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Automated DNA fingerprinting analysis of
Mycobacterium tuberculosis using fluorescent
detection of PCR products.
W R Butler, W H Haas and J T Crawford
J. Clin. Microbiol. 1996, 34(7):1801.
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JOURNAL OF CLINICAL MICROBIOLOGY, July 1996, p. 1801–1803
0095-1137/96/$04.0010
Copyright q 1996, American Society for Microbiology
Vol. 34, No. 7
Automated DNA Fingerprinting Analysis of Mycobacterium tuberculosis
Using Fluorescent Detection of PCR Products
W. RAY BUTLER,1* WALTER H. HAAS,2
AND
JACK T. CRAWFORD1
Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta Georgia 30333,1 and
University Childrens Hospital, Heidelberg University, Germany2
DNA fingerprints of Mycobacterium tuberculosis are produced by restriction fragment length polymorphism
analysis of the insertion element IS6110. We modified a PCR-based subtyping method, mixed-linker PCR with
fluorescent-labeled IS6110-specific oligonucleotides, to demonstrate rapid, automated, and unattended electrophoretic analysis. Variation in band sizing (normally occurring with fragment mobility), an artifact of
lane-to-lane and gel-to-gel differences, was controlled with an internal lane standard, resulting in accurate and
precise DNA sizing. By using this method, fingerprint analysis can be performed using actual fragment length
rather than estimated position analysis.
ored electrophoretic image with corresponding tabular data on
band sizes.
Mycobacterium strains. We examined M. tuberculosis strains
from the collection at CDC. They represented isolates obtained from clinical laboratories as part of investigations of
outbreaks or suspected occurrences of laboratory cross-contamination.
PCR method. For the ML-PCR method, mycobacterial
DNA was isolated in an aqueous phase after aggressive shaking with glass beads with a Mickle apparatus (Brinkman Instruments, Westbury, N.Y.) as described by Plikaytis et al. (6).
The procedure for ML-PCR has been previously described by
Haas et al. (4). Briefly, DNA is digested with HhaI, and a
double-stranded oligonucleotide, designated mixed linker, is
ligated to the cut ends. The mixed linker is an unphosphorylated product with a GC overhang at the 39 end compatible
with the HhaI-restriction fragments. One strand of the linker
contains uracil instead of thymidine and is subsequently removed by treatment with uracil N-glycosylase. Restriction fragments containing the IS6110 sequence are amplified by PCR
by using an IS6110-specific primer and a linker primer. Subsequently, a nested amplification with a second primer specific
for IS6110 increases the specificity of the reaction. Substitution
of fluorescently labeled primers for this second IS6110-specific
primer resulted in production of fluorescence-labeled PCR
products. For clarity, the sequences are shown in Table 1. With
the exception of the fluorescence-labeled primers, primers
were synthesized at the Biotechnology Core Facility, CDC, on
a DNA synthesizer (Model 381A; Applied Biosystems, Foster
City, Calif.).
Fluorescent tags. Four different fluorescent labels were
used: FAM (blue), JOE (green), TAMRA (yellow), and ROX
With the resurgence of tuberculosis, there has been a concurrent increase in basic molecular investigative research on
Mycobacterium tuberculosis. Some of this basic research has
been applied in mycobacterium reference laboratories, and
molecular approaches are fast becoming standard methods of
analysis. One such method is the molecular subtyping of M.
tuberculosis isolates by DNA fingerprinting. A standardized
restriction fragment length polymorphism (RFLP) procedure
has been adopted by the Centers for Disease Control and
Prevention (CDC) to track strains of M. tuberculosis associated
with epidemiological investigations (8). The procedure is based
on variability in copy number and sites of insertion of IS6110.
The DNA fingerprint patterns generated by this method are
identified by arbitrarily assigned series numbers and have been
shown to be reliable for tracking outbreaks of M. tuberculosis
(2, 8). Compared with the earlier phage-typing method (7),
IS6110 RFLP is a rapid method that yields results in several
weeks. However, compared with other molecular methods, the
IS6110 RFLP procedure is slow. Alternatively, PCR methods
produce DNA fingerprint results quickly. One such procedure
is the mixed-linker PCR (ML-PCR). This method does not rely
upon growing cultures or viable cells and reliably detects fragments containing IS6110 (4). Patterns generated by using MLPCR have shown a direct correlation to those obtained with
the standard RFLP method, providing the same clustering of
isolates (1, 3, 4, 5). Electrophoretic separation of the DNA
fragments on gels is used for both methods, and the sizes of the
fragments are estimated by their migration distances relative to
standard fragments of known size. However, standardization of
fingerprints can be problematic because lane-to-lane and gelto-gel variation caused by electrophoretic separation reduces
the accuracy of estimated fragment sizing. We report here a
procedure in which fluorescence-labeled oligonucleotide primers are used in the ML-PCR method to generate fluorescencetagged PCR products for rapid, accurate analysis using the
373A DNA sequencer (ABI) with Genescan 672 software.
Output from the DNA sequencer is in the form of a multicol-
TABLE 1. Modified oligonucleotides for ML-PCR
Purpose
Linker construction
Linker amplification
IS6110 amplification
IS6110 nested amplification
* Corresponding author. Mailing address: Mailstop F08, NCID/
DASTLR, CDC, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404)
639-1280. Fax: (404) 639-1287.
1801
Sequence
59-AGA ACT GAC CTC GAC TCG CAC G-39
39-TCU UGA CUG GAG CUG AGC GU-59
59-AGA ACT GAC CTC GAC TCG CA-39
59-TCG ACT GGT TCA ACC ATC GCC G-39
59-dye-ACC AGT ACT GCG GCG ACG TC-39
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Received 16 January 1996/Returned for modification 19 March 1996/Accepted 1 April 1996
1802
NOTES
J. CLIN. MICROBIOL.
Downloaded from http://jcm.asm.org/ on February 26, 2014 by PENN STATE UNIV
FIG. 1. Polyacrylamide gel analysis of PCR-amplified HhaI RFLP from representative strains of M. tuberculosis. (A) Precise base sizing of M. tuberculosis isolate
CA 93-8287, series 134, fluorescently labeled with yellow tag (lanes 1 to 10). Overlapping PCR fragments from electrophoresis of three M. tuberculosis strains per lane
shown as purple (lanes 11 to 15). (B to D) Detection of individual fluorescent dyes as discriminated by analysis software. The strains and the respective fluorescent tags
used in lanes 11 through 15 are as follows. Lane 11, CA 93-8408, series 141 (blue); NY 94-8256, series 021 (green); and TN 93-8263, series 132 (yellow). Lane 12, CA
93-8407, series 141 (blue); GA 93-8013, series 147 (green); and CA 93-8287, series 134 (yellow). Lane 13, TN 94-8003, series 146 (blue); CA 93-8003, series 079 (green);
and CA 93-8223, series 127 (yellow). Lane 14, TN 94-8002, series 146 (green); TN 93-8262, series 132 (blue); and CA 93-8223, series 127 (yellow). Lane 15, CA 93-8286,
series 134 (blue); MN 93-8405, series 068 (green); and TN 93-8227, series 126 (yellow).
VOL. 34, 1996
1803
ping PCR fragments electrophoresed in these lanes are displayed as purple in Fig. 1A. Panels B, C, and D show detection
of the individual fluorescent dyes as discriminated by the analysis software. In total, 15 different M. tuberculosis strains, representing 10 different CDC epidemiological investigations,
were easily separated in lanes 11 through 15. Different strains
representing the same RFLP series produced identical PCR
restriction fragments. The analysis demonstrated the separation of DNA restriction fragments from strains coloaded into
lanes 11 to 15 (Fig. 1B, C, and D). Coloading of multiple
samples into a single lane did not affect migration distances of
the fragments for individual samples.
The ML-PCR procedure can be completed in 3 days. Because detection of the three fluorescent tags is independent of
each other, 72 DNA samples could be analyzed in the 24 lanes
of a single gel with this technique. The number of HhaI restricted amplification fragments produced with the ML-PCR
does not always match the number of genomic DNA bands
produced from the same strain of M. tuberculosis digested with
PvuII for standard RFLP fingerprinting analysis. However, this
variation was never more than a single band difference for the
samples analyzed. Moreover, the method is suitable for epidemiological studies, since the grouping of the fingerprints by
ML-PCR is consistent for repetitive isolates and is in agreement with the clusters determined by standard RFLP results.
For the standard RFLP analysis method, DNA is isolated
from 1-week-old 7H9 broth cultures of M. tuberculosis and
digested with PvuII as previously described (8). PCR-based
analysis of DNA has the advantage that it does not rely upon
growing cells.
REFERENCES
1. Braden, C. R., and an Investigative Team. 1995. Infectiousness of a university
student with laryngeal and cavitary tuberculosis. Clin. Infect. Dis. 21:565–570.
2. Cave, M. D., K. D. Eisenach, P. F. McDermott, J. H. Bates, and J. T.
Crawford. 1991. IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol. Cell. Probes
5:73–80.
3. Cleveland, J. L., J. Kent, B. F. Gooch, S. E. Valway, D. W. Marianos, W. R.
Butler, and I. M. Onorato. 1995. Multidrug-resistant Mycobacterium tuberculosis in an HIV dental clinic. Infect. Control Hosp. Epidemiol. 16:7–11.
4. Haas, W. H., W. R. Butler, C. L. Woodley, and J. T. Crawford. 1993. Mixedlinker polymerase chain reaction: a new method for rapid fingerprinting of
isolates of the Mycobacterium tuberculosis complex. J. Clin. Microbiol. 31:
1293–1298.
5. Horn, D. L., D. Hewlett, W. H. Haas, W. R. Butler, J. T. Crawford, C. Alfalla,
E. Tan, A. Levine, A. Nayak, S. Peterson, and S. M. Opal. 1994. Superinfection with rifampin-isoniazid-streptomycin-ethamabutol (RISE)-resistant tuberculosis in three AIDS patients: confirmation by polymerase chain reaction.
Ann. Intern. Med. 121:115–116.
6. Plikaytis, B. B., R. H. Gelber, and T. M. Shinnick. 1990. Rapid and sensitive
detection of Mycobacterium leprae using a nested-primer gene amplification
assay. J. Clin. Microbiol. 28:1913–1917.
7. Snider, D. E., W. D. Jones, Jr., and R. C. Good. 1984. The usefulness of phage
typing Mycobacterium tuberculosis isolates. Am. Rev. Respir. Dis. 130:1095–
1099.
8. van Embden, J. D. A., M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach,
B. Gicquel, P. W. M. Hermans, C. Martain, R. McAdam, T. M. Shinnick, and
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Downloaded from http://jcm.asm.org/ on February 26, 2014 by PENN STATE UNIV
(red). Applied Biosystem’s FluoroProbes Service synthesized
the nested primer and 59-labeled it with one each of the different dyes, except red. The red dye was used for the internal
lane size standard (i.e., GS2500 ROX-labeled standard) designed for detection of fragments from 37 to 400 bp. The
Genescan software used the internal standard to create a calibration curve of peak detection time and used the curve to
automatically calculate the sizes of the PCR products.
Analysis of fluorescence-labeled sample. For loading, 2 ml
of fluorescence-labeled PCR products was rapidly evaporated
in a Savant SpeedVac (Savant Instruments, Inc., Farmingdale,
N.Y.) at ambient temperature. Each sample or multiple sample mixture was suspended in 5 ml of loading mixture, consisting of 4.3 ml of deionized formamide, 0.5 ml of GS2500 (ROX)
standard, and 0.2 ml of an aqueous solution of blue dextran (8
mg/ml). Samples were denatured by heating at 928C for 5 min
and snap cooled before loading. Multiple samples with different fluorescent labels were coloaded in the same well for analysis. PCR products were separated on a 6% polyacrylamide
denaturing gel with a 24 cm well-to-read distance for 7 h at 800
V. Electrophoretic data were automatically analyzed and sized
in base pairs with the Genescan software and reconstructed as
a gel image.
Automated fluorescence detection, following ML-PCR amplification of HhaI restriction fragments of IS6110, was accomplished by substitution of a 59-fluorescence-labeled oligonucleotide for the nested amplification primer (Table 1, Fig. 1). To
show accuracy of the sizing, the cleavage site for HhaI was
determined for the published sequence of the flanking region
for the single copy of IS6110 in Mycobacterium bovis BCG. This
was calculated to result in a fragment of 257 bp; the observed
band size was 257.4 bp.
To demonstrate the precise base size reproducibility of a
single M. tuberculosis isolate, CA 93-8287, series 134, was fluorescently labeled with the yellow tag and separated in lanes 1
through 10 (Fig. 1A). The Genescan software utilized the 14
DNA fragments produced from the GS2500 ROX-labeled internal lane size standard to compensate for variability in laneto-lane band migration and allowed for precise base sizing
(Fig. 1A). Seven bands were detected for this sample in the 10
lanes. The band at 129 bp is an artifact produced by all samples
analyzed with this method. However, this fragment was used as
a reference marker for the start of the fragments of interest.
Reproducibility was demonstrated by calculating the following
mean 6 standard deviation base pair values (n 5 10) for the
remaining six PCR fragments: 161.99 6 0.07, 178.22 6 0.09,
227.49 6 0.05, 253.86 6 0.05, 404.97 6 0.08, 446.55 6 0.36.
Similar results were demonstrated with different gels, and the
base sizing was not affected by gel-to-gel differences (data not
shown).
In 5 lanes, 11 through 15, multiple samples (n 5 15) were
electrophoresed in a single gel lane. Each gel lane contained
three different strains. These three M. tuberculosis strains were
fluorescently labeled with different dyes (yellow, blue, or
green) and coloaded into a single well for analysis. Overlap-
NOTES