Veterinary Reseurch Communications, 14 (D90) 447-451

Copyright 0 Khwer Academic Publishers bv - Printed in the Netherlands

Short Communication

A RAPID AND SIMPLIFIED PROTOCOL FOR DNA ISOLATION

FROM BACTERIA

D.L. TROYER’, A. REED2, D. OBERS+ AND M.M. CHENGAPPA2

‘Department of Anatomy and Physiology, ‘Department of Laboratory Medicine and
‘Department of Pathology, 228 Veterinary Medical Science Building, Kansas State
University 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 DNA
isolation from bacteria. Vekrinq Reseurch Communications, 14 (6), 447-451

Keywords acid-fast, bacteria, DNA, extraction, Gram-negative

INTRODUCTION

The process of isolating and purifying undegraded and unfragmented DNA from
prokaryotic and eukaryotic organisms is central to many of the molecular biological
techniques being used today. In selecting a DNA isolation procedure, one must
consider factors such as yield, simplicity, speed, reproducibility, and the subsequent
manipulations of the DNA which will take place. Consideration must also be given to
the cost-effectiveness of the protocol.
Bacteria have a single, circular chromosome which is not segregated from the rest
of the cell as is the case in eukaryotic genomes. The chromosomal DNA of
Escherichia coli has approximately four million base pairs, and encodes for about 4000
proteins (Timoney er aZ., 1988).
Epidemiological tracing (typing) of bacterial strains is of considerable importance
in veterinary microbiology. The data can be used to monitor trends in the occurrence
of pathogenic strains or to identify possible sources of infection. Presently, the most
frequently used typing techniques rely on interstrain differences in phenotype as
revealed by serology and other means (De Lisle er aZ., 1987).
More recently, attention has shifted to alternative ways to characterize and
differentiate between isolates of the same serotype, relying on DNA sequence
differences (Thiermann er aZ., 1986). Specifically, restriction endonucleases, which are
enzymes that cleave double stranded DNA only at certain palindromic sites, can be
used to detect these differences. Restriction endonuclease analysis is a sensitive
method for detection of minor genotypic differences in closely related bacteria
(DeLisle er &., 1987). However, the time-consuming and laborious preparation of
good quality DNA suitable for digestion with restriction enzymes from large numbers
of bacterial cultures is a drawback of this method.
In this study, we investigated whether a recently reported protocol for pur%cation
of plasmid DNA (Alter and Subrammian, 1989) could be modified for the isolation of
448

bulk DNA from several bacteria, including some which are particularly diflicult to
purify, such as Mycobactetium paratubexulosis (Baess, 197% Whipple et d., 1987
1989).

MATERIALS AND METHODS

An M. paratubenxdosis field strain was grown in Herrold’s medium (Remel, Lenexa,

Kansas) at 37°C for 8 weeks. D-Cycloserine (Sigma Chemical Co., St Louis) was
added to a fmal concentration of 1 mg/ml 24 h prior to harvesting the cells
(Shoemaker et aI., 1986). Pasteurella haemolytica (Al), Pasteurella multoci& (A3), a
Pasteurella-like organism and Actinobacillus pleuropneumoniae (type 1) were grown
on brain-heart infusion agar at 37T for 18 h. Campylobacter hyointestinalis was
grown on Mueller-Hinton agar containmg 5% sheep blood at 36’C in a micro-
aerophilic atmosphere (Gebhart et al., 1983). In all cases, the cells were harvested by
centrifugation at 5000 g for 20 min at 4’C, and the resulting pellet was transferred to a
microcentrifuge tube to be stored at -80°C until used.
After the sample was thawed, it was resuspended in 500 ~1 of buffer (TE, 10
mmol/L Tris HCl, pH 7.q 1 mmol/L EDTA), transferred to a microcentrifuge tube
containmg 400 ~1 of a 1:l mixture of phenol:chloroform (Mania& et al, 1982),
shaken vigorously for 24) s, held at -2O’C for 30 mm, and centrifuged at 12 OOOgfor 15
min at 4’C. The aqueous phase was transferred to another tube, and the
phenolxhloroform 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 curved
Pasteur pipette, after which it was washed twice with 70% ethanol, dried in vacua, and
dissolved in TE. As a general rule, about 10 pg of intact chromosome DNA was
recovered from 20 mg of cells (wet weight). The entire procedure took less than 2 h.
Puritied DNA (2 pg) was mixed with 5 units of restriction enzyme (EcoRI) in a
20 ~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 in
0.8% agarose gels at 2 V/cm for 16 h in TBE (20x TBE = 1 mmol/L Tris base, 1
mmol/L boric acid, 20 mmol/L EDTA) buffer. They were then stained with ethidium
bromide (0.25 pg/ml) for 45 min and photographed under UV light.

RESULTS

Using thii modiied protocol, we were able to considerably shorten the time required
to extract biochemically useful total DNA from all the micro-organisms tested in the
study. Yields were consistently greater than those obtained using other methods based
on more cumbersome techniques such as detergent, lysozyme, and pronase treatment.
Figure 1 shows bulk DNA extracted from M. paratubexdosis (lane 2) and a PstI
digest of a small amount of the extract (0.5 pg) (lane 3). Lanes 2 and 3 of Figure 2
compare 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 the
same bacteria, in the same order. Lanes 6 and 7 are EcoRI digests of diiferent strains
of PasteurelZa-liie organisms, (both different from that in lane 2). Lanes 8-11 are
EcoRI digests of ActinobaciIius pieuropneumoniae (lane 8), Pasteurella multocida
(lane 9), PasteureZfa mzdtocida of a diiferent isolate than lane 9 (lane lo), and
PasteureZla haemoZytica (lane 11). Most of the fragments comprising the restriction
types are between 3 and 20 kb in size. The bands which are below 2.0 kb represent
intact RNA and could be subsequently removed with RNAase.

I2 34

kb

- 23
-9
-6

- 2.3
- 2.0

Figure 1. Whole genomic DNA extracted from Mycobactetium paratubexulosis (lane

2), PSTI digest of 0.5 pg M. paratubextdosis DNA (lane 3), Him-III1 digested Lambd .,
phage size markers (lanes 1 and 4)
450

DISCLJSSION

The 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 chemical
treatment. Since virtually all the enzymatic steps have been eliminated, substantial
savings in time and cost are realiied. We have used this protocol to routinely isolate
genomic DNA from other bacteria, including those mentioned in this study, and it has
proven to be equally consistent with all these micro-organisms.

123456789 IO II
kb

23-

9-

6-

2.3 -
2.0-

Figure 2. All lanes show restriction fragments which had been digested with EcoRI
and are: Pu,rreure&z-like strains (lanes 2,6 and 7); Pus&u&u huemoZytica (lanes 3 and
11); Actinobadus pleuropneumoniae (lane 8), and Pasteurella multocida (lanes 9 and
10). Lanes 4 and 5 show undigested PasteureZla-like and P. haemoiytica strains,
respectively. Lane 1, size markers.
 451

The procedure does not separate DNA and RNA, thus necessitating the addition
of RNAase to the restriction enzyme reaction mixture or treatment with it after the
digestion. The bacterial cells are lysed and proteins are extracted during the
phenol-chloroform step. Cell debris and proteins are seen as a mass at the
organic-aqueous interface after centrifugation.
Pu.rrarelZu-like organisms have been recovered from various diseases in animals
and domestic birds but the clinical significance and taxonomic position of these
organisms are not firmly established. The technique described will help in the
molecular characterization of these strains, since it is simple and rapid. Strategies
such as restriction typing and blot analysis, which will allow the genomes of these and
other microbes to be better characterized, should also be facilitated by the procedure.
This may encourage the routine application of these methods to monitor over time
the progression of microbial genotypic changes, which may result in differences in the
host-pathogen relationship.

ACKNOWLEDGEMENTS

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

REFERENCES

Aher, DC. and Subrammian, KN., 1989. A one step, quick step, mini prep. Biorechniques, 7 (S), 455-457
Baess, I., 1974. Isolation and puritication of deoxyribonucleic acid fmm mycobacteria. ,4&r PuUm~o&r
Microbiologica Scandinavia Section B, 82,780-784
DeLisle, G.W., Pettett, A.M., Wall, E.O. and Collins, D.M., 1987. An examination of Campylobactw ferus
S.S.feau by restriction endonuclease analysis and semlogy. Veterinary Microbiology, 14, !kiO ~
Gebhart. C.J.. Ward. GE.. Chants. K. and Kurtz H.J.. l!Z33. Camtwbbaczer hvointesrinalis (new snecies)
isolated fmm swine with lesi&s of pmliferative ileitis. Ame&& Journal >f Veterinury keseakzh, 4;
361-367
Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Mo/ecuku 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. Restriction fragment analysis of
chmmosomal DNA defines different strains of Mycobacrtnkm derculosis. Annual Review of
Respira&ny Disease, 134,210-213
Thiermann, A.B., Handsaker, A.L., Foley, J.W,, White, F.H. and Kinseate, B.F., 1986. Reclassification of
North American leptospiral isolates belonging to semgroups Mini and Sejoroe by restriction
endonuclease analysis. American Journal of Veterinary Research, 47 (l), 61-66
Timoney, J.F., Gillespie, J.H., Scott, F.W. and Barlough, J.E., 1988. Hagan and Bruner’s Microbiology und
Infetious L&eases of Domestic Animals, 8th edn., (Cornell University Press, Ithaca, NY), p. 24
Whipple, D.L., LeFebre, RB., Andrews, RE. and Thiermann, A.B., 1987. Isolation and analysis of
restriction endonuclease digestive patterns of chmmosomal DNA fmm Myobacrekm pru-
h&x~osis and other Mycobacterium species. Journal of C&ricaI Mtiobiolngr, 25 (8), 1511-1515
Whipple, D.L., Kapke, PA. and Andrews, RE., 1989. Analysis of restriction endonuclease fragment
patterns of DNA fmm Mjvobactwiumparaa&erculosk. Veterimny Microbioiogy, 19,189-194

(Accepted: 26 June 1990)