We thank Jack T.Crawford for providing us with the pDC73 plasmid. REFERENCES. 1. Hennans,P.W.M., van Soolingen,D., Dale,J.W., Schuitema,A.R.J.,.
Nucleic Acids Research, 1993, Vol. 21, No. 3 761-762
DNA fingerprinting of Mycobacterium tuberculosis isolates by ligation-mediated polymerase chain reaction Prasit Palittapongarnpim, Sylvia Chomyc, Anne Fanning and Dennis Kunimoto* Department of Medical Microbiology and Infectious Diseases, Provincial Laboratory of Public Health for Northern Alberta, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada Received November 19, 1992; Revised and Accepted January 11, 1993 Strain identification of M. tuberculosis has been performed by probing Southern blots of restriction digested chromosomal DNA with the insertion sequence (IS) IS986 or IS6110 (1, 2) to detect restriction fragment length polymorphisms (RFLP). The resulting 'fingerprint' reflects the polymorphism of the sequences which flank the IS. We have designed a ligation-mediated polymerase chain reaction (LMPCR) procedure to amplify the flanking sequences on both sides of the IS (Figure la). Our method makes use of a nonphosphorylated BamHI-compatible linker which was designed to contain a sequence identical to that found in the IS6110 (3) itself (Figure lc). This strategy allows us to perform LMPCR using only two rather than three primers which are more typically required. We used this method to characterize the H37Rv and H37Ra strains and 26 clinical isolates of M. tuberculosis. DNA samples were diluted to a concentration of 20 ng4ld and digested with 20 units of BamHI or BgII per Ag of DNA in M buffer (Boehringer Mannheim) for one hour. A mixture containing 5 ,tl of the digested DNA and 10 /d of the empirically determined optimum of 50 pg4ld of the linker was warmed to 450C for 5 min, then chilled in an ice bath. Five microlitres of a
W1ke,
a buffer containing 200 mM Tris HCI pH 7.6, 40 mM MgCl2, 4 mM ATP, 4 mM dithiothreitol, 20% w/v PEG-8000 and 0.2 units of T4 DNA ligase were added to the mixture which was then incubated at 15°C for 4 hours. Amplification was perfonned in a total volume of 50 ,1 containing 2.5 units of Taq polymerase, 0.5 yd of the ligated DNA mixture, 5 yd of l0xbuffer (100 mM Tris HCl pH 9.0, 12.5 mM MgCl2, 500 mM KCl, 1% triton X-100, 0.1% w/v gelatin), 200 ,uM of each dNTP and 0.5 ,uM of each primer (dku53 and dku55, Figure lc). The reaction mix was overlaid with 50 IAI of mineral oil, incubated for 3 minutes at 94°C, and subjected to 30 cycles of PCR (1 min at 94°C, 1 min at 62°C and 2 min at 72°0C), using a PHC-2 thermocycler (Techne Incorporated, Princeton, NJ). The products were visualized by ethidium bromide staining after electrophoresis in a gel containing 0.7% agarose and 0.4% Synergel (Diversified Biotech, Newton Centre, MA). In this case the Synergel was used to enhance the visualization of the DNA. The amplified DNA fragments usually consisted of 5 to 12 bands, ranging from about 100 bp to 2 kb long (Figure ld). Using BamHI digested DNA, it was found that M. tuberculosis isolates from different case clusters or solitary cases always gave different
LIGATION
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IST CYCLE
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Figure 1. a. Fate of a Bglll digested fragment containing IS6110 during LMPCR. The insertion sequence IS6110 is represented by the dark grey area and the linkers are represented in light grey. The primers are represented by horizontal arrows. Bgl digestion creates BamHI-compatible cohesive ends. b. Fate of a restriction fragment not containing an insertion sequence. Since the linker is not phosphorylated, only the 5' ends of the fragment are ligated. The ligated products cannot serve as targets for PCR. c. The sequences of the linker and both primers. The sequence of dku55 is identical to the shorter strand of the linker. The linker was designed so that neither BamHI nor BgM can digest the ligated products, therefore, removal of the restriction enzymes is unnecessary. d. Ligation-mediated PCR
products of Bgtl digested chromosomal DNA. Lanes 2 and 3 are the products of H37Ra and H37Rv strains whereas lanes 4-11 are the PCR products of different clinical isolates of M. tuberculosis. The samples represented in lanes 10 and 11 were isolated from a baby and his mother. The DNA marker in lane 1 is the replicative form of tX174 digested with HaeEII. *
To whom correspondence should be addressed
762 Nucleic Acids Research, 1993, Vol. 21, No. 3 banding patterns, whereas isolates from the same case cluster usually gave the same banding pattern. The two closely related type strains, H37Rv and H37Ra, had similar but distinctive banding patterns. The results were confirmed by using Bgll digested DNA. In addition, these LMPCR results were confirmed by Southern blotting and probing with a BamHI -SalI fragment of pDC73, which contains a part of the IS6110 (2). Five isolates could not be initially characterized clearly by Southern hybridization due to the sheared state of the chromosomal DNA samples and their DNA had to be extracted a second time to be successfully fingerprinted by Southern hybridization. Blunt end LMPCR has been used to amplify DNA fragments between a known and unknown sequence (4, 5). The method described here is different as cohesive end ligation was performed. One might expect that the linker would be ligated to both ends of all fragments and therefore produce a smear after PCR. This does not occur as the linker is not phosphorylated, and hence the longer linker strand cannot be ligated to the genomic DNA and act as a PCR priming site for the dku55 primer (Figure Ib). The LMPCR method is technically easy and can be finished in 15 hours, as purification of DNA is unnecessary after restriction digestion or ligation. It also requires less DNA and tolerates greater shearing than Southern blotting and hybridization. Since the results were confirmed by RFLP detected by an IS6110 probe, we feel that the LMPCR method is an attractive alternative in case-tracing and epidemiologic studies of M.tuberculosis. In addition this methodology should be applicable to any organisms containing known insertion sequences.
ACKNOWLEDGMENT We thank Jack T.Crawford for providing us with the pDC73 plasmid.
REFERENCES 1. Hennans,P.W.M., van Soolingen,D., Dale,J.W., Schuitema,A.R.J., McAdam,R.A., Catty,D. and van Embden,J.D.A. (1990) J. Clin. Microbiol. 28, 2501-2508. 2. Mazurek,G.H., Cave,M.D., Eisenach,K.D., Wallace,R.J.,Jr, Bates,J.H. and Crawford,J.T. (1991) J. Clin. Microbiol. 29, 2030-2033. 3. McAdam,R.A., Hermans,P.W.M., van Soolingen,D., Zainuddin,Z.F., Cany,D., van Embden,J.D.A. and Dale,J.W. (1990) Moi. Microbiol. 4,
1607-1613. 4. Mueller,P.R. and Wold,B. (1989) Science 246, 780-786. 5. Pfeifer,G.P., Steigerwald,S., Mueller,P.R., Wold,B. and Riggs,A.D. (1989) Science 246, 810-812.
