Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays.

BACKGROUND: Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. RESULTS: The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. CONCLUSION: The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori.

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.