JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1993, p. 406-409
Strain Identification of Mycobacterium tuberculosis by DNA
Fingerprinting: Recommendations for a
JAN D. A. VAN EMBDEN,1* M. DONALD CAVE,2 JACK T. CRAWFORD,3 JEREMY W. DALE,4
KATHLEEN D. EISENACH,2 BRIGIYIrE GICQUEL,s PETER HERMANS,6 CARLOS MARTIN,7
RUTH McADAM,8 THOMAS M. SHINNICK,3 AND PETER M. SMALL9
Unit Molecular Microbiology, National Institute ofPublic Health and Environmental Protection, P.O. Box 1, 3720 BA
Bilthoven, The Netherlands'; University ofArkansas for Medical Sciences, Little Rock, Arkansas 722052; Division
ofBacterial and Mycotic Diseases, National Centerfor Infectious Diseases, Centers for Disease Control,
Atlanta, Georgia 303333; Department ofMicrobiology, University ofSurrey, Guildford GU2 5XH,
United Kingdom4; Microbial Engineering Unit, Institut Pasteur, 75724 Paris, Cedex 15,
Frances; Armauer Hansen Research Institute, Addis Ababa, Ethiopia6; Area
Microbiologia, Faculty ofMedicine, University ofZaragoza, Zaragoza,
Spain7; Albert Einstein College ofMedicine, Yeshiva University,
Bronx, New York 104618; and Beckman Center B251, Howard
Hughes Medical Institute, Stanford University,
Stanford, California 94305-54259
Received 26 August 1992/Accepted 26 October 1992
DNA fingerprinting ofMycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We
propose a standardized technique which exploits variability in both the number and genomic position ofIS6110
to generate strain-specific patterns. General use of this technique will permit comparison of results between
different laboratories. Such comparisons will facilitate investigations into the international transmission of
tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug
Epidemiologic studies of tuberculosis can be greatly facil-
itated by the application of strain-specific markers. Unusual
antibiotic susceptibility patterns and phage typing, which
have been used for this purpose, have significant limitations.
The recently discovered transposable elements in Mycobac-
terium tuberculosis have been shown to be of great potential
for use in strain differentiation. M. tuberculosis strain typing
has already proven to be extremely useful in outbreak
investigations (2, 7, 13) and is being applied to a variety of
epidemiologic questions in numerous laboratories.
The existence of repetitive DNA elements in M. tubercu-
losis and their potential for use in fingerprinting of M.
isolates was recognized independently by
Eisenach et al. (4), Zainuddin (14), and Zainuddin and Dale
(15). The sequence of one of these elements, designated
IS6110, was first reported by Thierry et al. (11, 12) and was
shown to be related to the IS3 family of insertion sequences
which were discovered in members of the family Enterobac-
teriaceae. McAdam et al. (9) independently sequenced the
element isolated by Zainuddin and Dale (15), which was
designated IS986. Subsequently, a related element from
Mycobacterium bovis BCG was sequenced by Hermans et
al. (6) and is referred to as IS987. These three sequences
differ in only a few base pairs and therefore can be consid-
ered essentially the same element. To avoid confusion, we
recommend the designation IS6110 for these elements, ex-
cept when a specific copy is concerned.
The effectiveness of this insertion sequence (IS) typing
system for epidemiological analysis of M. tuberculosis iso-
lates has been demonstrated in a number of studies (1-3, 5,
7, 8, 10, 13). In principle, the results obtained by testing large
numbers of strains in different laboratories could be com-
pared. This would allow strains from different geographic
areas to be compared and the movement of individual strains
to be tracked. Such data may provide important insights into
the global transmission of tuberculosis and identify strains
with particular properties, such as high infectivity, high
virulence, and/or multidrug resistance. Analysis of large
numbers of isolates may provide answers to long-standing
questions regarding the efficacy of BCG vaccination and the
frequency of reactivation versus reinfection, which are in-
creasingly important in light of the AIDS pandemic. These
large-scale studies will require the use of computer-assisted
analysis and comparison of DNA fingerprints. This report
describes such a standard method, which will be adopted in
our laboratories, and recommends its use to other laborato-
ries so that the results obtained by different laboratories can
Bacterial growth, DNA extraction, digestion ofDNA, and
Southern blotting were done as described previously (13).
Agarose gels were loaded with a mixture of 1 ,g of PvuII-
digested genomic M. tuberculosis DNA and molecular size
marker DNA. PvuII-digested supercoiled ladder DNA (4 ng;
Vol. 31, No. 2
FIG. 1. Physical map of the 1.35-kb M. tuberculosis insertion element IS6110 (9). The cleavage sites of several restriction enzymes are
depicted. PvuII cleaves the element at base pair 461. Therefore, any chromosomal mycobacterial DNA fragment obtained by the
recommended standard typing method is larger than 0.9 kb. The closed bars represent the 28-bp inverted repeats bordering IS6110 DNA. The
lines to the left and right denote chromosomal DNA.
Bethesda Research Laboratories, Gaithersburg, Md.) and
1.2 ng of HaeII-digestedOX174DNA (Clontech, Palo Alto,
Calif.) were used as molecular size standards. The molecular
sizes of these two reference markers ranged from 16.2 to
0.603 kb, respectively (see Fig. 2B).
The mycobacterial IS probe was prepared by peroxidase
labeling of a 245-bp fragment obtained by amplification by
the polymerase chain reaction (PCR) described previously
(7). Briefly, the oligonucleotides INS-1 (5'-CGTGAGGGCA
TCGAGGTGGC) and INS-2 (5'-GCGTAGGCGTCGGTGA
CAAA) were used to amplify a 245-bp fragment from puri-
fied chromosomal M. bovis BCG DNA by PCR. This frag-
ment was purified by Sephadex G50 chromatography. The
DNA was precipitated with ethanol, and after solubilization
the DNA was labeled with peroxidase as described previ-
Standard method of fingerprinting. DNA typing of M.
tuberculosis complex strains is based on polymorphisms
generated by variabilities in both the copy numbers and the
chromosomal positions of IS6110 among clinical isolates of
M. tuberculosis (1-3, 5-8, 10, 13, 15). The technique of
fingerprinting entails the growth of M. tuberculosis, extrac-
tion of DNA, restriction endonuclease digestion, Southern
blotting, and probing for the IS element. Only three param-
eters are critical for a standardized IS6110-based DNA
fingerprinting system: the specificity of the restriction en-
zyme, the nature of the DNA probe, and the use of appro-
priate molecular mass standards.
The physical map of the IS6110 sequence (Fig. 1) indicates
that various restriction enzymes cleave within the 1,355-bp
element. BamHI, SstII, PstI, BstEII, BssHII, and PvuII
have all been successfully used to generate restriction frag-
ment length polymorphisms (1-8, 13, 14). For the standard
method we recommend the use ofPvuII, because it has been
used by the majority of laboratories and it cleaves the IS6110
sequence only once. Because of this latter property, PvuII
digestion of IS6110-containing genomic DNA leads
IS6110-hybridizing fragments of at least 0.90 or 0.46 kb,
depending on the IS6110 probe that is used. Since M.
tuberculosis usually contains 8 to 20 IS6110 copies (13), the
use of a DNA probe which overlaps both sides of the PvuII
site would result in 16 to 40 bands. This large number of
bands would result in overcrowded lanes with overlapping
bands. Therefore, we arbitrarily chose a DNA probe to the
right of the PvuII site on the physical map, as shown in Fig.
1. This reduced the number of IS6110-containing bands in
the fingerprint to half of the maximum number possible. In
exceptional cases, when the differentiation of the patterns is
not adequate, the membranes could be reprobed with labeled
DNA containing only IS sequences to the left of the PvuII
site. For the standardized method, the exact DNA se-
quences of the probe do not matter as long as the IS
sequence to the right of the PvuII site is used. An illustration
of a Southern blot is given in Fig. 2A.
In order to compare fingerprints between M. tuberculosis
isolates run on different gels and in different laboratories, the
size of each IS6110-hybridizing fragment must be deter-
mined. This requires the use of molecular size markers
which span the 10- to 0.9-kb range of most IS6110-hybridiz-
ing fragments. We recommend use of a combination of
external and internal standards which provide a compromise
between technical ease and maximal precision. External
molecular size markers should be run in two or three lanes of
each gel. Furthermore, we recommend inclusion in each gel
a lane containing DNA from the reference strain of M.
tuberculosis Mt14323, which, when digested with PvuII and
probed with IS6110, gives 10 approximately evenly spaced
bands of known size (Fig. 2A). Although the use of external
markers is adequate for comparing small numbers of similar
strains (such as in outbreak investigations),
provide sufficient precision to permit computerized compar-
isons of hundreds or thousands of strains. For this reason we
recommend that molecular size standards which do not
hybridize with IS6110 also be added to the wells with the
cleaved M. tuberculosis DNA. After hybridization with
IS6110, the membrane can be reprobed with labeled molec-
ular size marker standards. This results in a second autora-
diograph with molecular size standards in each lane. The
second autoradiograph can be superimposed on the first
autoradiograph, resulting in extremely precise molecular
size determinations (Fig. 2B). We were able to obtain
standard deviations of the molecular sizes of less than 2% in
the 1- to 2-kb range and less than 5% in the 9- to 10-kb range
(2a). If internal standards are used, a single reference exter-
nal marker is sufficient.
Finally, to be able to compare DNA fingerprints made in
different laboratories, a minimal resolving power of the gels
is needed. At a given agarose concentration, the resolving
power mainly depends on the electrophoresis time. We
recommend conditions such that the distance between the
0.9-kb marker (the approximate size of the smallest PvuII
IS-containing fragment) and the 10-kb marker is at least 10
These recommendations will permit comparison of DNA
fingerprints ofM. tuberculosis made in different laboratories
that can use their own optimized procedures for DNA
it may not
VOL. 31, 1993
J. CLIN. MICROBIOL.
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FIG. 2. Fingerprints of M. tuberculosis strains obtained by the recommended standard method. Chromosomal DNAs of 22 different
mycobacterial isolates were digested with Pvull, and after mixing with marker DNA, the fragments were separated by overnight
electrophoresis. The fragments were transferred to filters and hybridized with peroxidase-labeled IS6110 DNA (A) and peroxidase-labeled
marker DNA (B). Lane 22, reference M. tuberculosis Mt14323, which is available on request; lanes 2 to 5, strains from an outbreak of
tuberculosis in Amsterdam during 1991. All other lanes contain DNAs from M. tuberculosis isolated from epidemiologically unrelated cases.
The strains corresponding to lanes 16 to 21 were selected from a collection of about 200 randomly chosen strains from patients in The
Netherlands. The internal DNA markers (B) werePvuII-digested supercoiled ladder DNAs with molecular sizes of 16.2, 14.2, 12.1, 10.1, 8.07,
7.04, 6.03, 5.01, 4.00, 2.97, and 2.07 kb and HaeIII-digested 4X174 with molecular sizes of 1,353, 1,078, 872, and 603 bp, respectively. A
detailed protocol on the procedures for DNA isolation and fingerprinting is available from one of us (J.D.A.V.).
electrophoresis, blotting, and hybridization, provided that
the resolving power of the electrophoresis procedure is
within the DNA fragment size range of 0.9 to 10 kb.
Dickvan Soolingen and Petra de Haas are acknowledged for ideas
and technical assistance. Janetta Top and Wilma van der Roest are
acknowledged for secretarial help.
This study was financially supported by the World Health Orga-
nization Program for Vaccine Development and the European
Community Program on Science Technology and Development.
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