JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1996, p. 3129–3137 Vol. 34, No.

12
0095-1137/96/$04.0010
Copyright q 1996, American Society for Microbiology

Differentiation of Bacterial 16S rRNA Genes and Intergenic

Regions and Mycobacterium tuberculosis katG Genes by
Structure-Specific Endonuclease Cleavage
MARY ANN D. BROW,1* MARY C. OLDENBURG,1 VICTOR LYAMICHEV,1 LAURA M. HEISLER,1
NATASHA LYAMICHEVA,1 JEFF G. HALL,1 NANCY J. EAGAN,1 D. MICHAEL OLIVE,1
LLOYD M. SMITH,2 LANCE FORS,1 AND JAMES E. DAHLBERG3
Third Wave Technologies, Inc.,1 and Department of Chemistry2 and Department of
Biomolecular Chemistry,3 University of Wisconsin, Madison, Wisconsin
Received 3 July 1996/Returned for modification 3 August 1996/Accepted 17 September 1996

We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonu-
clease, Cleavase I. We have applied this technique to the detection and localization of mutations associated
with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species, and
strains. The technique described here is based on the observation that single strands of DNAs can assume
defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase
I. The patterns of fragments produced are characteristic of the sequences responsible for the structures, so that

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each DNA has its own structural fingerprint. Amplicons containing either a single 5*-fluorescein or 5*-tetra-
methyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and
-sensitive M. tuberculosis, the 5* 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhi-
murium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, Staph-
ylococcus hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment
comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella
enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the struc-
tural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates
were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Band
patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated
from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differen-
tiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple,
and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.

The characterization of microbiological pathogens on the
basis of nucleic acid sequences is becoming increasingly im-
portant in clinical microbiology. In the future, detection of
mutations in genes such as the Mycobacterium tuberculosis rpoB
and katG genes may be used to predict resistance to rifampin
and isoniazid, respectively. As the number and spectrum of
nosocomial pathogens continue to expand, the ability to ac-
curately determine the relatedness of microbial isolates has
become increasingly important. Techniques for analyzing mi-
crobial relatedness have become critical (i) for identifying out-
breaks of infection, (ii) for determining the mode of acquisi-
tion of a pathogen, (iii) for analyzing individual patients to
determine if a series of isolates obtained over time represents
relapse of infection due to a single strain or separate episodes
of disease from different infections, and finally (iv) for defining
effective preventive and therapeutic measures.
The advent of the PCR and improvements in DNA sequenc-
ing technology have led to the rapid accumulation of primary
sequence information for a number of genes, gene systems,
and whole organisms. The understanding of the significance of
any particular locus, however, requires the comparison of
newly determined sequences to those of similar nucleic acids of
known function, so that differences in phenotype may be cor-
* Corresponding author. Mailing address: Third Wave Technolo-
gies, Inc., 2800 S. Fish Hatchery Rd., Madison, WI 53711. Phone: (608)
273-8933. Fax: (608)-273-6989. Electronic mail address: Mary_Ann
_Brow@twt.com.
related with particular differences at the nucleic acid level.
When such sequence differences are confined to a few known
sites, it is possible to assess a new isolate by simple methods
such as allele specific oligonucleotide hybridization (6) or re-
striction fragment length polymorphism (RFLP) analysis. In
many genes, however, polymorphisms may occur anywhere
within a given region, rather than at one or a few easily exam-
ined positions. Genetic analysis of this type requires the use of
methods that indicate differences between nucleic acids re-
gardless of the identity or the position of change and without
prior knowledge of the precise sequence of the molecule.
While DNA sequencing (11, 18) is a reliable way of identifying
such mutations, resequencing thousands of bases in search of
single-base changes can be slow and prohibitively costly. The
development of alternative methods to efficiently detect and
characterize such changes will allow comparison studies to
proceed more rapidly, as well as enable their use in the clinical
diagnostic arena.
Methods such as RFLP (5), single-strand conformation poly-
morphism (8, 13), and dideoxy fingerprinting (19) have been
applied for the detection of mutations associated with drug
resistance in M. tuberculosis (5, 10, 22). All of these methods
are limited by either their inability to analyze long DNA frag-
ments, their ability to examine only a few genetic loci (i.e.,
RFLP), or their inability to differentiate phenotypically silent
mutations from mutations resulting in drug resistance.
Identification and typing of bacteria have been accomplished
by both phenotypic and genotypic methods. Methods in which

3129
3130 BROW ET AL.
FIG. 1. Schematic representation of steps of CFLP pattern generation. La-
beled fragments of DNA are heated to separate the complementary strands.
When the samples are cooled, the single strands of DNA assume folded hairpin-
like structures; subtle differences in the sequences of the fragments can cause
formation of different structures. The Cleavase I enzyme cleaves at the 59 side of
these structures, at the junction between duplexed and single-stranded regions.
Separation and detection of the resulting fragments create signature banding
patterns that can be compared to detect differences between the test molecules.
phenotypic characteristics are used to assess relatedness are
inherently limited. These include biotyping, antimicrobial sus-
ceptibility testing, serotyping, and multilocus enzyme electro-
phoresis. Characteristics which define a particular strain may
be expressed differently under different environmental condi-
tions. For example, some toxins involved in virulence due to
Escherichia coli are expressed only in a host animal but are
repressed on solid growth media. In other cases, point muta-
tions which result in the abnormal expression of a gene respon-
sible for an observed phenotype can cause related bacteria to
appear different when in fact they are closely related (2).
Because of the problems associated with phenotypic typing
techniques, interest in the development of DNA-based or ge-
notypic typing methods has been stimulated. Ribotyping (15,
21) has moderate discriminatory power for organisms such as
E. coli and Klebsiella, Haemophilus, and Staphylococcus spp.
which contain multiple ribosomal operons. Nevertheless, epi-
demiologically unrelated isolates frequently demonstrate the
same pattern, limiting the utility of this method (4, 14, 23). For
bacterial species with only a single ribosomal operon such as
mycobacteria, ribotyping detects only one or two bands and has
limited utility for epidemiologic purposes (3).
Pulsed-field gel electrophoresis has been used extensively as
a means of identifying bacterial strain differences (20). While
pulsed-field gel electrophoresis has been applied to a number
of bacterial species, pathogens such as E. coli or methicillin-
resistant Staphylococcus aureus represent a genetically re-
stricted subset of strains within a species and consequently may
have similar genotypes, which often make them indistinguish-
able (2).
Several methods based on PCR amplification of either short
extragenic repetitive sequences or random sequences have
been applied to strain identification (2). However, the PCR-
based methods have shown either poor reproducibility or lim-
ited discriminatory power (19, 26).
We describe here a more robust and informative method of
differentiating the highly individual conformations assumed by
strands of nucleic acid, using enzymes that recognize and
cleave these structures. The method comprises steps of (i)
separation of DNA strands by heating, (ii) formation of intra-
strand structures on cooling, (iii) rapid enzymatic cleavage of
these structures before they are disrupted by reannealing of
the complementary strands, and (iv) separation and visualiza-
tion of the resulting structural fingerprint (Fig. 1). By detecting
conformational changes with the use of enzymatic cleavage
rather than electrophoretic mobility, sequence differences in
much larger molecules are detectable. Moreover, because
J. CLIN. MICROBIOL.
maintenance of the structure is not required after the cleavage,
resolution of the products can be done quickly and reproduc-
ibly by denaturing gel electrophoresis.
The data presented here show that sequence polymor-
phisms, whether single or multiple base, cause sufficient alter-
ation in the folded structures of large molecules to yield poly-
morphisms in the structural fingerprint patterns. By analogy to
RFLP and single-strand conformation polymorphism analyses,
we call this method Cleavase fragment length polymorphism
(CFLP) analysis. We demonstrate the utility of CFLP analysis
by using it for differentiating isoniazid-sensitive and -resistant
M. tuberculosis and for distinguishing bacteria at the levels of
genus, species, and strain.
MATERIALS AND METHODS
Cleavase I unit definition. Cleavase I is an engineered enzyme consisting of
the 59 nuclease domain (amino acids 1 through 281) of Thermus aquaticus DNA
polymerase. Its construction and characterization will be described elsewhere.
The activity of the Cleavase I enzyme was determined by digestion of an oligo-
nucleotide substrate (59-TGGTCGCTGTCTCGCTGAAAGCGAGACAGCGT
G-39) labeled at the 59 end with fluorescein, which folds to create a 12-bp stem
with a 3-nucleotide (nt) loop. Reaction mixtures contained enzyme and 20 pmol
of this substrate in 20 ml of 10 mM MOPS (morpholinepropanesulfonic acid [pH
7.5])–1 mM MnCl2. Reaction mixtures were incubated at 508C for 2 min. Release
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of a 5-nt fragment from the 59 end was monitored by electrophoresis through a
10% polyacrylamide gel (19:1 cross-link) with 7 M urea in 0.53 TBE buffer (45
mM Tris-borate [pH 8.3], 1.4 mM EDTA [17]), followed by visualization and
quantitation with an Hitachi model FMBIO-100 fluorescence image analyzer
fitted with a 505-nm filter. One unit of enzymatic activity is defined as the amount
that digests 3.7 pmol of this substrate under conditions of vast substrate excess in
1 min. The enzyme is stored in 20 mM Tris-Cl (pH 8.0) with 50 mM KCl, 100 mg
of bovine serum albumin per ml, 0.5% Tween 20, and 0.5% Nonidet P-40.
Substrate DNA fragments. The DNA fragments used in this study were gen-
erated by PCR. Primers were labeled on their 59 ends with either fluorescein,
tetramethyl rhodamine, or tetrachloro fluorescein or were used unlabeled, as indi-
cated. PCR mixtures (12, 16) contained 1 to 5 ng of plasmid, 25 ng of genomic
DNA or approximately 10 fmol of DNA fragment, 2.5 U of AmpliTaq DNA
polymerase (Perkin Elmer, Inc., Foster City, Calif.), 25 pmol of each primer, and
50 mM of each deoxyribonucleotide (Perkin Elmer) in 100 ml of 20 mM Tris-HCl
(pH 8.5)–1.5 mM MgCl2–50 mM KCl–0.5% Tween 20–0.5% Nonidet P-40.
Visualization of fluorescently labeled amplification products following elec-
trophoresis revealed a background smear of shorter products that were not
visible by staining. To remove these products, the PCR mixtures were treated
with 1 U of exonuclease I (Amersham International, Chicago, Ill.) for 20 min at
378C (9). The nuclease was inactivated by heating at 708C for 15 min. Reaction
mixtures were brought to 2 M ammonium acetate, and the DNAs were precip-
itated by the addition of 1 volume of isopropanol.
Amplification reactions that produced multiple double-stranded products
were resolved on polyacrylamide gels (6 to 10%, with 19:1 cross-linking) gels with
7 M urea, in 0.53 TBE buffer. Gels were stained with ethidium bromide, and the
bands of interest were excised and eluted by passive diffusion at 378C overnight
into a solution of 0.5 M ammonium acetate, 0.1 mM EDTA, and 0.1% sodium
dodecyl sulfate. Eluates were collected, and the DNA was recovered by the
addition of 2.5 volumes of 100% ethanol.
Precipitates were collected by microcentrifugation, washed briefly with 70%
ethanol, and dried under vacuum. All DNAs were dissolved in 10 mM Tris-Cl
(pH 7.5 to 8.0) with 0.1 mM EDTA.
Bacterial DNA preparation. The bacteria used in this study are listed in Table
1. E. coli O157:H7 isolates were obtained from Jeff Klinger (Vysis, Inc., Naper-
ville, Ill.). Bacteria were grown in Trypticase soy broth overnight at 378C with
constant agitation, and the DNA was obtained by phenol-chloroform extraction
(1:1) and ethanol precipitation. For PCR amplification of the 16S rRNA gene, a
350-bp amplicon was generated from each strain of bacteria with a set of con-
served eubacterial primers designated EB-R (59-AGAGTTTGATCCTGGCTC
AG-39), corresponding to nt 41 to 61, and EB-L (59-CTGCTGCCTCCCGTAG
GAGT-39), corresponding to nt 388 to 408 on the E. coli consensus sequence.
The intergenic regions of Salmonella and Shigella spp. were amplified with a 16S
sense primer (59-CTGGGGTGAAGTCGTAACAA-39) corresponding to nt
2768 to 2788 on the E. coli 16S rRNA consensus sequence and a 23S antisense
primer (59-GGGCATCCACCGTGTACGCT-39) corresponding to nt 26 to 46
on the E. coli consensus 23S rRNA sequence.
DNAs from isoniazid-sensitive and -resistant strains of M. tuberculosis were
the gift of Frank Cockerill (Mayo Clinic). Amplicons spanning a 620-bp region
of the M. tuberculosis katG gene (codons 302 to 507) of a wild-type and three
isoniazid-resistant variants (S at position 315 replaced by T [S315T], R463L, and
the double mutant S315T/R463L) were generated with primers katG904:59-AG
CTCGTATGGCACCGGAAC-39 and katG1523:59-TTGACCTCCCACCCGA
CTTG-39 (5).
VOL. 34, 1996 SEQUENCE ANALYSIS WITH A STRUCTURE-SPECIFIC ENDONUCLEASE
TABLE 1. Bacterial strains tested in this study
Bacterium ATCC no. Description
Escherichia coli O157:H7 Clinical isolate
Shigella sonnei 25931
Shigella dysenteriae 29026 Serotype 8
Shigella dysenteriae 29027 Serotype 1
Shigella dysenteriae 12021 Serotype 2
Salmonella typhimurium 23566
Salmonella enteritidis 13076
Salmonella arizonae 13314
Yersinia enterocolitica 27729
Campylobacter jejuni 33291
Staphylococcus aureus 13565 Enterotoxic
Staphylococcus aureus 33591 Methicillin resistant
Staphylococcus hominus 29885
Staphylococcus warneri 17917
Tyrosinase gene. Genomic DNAs and cDNA clones of a wild-type and a
mutant human tyrosinase gene were used as controls for the CFLP reaction and
were a gift from Richard Spritz (University of Wisconsin, Madison). The tyrosi-
nase mutant R422Q has a G-to-A substitution in codon 422 (7). A second mutant
version of the tyrosinase gene, R278X, was created from the wild-type cDNA by
a single base substitution 525 nt from the 59 end of the sense strand, by recom-
binant PCR methods (9). R278X has a C-to-T substitution that introduces a stop
codon at position 278 (24). A 516-bp fragment spanning exons 2 through 4 of the
wild-type gene was made through the use of primers Tyr1 (59-CTGAATCTTG
TAGATAGCTA-39) and Tyr3 (59-GCCATCAGTCTTTATGCAAT-39); 1,059-
bp fragments spanning exons 1 to 4 were created with primers Tyr1 and Tyr4
(59-GCAAGTTTGGCTTTTGGGGA-39).
Hepatitis C virus 5* noncoding region (NCR). A cDNA of the 59 noncoding
region (NCR) of the hepatitis C viral (HCV) genome was a gift from Manual
Altamirano (University of British Columbia, Vancouver) (1). The cDNA was
cloned into a TA vector (In Vitrogen, St. Louis, Mo.). A nested set of fragments
were amplified with the primers described below, from 10 ng of template frag-
ment. All fragments were labeled on their 59 ends with tetrachloro fluorescein-
labeled HCV-1 primer (59-CTGTCTTCACGCAGAAAGCG-39). Primers HCV-2
(59-CACGGTCTACGAGACCAC-39), HCV-3 (59-GCGGGGGCACGCCCA
AAT-39), HCV-4 (59-TCCAAGAAAGGACCCGGTC-39), and HCV-5 (59-AT
TCCGGTGTACTCACCGG-39), all unlabeled, were used with the HCV-1
primer to make products of 281, 186, 145, and 117 bp, respectively.
Cleavage reactions. Unless otherwise noted, each cleavage reaction mixture
was assembled as follows. Approximately 100 fmol of the DNA substrate in 15 ml
of water was heated to 958C for 5 to 10 s, cooled to the reaction temperature, and
mixed with 5 ml of a prewarmed solution containing 12.5 to 25 U of Cleavase I,
2 ml of 103 CFLP buffer (100 mM MOPS [pH 7.5], 0.5% Tween 20, 0.5%
Nonidet P-40), and 2 ml of 2 mM MnCl2. Reactions were stopped after 1 to 5 min
by the addition of 16 ml of 95% formamide with 10 mM EDTA (pH 8.0) and
0.02% methyl violet. The cleavage fragments were resolved by electrophoresis in
6 or 10% polyacrylamide gels (19:1 cross-link) containing 7 M urea in 0.53 TBE.
Detection of structural fingerprint patterns. Polyacrylamide gel cassettes were
transferred to an Hitachi model FMBIO-100 fluorescence analyzer and were
scanned. A 585-nm filter was used for scans of tetramethyl rhodamine-labeled
DNA; 505 nm was used for fluorescein-labeled DNA.
RESULTS
Examination of bacterial 16S rRNA genes by CFLP analy-
sis. The cleavage reactions used in CFLP analysis are partial
digests producing nested sets of products from each 59-end-
labeled DNA fragment. The ideal pattern, therefore, should
have both a full range of band representation, from short
products a few nucleotides long to residual undigested mate-
rial, and a relatively uniform distribution of signal across the
pattern.
The DNA sequences for the amplicons derived from the 16S
rRNA genes of some of the organisms examined here are
shown in Fig. 2. CFLP analysis of both DNA strands of eight
different isolates of E. coli O157:H7 are shown in Fig. 3 (lanes
1 through 8 of each panel). The CFLP fingerprints of these
closely related strains are indistinguishable from each other.
The four isolates of Shigella, which are evolutionarily close to
E. coli O157:H7 (Fig. 3, lanes 9 to 12) show a close relationship
3131
to each other and a strong familial resemblance to E. coli.
Sequence differences between the 16S rRNA genes of the
Shigella isolate and E. coli O157:H7 are reflected as changes in
several bands (e.g., mobility shifts, appearance or disappear-
ance of bands, or intensity changes). Salmonella species, which
are evolutionarily close to both E. coli O157:H7 and Shigella
spp., show a similar strong familial resemblance (Fig. 3, lanes
13 to 15) to both groups of organisms and yield related yet
clearly different structural fingerprints for each species. On the
antisense strand, a band of approximately 60 nt in length is
unique to S. typhimurium (Fig. 3A, lane 13), while the sense
strand S. arizonae pattern (Fig. 3B, lane 15) shows a charac-
teristic band shift at approximately 180 nt.
Structural fingerprints of staphylococcal species show a
strong relatedness to each other yet have distinct differences
from those of the gram-negative organisms. Finally, Campy-
lobacter jejuni and Yersinia enterocolitica show patterns indica-
tive of distinct genera, as would be expected based on the DNA
sequence.
Sensitivity of Cleavase I structural fingerprints to reaction
conditions. To assess the reproducibility of these patterns, we
examined reaction parameters that would influence the extent
of cleavage (enzyme amount and reaction time) and those that
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would influence the formation of the structures (temperature
and salt concentration). The effects of varying these reaction
conditions and constituents on the development of the struc-
tural fingerprint patterns are shown in Fig. 4.
Since the CFLP is a partial digest, it is desirable for the
patterns to show some residual uncut material to ensure that
the pattern includes the upper range of digestion products.
Therefore, the DNA is considered overdigested when no uncut
material remains. We examined the effects of varying the en-
zyme concentration and the reaction time and the degree to
which these variations would cause overdigestion. All of these
tests were performed with the 516-bp amplicon derived from
the R422Q tyrosinase mutant spanning exons 2 through 4 (7).
A standard reaction was defined as including 100 fmol of this
DNA digested with 25 U of Cleavase I enzyme at 558C for 2
min without added KCl. For each variable analyzed below, the
other parameters of the reaction were held to the standard.
As the amount of Cleavase I enzyme was increased, more
extensive digestion was observed (Fig. 4). However, optimal
levels of cleavage were obtained over a broad range between 5
and 25 U of enzyme, indicating that the reactions can tolerate
a fivefold variation in the amount of enzyme without significant
detriment to the pattern. Variations in the time of incubation
showed that the cleavage pattern development was very rapid,
being nearly complete within 1 min, and that the DNAs were
overdigested when the time exceeded 5 min.
These data show that while care must be taken, absolute
precision of time and enzyme measurement is not required to
achieve optimal digestion. Longer DNAs may be more easily
overdigested because they contain a greater number of poten-
tial cleavage sites. Overdigestion of such DNAs can be pre-
vented by including 1 mM MgCl2, in addition to the MnCl2, in
the reaction mixture to slow the reaction approximately five-
fold (data not shown). Alternatively, the amount of enzyme
may be reduced to 2 to 5 U per reaction or the cleavage time
may be shortened to 0.5 to 1 min.
When conditions likely to influence structure were exam-
ined, it was expected that changes in the banding patterns
would be observed, but we questioned whether the changes
would be abrupt and global, and therefore less reproducible, or
gradual, so that patterns would be reproducible even with
minor differences in the reaction conditions.
To examine the effects of temperature on the cleavage pat-
3132 BROW ET AL. J. CLIN. MICROBIOL.

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FIG. 2. DNA sequences of amplicons derived from amplification of 16S rRNA genes of representative bacteria used in this study. The sequence of the E. coli
O157:H7 amplicons is given as a reference. Deleted bases are indicated by asterisks.

tern, the reactions were performed at temperatures ranging stabilities of the causative structures. As would be expected,
from 35 to 758C (Fig. 4). These results show that the patterns bands were progressively less abundant at higher tempera-
change gradually with temperature variation, with particular tures, as less stable structures melted out. While these results
bands emerging and receding in accordance with the different do demonstrate that DNAs to be compared should be digested
VOL. 34, 1996 SEQUENCE ANALYSIS WITH A STRUCTURE-SPECIFIC ENDONUCLEASE 3133

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FIG. 3. CFLP structural fingerprinting of 16S rRNA genes of bacteria. A 350-bp amplicon was generated by PCR amplification of the 16S rRNA gene of each strain
as described in Materials and Methods. The amplicons were subjected to CFLP analysis. (A) Antisense strand (primer EB-L labeled). Lanes 1 to 8, E. coli O157:H7
strains; 9, Shigella sonnei; 10, Shigella dysenteriae type 2; 11, Shigella dysenteriae type 8; 12, Shigella dysenteriae type 1; 13, Salmonella typhimurium; 14, Salmonella
enteritidis; 15, Salmonella arizonae; 16, Staphylococcus aureus, (ATCC 13565); 17, Staphylococcus aureus, (ATCC 33591); 18, Staphylococcus hominus; 19, Staphylococcus
warneri; 20, C. jejuni; 21, Y. enterocolitica. (B) Sense strand (primer EB-R labeled). Lanes: 1 to 15, same as those in panel A; 16, Y. enterocolitica; 17, Staphylococcus
aureus (ATCC 13565); 18, Staphylococcus aureus, (ATCC 33591); 19, Staphylococcus hominus; 20, Staphylococcus warneri; 21, C. jejuni.
3134 BROW ET AL. J. CLIN. MICROBIOL.

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FIG. 4. Effect of reaction condition variation on cleavage pattern development. The integrity of the structural fingerprint patterns was tested under a variety of
solution conditions. The DNA template was the 516-bp tyrosinase gene fragment spanning exons 2 through 4 of the R422Q mutant human tyrosinase gene, labeled
on the 59 end of the antisense strand with tetrachlorofluorescein. In each panel, the lane labeled S indicates that the standard reaction was reproduced precisely. (A)
Reactions were performed with 1, 5, 10, 25, 50, 100, or 250 U of Cleavase I. (B) In separate reactions, the incubations were stopped after 0.1, 1, 2, 5, 10, 15, 20, 30,
60, or 120 min. In the lane labeled 0.1 min, the stop solution was added immediately after the enzyme addition. (C) Following denaturation at 958C, the DNAs were
cooled and the cleavage was performed at 35 to 758C in 58C increments. (D) The reaction mixtures were supplemented with 0, 1, 5, 10, 15, 20, 25, 30, or 50 mM KCl
(in addition to the 2.5 mM derived from the enzyme storage buffer). The positions of the uncut DNA and the 72-nt band are indicated.

at the same reaction temperatures, they also suggest that minor
changes in temperature, such as might be caused by instrument
variation, would have little impact on the reproducibility of the
pattern.
The temperature also had an influence on the extent of
digestion. As the reaction temperatures were increased from
35 to 508C, the patterns showed progressively more extensive
digestion. This is consistent with our observation that the ac-
tivity of this enzyme increases as the temperature is raised.
Overdigestion at higher temperatures is prevented by the grad-
ual melting out of the cleavage structures, leading to a well-
balanced pattern.
The effect of salt concentration on the cleavage patterns was
also examined (Fig. 4). An inhibitory effect is reflected in the
reduced cleavage seen with increasing KCl concentrations
(Fig. 4). The rate of cleavage by Cleavase I decreases with
increasing concentrations of KCl. The cleavage activity is re-
duced by approximately 65% at 50 mM KCl, when assayed with
an oligonucleotide whose structure should not be affected by
salt (see activity assay in Materials and Methods) (unpublished
data). Reannealing of the strands may also contribute to the
suppression of cleavage, as suggested by the appearance of
increasing amounts of reassociated material, seen as a band
migrating above the single-stranded uncut material at the
higher salt concentrations. Strikingly, the addition of salt did
not cause perceptible reorganization of the structures recog-
nized by the enzyme, indicating that minor differences in the
salt content of these reaction mixtures will not adversely affect
reproducibility. Taken together, the results shown in Fig. 4
demonstrate that the structural fingerprint patterns generated
by the Cleavase I enzyme are quite resistant to minor varia-
tions in the reaction conditions.
Identification and positioning of mutations associated with
isoniazid resistance in M. tuberculosis. CFLP patterns of three
variants of the M. tuberculosis katG gene were compared with
that of the wild type gene. Analysis of the antisense strand
revealed the appearance of two prominent bands (A and B)
due to cleavage approximately 127 and 130 nt from the 59 end
which are representative of the R463L mutation in both the
R463L and S315T/R463L mutants (Fig. 5a). The actual nucle-
otide change is located 135 nt from the 59 end. In addition, the
S315T mutation is visible as a faster migrating band due to
cleavage approximately 580 nt from the 59 end of the S315T
and S315T/R463L mutants, again corresponding closely with
the actual position of the mutation located at 579 nt (Fig. 5b).
Similar data on the detection and localization of the mutations
were obtained for the sense strand (data not shown).
When determined alongside the corresponding sequencing
ladder, the pattern variations observed in the CFLP analysis
have been within about 50 nt of the actual sites of mutation
(sequence data not shown). This localization effect suggests
that most or all of the structures that are cleaved are formed by
local interactions, rather than by interactions between distant
regions of the nucleic acid strand.
Disruptions in local structures result in the observed dif-
ferences in CFLP structural fingerprints. If the structures re-
VOL. 34, 1996 SEQUENCE ANALYSIS WITH A STRUCTURE-SPECIFIC ENDONUCLEASE
FIG. 5. Identification and positioning of mutations associated with isoniazid
resistance in M. tuberculosis. (a) Amplicons, generated with a 59 labeled primer
and an unlabeled primer, were heat denatured for 15 s, rapidly cooled to 608C,
and digested with 25 U of Cleavase I for 2 min. The samples are shown, with M
indicating DNA size markers (the sizes are given on the left). Variant bands
distinguishing mutant from wild-type (WT) DNA are marked A and B. (b) A
second gel electrophoresis was performed to expand the 500- to 600-nt region.
Bands C in the wild type and R463L mutant are similar while the same band
migrates faster in variants containing the S315T mutation.
cognized by the enzyme are in fact local, their formation
should be relatively insensitive to the presence of remote flank-
ing sequences. To examine this point, we compared the CFLP
patterns of the 516- and 1,059-bp tyrosinase gene fragments
which start from the same labeled 59 end but which extended
either 516 or 1,059 nt to their 39 ends (Fig. 6). Comparison of
the banding patterns of these samples showed that they were
identical up to the point at which the shorter pattern ends. This
result supports the model that the bands generated in this
reaction are primarily due to close interactions along the DNA
strand and that the extra length on the 39 end of the 1,059-mer
has little influence on these 59-proximal structures.
FIG. 6. Nested fragments with a common 59 end give nested cleavage pat-
terns. Fragments with different sizes were amplified from a human tyrosinase
cDNA clone. A 516-bp fragment, spanning exons 2 to 4, and an overlapping
1,059-bp fragment, covering exons 1 to 4, were amplified from cDNA clones of
the wild-type human tyrosinase gene. The antisense strand of each fragment was
labeled on the 59 end with TET. Lane M, size markers, with fragment sizes (in
nucleotides) indicated on the left.
3135
FIG. 7. Individual regions of DNA can be involved in multiple cleavage
structures. (A) Differential responses of proximal bands to deletion of flanking
sequences. A nested set of fragments were amplified from the 59 NCR of the
HCV genome (1). All fragments were labeled on their 59 ends with a TET-
labeled HCV-1 primer. The sizes of the uncut fragments (281, 186, 145, and 117
Downloaded from jcm.asm.org by on September 19, 2009
nt) and the predominant cleavage products (95, 71, and 69 nt) are indicated on
the left. (B) Multiple band changes caused by a single-base sequence change. The
cleavage pattern from the 1,059-nt segment of the wild-type (wt) human tyrosi-
nase gene is compared with the pattern from the R278X mutant, which is
different at a single site. Sense and antisense strand patterns were compared. The
distance of the mutation from the 59 end of each labeled strand is indicated
below each mutant-containing lane. Lane M contains markers, with the uncut
DNA and marker fragment sizes (in nucleotides) indicated on the left.
Several aspects of the data presented here suggest that the
pool of molecules in the cleavage reaction assume a multiplic-
ity of competitive structures and that the cleavage pattern is a
composite picture of individual cleavage events. This is sug-
gested by the proximity of the bands in some patterns, which
are closer than the cleavage of colinear structures would allow.
The existence of multiple, competing structures is more strong-
ly indicated by the observation that single-base changes can
cause multiple band changes within the structural fingerprint
patterns.
To test if close bands in a fingerprint pattern arise from
multiple structures, a nested set of DNA fragments sharing a
common labeled 59 end were amplified from the 59 NCR of
HCV. Cleavage in this region produces strong bands only 2 nt
apart at 69 and 71 nt (Fig. 7A). We questioned whether these
products arose from two points of cleavage on the same struc-
ture or from single cuts at the ends of two competing struc-
tures. The banding pattern of the 186-nt fragment is indistin-
guishable from the full-length pattern over the region shared
by the two fragments. As the fragment is truncated further to
145 nt, two new bands occur, presumably because of the dele-
tion of previously preferred secondary structural sequences in
the 186- and 281-nt fragments and subsequent creation of
alternative secondary structures in the 145-nt fragment. When
the 69- and 71-nt bands are compared in the fingerprint pat-
terns of the successively shorter DNAs, it is clear that deletion
of the sequences beyond 145 nt causes a substantial reduction
in the band at 71 nt and an enhancement of the band at 69 nt.
This differential response to the deletion of flanking sequences
shows that multiple coexisting structures are being cleaved.
The point is further illustrated by examination of Fig. 7B,
which shows the cleavage patterns for the sense and antisense
strands of two 1,059-nt fragments of the tyrosinase DNA that
differ at a single base near the middle of the fragment. The
3136 BROW ET AL.
FIG. 8. Examination of 16S-23S intergenic region of Salmonella and Shigella
spp. by CFLP analysis. The intergenic regions were amplified with a 16S sense
primer and a 23S antisense primer to generate an amplicon of approximately 550
bp. The CFLP reactions were performed under identical conditions for each
DNA strand. CFLP reactions were performed at 558C for 2 min. Lanes: M, size
marker DNA (with sizes (in nucleotides) indicated on the left); 1 through 7
(sense strand analysis) S. dysenteriae type 8, S. sonnei, S. dysenteriae type 1, S.
dysenteriae type 2, S. typhimurium, S. arizonae, and S. enteritidis, respectively; 8
through 14 (antisense strand), S. dysenteriae type 8, S. sonnei, S. dysenteriae type
1, S. dysenteriae type 2, S. typhimurium, S. arizonae, and S. enteritidis, respectively.
fingerprints of the sense and antisense strands of the R278X
fragment have several differences in the 500-nt region, com-
pared with the wild-type patterns. Thus multiple changes in the
CFLP pattern can result from a single point mutation.
Intergenic region. We examined the potential of the CFLP
method for differentiating bacteria. In order to provide the
greatest utility, a bacterium-typing method should be able to
differentiate both species and strains. The genotype of the 16S
rRNA genes, while useful for general classification, would not
be expected to necessarily allow identification to the species
and strain levels. For these purposes, we examined a 550-bp
amplicon derived from the intergenic region of several isolates
of Shigella and Salmonella spp. (Fig. 8). The structural finger-
prints generated by Cleavase I treatment clearly differentiated
all species of Salmonella and Shigella (Fig. 8). Again, a familial
resemblance is clear in all of these evolutionarily related bac-
teria, yet differences in the structural fingerprints of each genus
and species are clearly visible. The power of CFLP analysis is
even more striking when the analysis of Shigella spp. is exam-
ined (Fig. 8, lanes 1 to 4 and 8 to 11). Shigella sonnei was clearly
differentiated from the three Shigella dysenteriae isolates exam-
ined. Furthermore, although serotypes 1, 2, and 8 of Shigella
dysenteriae yielded related structural fingerprints, the antisense
patterns for the individual serotypes could, however, be distin-
guished. Therefore, CFLP analysis can potentially differentiate
microorganisms to the genus, species, and strain levels.
DISCUSSION
We have shown here that structure-specific endonucleases
can be used as sensitive tools for the analysis of the highly
J. CLIN. MICROBIOL.
individual conformations assumed by single strands of DNA.
Cleavage of the folded structure produces a fixed and repro-
ducible record of the conformation of local regions of DNA.
Because such structures are responsive to the sequence of the
DNA, the pattern of the cleavage products serves as a struc-
tural fingerprint of the molecule. Comparison of the structural
fingerprints of related DNAs is an extremely sensitive and
rapid means of determining if they are identical.
We have been able to distinguish a number of bacterial
genera and species on the basis of CFLP analysis of appropri-
ate markers. The method is suitable for analysis of 16S rRNA
genes, intergenic regions, or virulence factors such as toxin,
pilin, or antibiotic resistance genes. CFLP analysis of the 16S
rRNA genes clearly distinguished E. coli and Salmonella, and
Shigella spp. in contrast to a recent report by Widjojoatmoto et
al. (25) in which analysis of this same amplicon by single-strand
conformation polymorphism could not distinguish E. coli and
Shigella spp.
Because the structural fingerprint relies on the cleavage of
intrastrand structures, we tested the influence of changes in
reaction conditions that are likely to influence these structures.
The patterns were remarkably robust, changing relatively little
in response to two- to threefold changes in such variables as
Downloaded from jcm.asm.org by on September 19, 2009
the time of incubation, enzyme concentration, and salt concen-
tration. As expected, alterations in temperature did change the
pattern of fragments, but the change was gradual, requiring
several degrees shift to alter any particular cleavage structure.
The fact that the influence of these different conditions is not
abrupt makes the patterns reproducible, even when carried out
on different days or in different laboratories.
The use of a thermostable endonuclease, Cleavase I, allows
the cleavage reactions to be performed at elevated tempera-
ture, which is fundamental to realizing the full benefit of this
assay. For example, if a particularly stable secondary structure
is assumed by the DNA, a single nucleotide change is unlikely
to significantly alter that structure or the cleavage pattern it
produces. Elevated temperatures can be used to bring struc-
tures to the brink of instability, so that the effects of small
changes in sequence are maximized and revealed as alterations
in the cleavage pattern. Furthermore, the use of high temper-
ature reduces long-range interactions along the DNA strands,
thereby increasing the likelihood that observed cleavage dif-
ferences will reflect the locations of the base changes.
The potential for multiple localized pattern changes in re-
sponse to a single sequence change, as shown in Fig. 7B,
presents an additional advantage of this method of mutation
detection over other methods in use. Direct sequencing of this
region would provide a single variant peak on which to base a
conclusion. In contrast, the multiplicity of effects seen here
provides redundant confirmation of the base change, allowing
multiple checkpoints within the pattern to be compared. This
is especially useful in the cases of heterozygosity, or the pres-
ence of drug-sensitive and drug-resistant bacteria in a single
sample.
While the analyses shown above have been done with non-
isotopic labels, the method is fully compatible with radiola-
beled DNAs and standard autoradiography. When staining or
uniform labeling are used, the patterns produced are more
complex, as all fragments are visible, and the localization fea-
ture is lost because bands cannot be measured from a discrete
labeled end, yet variations in the cleavage patterns are readily
detected. We have also found that CFLP can be performed on
differently labeled wild-type and mutant alleles in the same
reaction, with no discernible effect of the patterns of each.
When fragment size analyses are performed on a fluorescence-
based DNA sequencer, the wild-type pattern can serve as an
VOL. 34, 1996 SEQUENCE ANALYSIS WITH A STRUCTURE-SPECIFIC ENDONUCLEASE
internal control for enzyme digestion and as an internal refer-
ence that allows accurate discrimination of pattern differences
in the mutant samples. When calibrated against a sequence
ladder or other known markers, the wild-type pattern also
allows precise sizing of the mutant bands. With the CFLP
patterns normalized in this way, experimental samples can be
compared day-to-day and lab-to-lab, with a high degree of
confidence. Lastly, the ability to rapidly perform an indepen-
dent analysis of both DNA strands with the use of differently
labeled PCR primers facilitates complementary confirmatory
information analogous to sequencing both DNA strands.
Analysis of DNAs through the probing of their structures
with the Cleavase I enzyme promises to be a powerful tool in
the comparison of sequences. As demonstrated here, the
method is able to detect single base changes in DNA molecules
over 1 kb long. In addition to the work discussed here, we have
been able to apply CFLP analysis to detection of mutations
responsible for rifampin resistance in M. tuberculosis and for
determining the genotype of HCVs and have successfully an-
alyzed DNAs as long as 2.7 kb. The low cost, rapidity, and
reliability of the analysis make this method very suitable for the
rapid screening of DNAs, not only for the examination of
disease-associated mutations but also in applications such as
tissue typing, genetic identity, bacterial and viral typing, and
mutant screening in genetic crosses.
ACKNOWLEDGMENTS
We thank Frank Cockerill, Jeff Klinger, Richard Spritz, and Manual
Altamirano for DNA samples and bacterial cultures. Cleavase I re-
agents may be obtained from either Boehringer Mannheim Biochemi-
cals (Indianapolis, Ind.) for CFLPScan and CFLPScan-XL kits or Life
Technologies, Inc. (Gaithersburg, Md.) for PowerScan kits.
This work was supported by grants from the National Institutes of
Health (1R43 GM51704-01) and the National Institutes of Standards
and Technology Advanced Technology Program (94-05-0012).
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