Single-molecule polymerase chain reaction reduces bias: application to DNA methylation analysis by bisulfite sequencing.

The treatment of DNA with bisulfite, which converts C to U but leaves 5-methyl-C unchanged, forms the basis of many analytical techniques for DNA methylation analysis. Many techniques exist for measuring the methylation state of a single CpG but, for analysis of an entire region, cloning and sequencing remains the gold standard. However, biases in polymerase chain reaction (PCR) amplification and in cloning can skew the results.

Attenuation of late-stage disease in mice infected by the Mycobacterium tuberculosis mutant lacking the SigF alternate sigma factor and identification of SigF-dependent genes by microarray analysis.

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis DeltasigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type.

Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.

The pathogenesis of tuberculosis involves multiple phases and is believed to involve both a carefully deployed series of adaptive bacterial virulence factors and inappropriate host immune responses that lead to tissue damage. A defined Mycobacterium tuberculosis mutant strain lacking the sigH-encoded transcription factor showed a distinctive infection phenotype. In resistant C57BL/6 mice, the mutant achieved high bacterial counts in lung and spleen that persisted in tissues in a pattern identical to those of wild-type bacteria.

Improved quantitation and reproducibility in Mycobacterium tuberculosis DNA microarrays.

We show here that the amount of labeled cDNA and its specific activity can play a significant role in the quantitation of microarray experiments. Standard reverse transcription of 2 microg total bacterial RNA with concomitant incorporation of cyanine dye-conjugated nucleotides did not produce enough label for optimal hybridization results in our Mycobacterium tuberculosis DNA microarray. Therefore we turned to an alternative labeling method using the incorporation of aminoallyl nucleotides followed by conjugation to Cy-dye.