Comparative genomics of BCG vaccines by whole-genome DNA microarray

Science

Volumn 284, Issue 5419, 1999, Pages 1520-1523

  Behr, M A ^a   Wilson, M A ^b   Gill, W P ^b   Salamon, H ^b   Schoolnik, G K ^b   Rane, S ^b   Small, P M ^b  

^a MCGILL UNIVERSITY HEALTH CENTRE   (Canada)

^b STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BCG VACCINE;

ARTICLE; BACTERIAL GENE; BCG VACCINATION; COMPARATIVE GENOMIC HYBRIDIZATION; DNA DETERMINATION; GENE DELETION; MYCOBACTERIUM BOVIS; MYCOBACTERIUM TUBERCULOSIS; NONHUMAN; OPEN READING FRAME; PRIORITY JOURNAL; STRAIN DIFFERENCE; TUBERCULOSIS;

BCG VACCINE; DNA, BACTERIAL; EVOLUTION, MOLECULAR; GENE DELETION; GENOME, BACTERIAL; HUMANS; MYCOBACTERIUM BOVIS; MYCOBACTERIUM TUBERCULOSIS; NUCLEIC ACID HYBRIDIZATION; OLIGONUCLEOTIDE ARRAY SEQUENCE ANALYSIS; OPEN READING FRAMES; POLYMERASE CHAIN REACTION; VACCINES, ATTENUATED; VARIATION (GENETICS); VIRULENCE;

EID: 0033612136     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5419.1520     Document Type: Article

References (29)

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5. Strains used were M. tuberculosis H37Rv, BCG-Russia (ATCC 35740), BCG-Moreau, BCG-Japan, BCG-Sweden, BCG-Prague, BCG-Phipps (ATCC 35744), BCG-Denmark 1331 (ATCC 35733), BCG-Tice (ATCC 35743), BCG-Frappier (ATCC 35735), BCG-Connaught, BCG-Birkhaug (ATCC 35731), BCG-Glaxo (ATCC 35741), and BCG-Pasteur 1173.
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8. Using the annotated sequence of M. tuberculosis H37Rv made available by the Sanger Centre (ftp://ftp.sanger. ac.uk/pub/tb/sequences), we designed PCR primer pairs to amplify 250-to 1000-bp internal fragments of predicted ORFs (details about primers used are available at http://molepi.stanford.edu/bcg). Primer design was automated using Primer 3 [release 0.6, S. Rozen and H. J. Skaletsky (1996, 1997), code available at www-genome. wi.mit.edu/genome_software/other/primer3.html]. PCR reactions were prepared in 65-μl reactions in 96-well plates; amplicons were evaluated to ensure they were the expected size and then robotically applied to poly-L-lysine-coated microscope slides as described (protocols available at http://cmgm.stanford.edu/pbrown/ mguide/index.html). After printing, free amino groups of the poly-L-lysine-coated slides were acetylated with 5.5 g of succinic anhydride in 350 ml of borate-buffered 1-methyl-2-pyrrolidinone (pH 8).
9. 2 cover slip. The slide was then placed in a waterproof hybridization chamber for hybridization in a 65°C water bath for 3 hours. After hybridization, slides were washed 2 min in 1× SSC buffer with 0.06% SDS followed by 2 min in 0.06× SSC buffer.
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12. Slides were scanned with a custom-built scanning laser microscope as described [J. L. DeRisi, V. R. Iyer, P. O. Brown, Science 278, 680 (1997)]. Fluorescent spot intensities were quantified using custom software as described by DeRisi and available at http:// rana.Stanford.EDU/software. For each spot, background fluorescence was subtracted from the average spot fluorescence to produce a channel-specific value. Spots were excluded from analysis when background intensity was greater than the spot intensity. The ratio of Cy3/Cy5 hybridization was calculated and log-transformed for representation according to genomic location implementing ProbeBrowser software (designed and written in our laboratory and available at http://molepi.stanford.edu/free_software.html). This software integrates microarray experimental results with the genomic positions of the hybridization targets and displays corresponding ORF annotations (see Fig. 1, B and C).
13. For any single experimental spot a mismatched hybridization could represent (i) a repeated element more numerous in H37Rv, such as IS6110, (ii) an element present in H37Rv and absent from BCG, or (iii) a false positive result that may occasionally occur during thousands of simultaneous hybridizations. We observed that spots representing IS6110 reliably resulted in high ratios of H37Rv:BCG, corresponding to their known 16:1 genomic presence. For assigning deletions, we restricted our investigation to regions where probes from at least two adjacent ORFs exhibited mismatched hybridization. This strategy was invoked to maximize the likelihood of detecting true deletions at the expense of decreased resolution. For putative deletions identified by the array experiments, we performed Southern (DNA) hybridization using probes independent of those on the array to confirm their absence In BCG strains. Then, to characterize confirmed deletions, we designed PCR primers to approach the predicted edges of each deletion so that the amplified region would span the missing genomic locus for those strains in which it is deleted. These amplicons were sequenced using the PCR primers at the Protein and Nudeic Acid (PAN) Core Facility, Stanford University, by ABI dye terminator chemistry. Sequences obtained were aligned to the known sequence of H37Rv by BLAST search [S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997)] to determine the precise site of each deletion (details available at http://molepi.stanford.edu/bcg). Based on the site and the predicted ORFs from the H37Rv, we documented which reading frames were partially or completely missing from BCG strains. To classify deleted regions as BCG-specific or as differences between H37Rv and virulent M. bovis, we repeated PCR with the primers spanning the missing genetic region using DNA from virulent M. bovis (four human and four bovine isolates).
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19. In the previous work, there were two discrepancies between genotype data and the written record; in both we have subsequently obtained new information indicating that our historical information was incorrect. During the American Indian trial, Aronson obtained BCG-Pasteur (passage) 575 in 1938 [J. D. Aronson, E. I. Parr, R. M. Saylor, Am. Rev. Tuberc. 42, 651 (1940)]; this likely became the stock from which BCG-Phipps was derived. According to Birkhaug himself, BCG-Birkhaug appears to have been obtained from the Institut Pasteur in 1927 [K. Birkhaug, Psychiatr. Q. 21, 453 (1948)].
20. In the previous work, there were two discrepancies between genotype data and the written record; in both we have subsequently obtained new information indicating that our historical information was incorrect. During the American Indian trial, Aronson obtained BCG-Pasteur (passage) 575 in 1938 [J. D. Aronson, E. I. Parr, R. M. Saylor, Am. Rev. Tuberc. 42, 651 (1940)]; this likely became the stock from which BCG-Phipps was derived. According to Birkhaug himself, BCG-Birkhaug appears to have been obtained from the Institut Pasteur in 1927 [K. Birkhaug, Psychiatr. Q. 21, 453 (1948)].
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29. For strains and DNA of M. tuberculosis, M. bovis, and M. bovis BCG, we thank M. Gheorghiu and S. Cole, Institut Pasteur, Paris (BCG-Pasteur 1173); A. Ladefoged, Statens Serum Institut, Copenhagen (BCG-Sweden and BCG-Prague); M. Takahashi, Japanese Anti-Tuberculosis Association (BCG-Japan); P. Rousseau, Institut Armand Frappier, Canada (BCG-Moreau); U. Kosecka, Connaught Laboratories, Canada (BCG-Connaught); M. D. Cave, University of Arkansas; and G. Hewinson, Central Veterinary Laboratories, Surrey, UK. For technical assistance we thank J. DeRisi and members of P. Brown's laboratory. For assistance with the gene annotation database, we thank S. Cole and his laboratory at the Institut Pasteur.