Kansas StateUniversity College of Veterinary Medicine, Manhattan, Kansas 66506, USA

Troyer, D.L., Reed, A., Oberst, D . and Chengappa, M .M ., 19!90.A rapid and simplified protocol for DNAisolation from bacteria. Vekrinq Reseurch Comm unications, 14 (6), 447-451Keywords acid-fast, bacteria, DNA , extraction, Gram-negative

INTRODUCTIONThe process of isolating and purifying undegraded and unfragmented DNA fromprokaryotic and eukaryotic organisms is central to many of the molecular biologicaltechniques being used today. In selecting a DNA isolation procedure, one mustconsider factors such as yield, simplicity, speed, reproducibility, and the subsequentmanipulations of the DNA which will take place. Consideration must also be given tothe cost-effectiveness of the protocol.

Bacteria have a single, circular chromosome which is not segregated from the restof the cel l as is the case in eukaryotic genomes. The chromosomal DNA ofEscherichia col i has approximately four million base pairs, and encodes for about 4000proteins (Timoney er aZ., 1988).Epidemiological tracing (typing) of bacterial strains is of considerable importancein veterinary microbiology. The data can be used to monitor trends in the occurrenceof pathogenic strains or to identify possible sources of infection. Presently, the mostfrequently used typing techniques rely on interstrain differences in phenotype asrevealed by serology and other means (De Lisle er aZ., 1987).More recently, attention has shifted to alternative ways to characterize anddifferentiate between isolates of the same serotype, relying on DNA sequencedifferences (Thiermann er aZ., 1986). Specifically, restriction endonucleases, which areenzymes that cleave double stranded DNA only at certain palindromic sites, can beused to detect these differences. Restriction endonuclease analysis is a sensitivemethod for detection of minor genotypic differences in closely related bacteria

(DeLisle er &., 1987). However, the time-consuming and laborious preparation ofgood quality DNA suitable for digestion with restriction enzymes from large numbersof bacterial cultures is a drawback of this method.In this study, we investigated whether a recently reported protocol for pur%cationof plasmid DNA (Alter and Subrammian, 1989) could be modified for the isolation of


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bulk DNA from several bacteria, including some which are particularly diflicult topurify, such as Mycobactetium paratubexulosis (Baess, 197% Whipple et d., 19871989).

MATERIALS AND METHODSAn M. paratubenxdosis field strain was grown in Herrolds medium (Remel, Lenexa,Kansas) at 37C for 8 weeks. D-Cycloserine (Sigma Chemical Co., St Louis) wasadded to a fmal concentration of 1 mg/ml 24 h prior to harvesting the cel ls(Shoemaker et aI., 1986). Pasteurella haemolytica (Al), Pasteurella multoci& (A3), aPasteurella-like organism and Actinobacillus pleuropneumoniae (type 1) were grownon brain-heart infusion agar at 37T for 18 h. Campylobacter hyointestinalis wasgrown on Mueller-Hinton agar containmg 5% sheep blood at 36C in a micro-aerophilic atmosphere (Gebhart et al., 1983). In all cases, the cel ls were harvested bycentrifugation at 5000 g for 20 min at 4C, and the resulting pellet was transferred to amicrocentrifuge tube to be stored at -80C until used.After the sample was thawed, it was resuspended in 500 ~1 of buffer (TE, 10mmol/L Tris HCl, pH 7.q 1 mmol/L EDTA), transferred to a microcentrifuge tubecontainmg 400 ~1 of a 1:l mixture of phenol:chloroform (Mania& et al, 1982),shaken vigorously for 24) s, held at -2OC for 30 mm, and centrifuged at 12 OOOg or 15min at 4C. The aqueous phase was transferred to another tube, and thephenolxhloroform step was repeated once. DNA was precipitated by adding 0.1 vol.of 3 mmol/L sodium acetate and 1 vol. of isopropanol and removed with a curvedPasteur pipette, after which it was washed twice with 70% ethanol, dried in vacua, anddissolved in TE. As a general rule, about 10 pg of intact chromosome DNA wasrecovered from 20 mg of cel ls (wet weight). The entire procedure took less than 2 h.Puritied DNA (2 pg) was mixed with 5 units of restriction enzyme (EcoRI) in a20 ~1 reaction mixture, and the reaction carried out for 4 h under conditions recom-mended by the manufacturer (Bethesda Research Laboratories Inc., Gaithersburg,MD). The digested samples were mixed with loading buffer and electrophoresed in0.8% agarose gels at 2 V/cm for 16 h in TBE (20x TBE = 1 mmol/L Tris base, 1mmol/L boric acid, 20 mmol/L EDTA) buffer. They were then stained with ethidiumbromide (0.25 pg/ml) for 45 min and photographed under UV light.

RESULTSUsing thii modiied protocol, we were able to considerably shorten the time requiredto extract biochemically useful total DNA from all the micro-organisms tested in thestudy. Yields were consistently greater than those obtained using other methods basedon more cumbersome techniques such as detergent, lysozyme, and pronase treatment.Figure 1 shows bulk DNA extracted from M. paratubexdosis (lane 2) and a PstIdigest of a small amount of the extract (0.5 pg) (lane 3). Lanes 2 and 3 of Figure 2compare restriction patterns of a Pu.rtareZla-like bacterium and PasteurelZa haemo-Zytica respectively, both digested with EcoRI; lanes 4 and 5 are uncut DNA from thesame bacteria, in the same order. Lanes 6 and 7 are EcoRI digests of diiferent strainsof PasteurelZa-liie organisms, (both different from that in lane 2). Lanes 8-11 are


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EcoRI digests of ActinobaciIius pieuropneumoniae (lane 8), Pasteurella multocida(lane 9), PasteureZfa mzdtocida of a diiferent isolate than lane 9 (lane lo), andPasteureZla haemoZytica (lane 11). Most of the fragments comprising the restrictiontypes are between 3 and 20 kb in size. The bands which are below 2.0 kb representintact RNA and could be subsequently removed with RNAase.

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Figure 1. Whole genomic DNA extracted from Mycobactetium paratubexulosis (lane2), PSTI digest of 0.5 pg M. paratubextdosis DNA (lane 3), Him-III1 digested Lambd .,phage size markers (lanes 1 and 4)


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DISCLJSSIONThe ease and rapidity of this manipulation was surprising, especially in the case of M.puratuberdosis, given the fact that this bacterium is somewhat resistant to chemicaltreatment. Since virtually all the enzymatic steps have been eliminated, substantialsavings in time and cost are realiied. We have used this protocol to routinely isolategenomic DNA from other bacteria, including those mentioned in this study, and it hasproven to be equally consistent with all these micro-organisms.

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Figure 2. All lanes show restriction fragments which had been digested with EcoRIand are: Pu,rreure&z-like strains (lanes 2,6 and 7); Pus&u&u huemoZytica (lanes 3 and11); Actinobadus pleuropneumoniae (lane 8), and Pasteurella multocida (lanes 9 and10). Lanes 4 and 5 show undigested PasteureZla-like and P. haemoiytica strains,respectively. Lane 1, size markers.


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451The procedure does not separate DNA and RNA, thus necessitating the additionof RNAase to the restriction enzyme reaction mixture or treatment with it after the

digestion. The bacterial cel ls are lysed and proteins are extracted during thephenol-chloroform step. Cell debris and proteins are seen as a mass at theorganic-aqueous interface after centrifugation.Pu.rrarelZu-like organisms have been recovered from various diseases in animalsand domestic birds but the clinical significance and taxonomic position of theseorganisms are not firmly established. The technique described will help in themolecular characterization of these strains, since it is simple and rapid. Strategiessuch as restriction typing and blot analysis, which will allow the genomes of these andother microbes to be better characterized, should also be facilitated by the procedure.This may encourage the routine application of these methods to monitor over timethe progression of microbial genotypic changes, which may result in differences in thehost-pathogen relationship.

ACKNOWLEDGEMENTSWe thank Pam Say for secretarial assistance and Dan Howard for technicalassistance.This work was supported by USDA formula funds and the Kansas AgriculturalExperiment Station; published as RAES contribution no. 90-414-J.

REFERENCESAher, DC . and Subrammian, KN ., 1989. A one step, quick step, mini prep. Biorechniques, 7 (S), 455-457Baess, I. , 1974. Isolation and puri tication of deoxyribonucleic acid fmm mycoba cteria. ,4&r PuUm~o&rMicrobiologica Scandinavia Section B, 82,780-784DeLisle, G.W ., Pettett, A.M., Wa ll , E.O. and Coll ins, D.M ., 1987. An examination of Cam pylobactw ferusS.S.eau by restriction endonuclease analysis and semlogy. Veterinary Microbiology, 14, !kiO ~Gebhart. C.J .. Ward. GE.. Chants. K. and Kurtz H.J .. l !Z33. Cam twbbaczer hvointesrinalis (new snecies)isolated fmm swine with lesi&s o f pmliferative i leit is. Ame&& Journal >f Veterinury keseakzh, 4;361-367Maniatis, T., Fri tsch, E.F . and Sambrook, J., 1982. Mo/ecu ku Cloning: A Luboratoty Manual. (Cold Spring

Harbor Laboratory, Cold Spring Harbor, New York)Shoemaker, SA., Fisher, J.H., Jones, W .D. and Scoggin, C.H., 1986. Restrict ion fragment analysis ofchmm osoma l DNA defines different strains of Myco bacrtnkm derculosis. Annual Review ofRespira&ny Disease, 134,210-213Thiermann, A.B., Handsa ker, A.L., Foley, J.W ,, White, F.H . and Kinseate, B .F., 1986. Reclassif ication ofNorth Am erican leptospiral isolates belonging to sem groups Mini and Sejoroe by restrictionendonuclease analysis. American Journal of Veterinary Resea rch, 47 (l), 61-66Timone y, J.F ., Gil lespie, J.H ., Scott, F.W . and Barlough, J.E., 1988. Hagan and Bruners Microbiology undInfetious L&eases of Dom estic Animals, 8th edn., (Cornell University Press, Ithaca, NY), p. 24Whipple, D.L., LeFebre, RB ., Andrew s, RE. and Thiermann, A.B., 1987. Isolation and analysis ofrestriction endonuclease digestive patterns of chmm osoma l D NA fm m Myoba crekm pru-h&x~osis and other Mycobacterium species. Journal of C& ricaI Mtiobiolngr, 25 (8), 1511-1515Whipple, D.L., Kapke, PA. and Andrews, RE ., 1989. Analysis of restrict ion endonuclease fragmentpatterns of DNA fmm Mjvobactwiumparaa&e rculosk. Veterimny Microbioiogy, 19,189-194

(Accepted: 26 June 1990)

its protocol for dna isolation

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