Four-color DNA cycle sequencing was performed on an M13mp18 template using dye-labeled primers. Sequencing fragments were separated by capillary electrophoresis at 60 degrees C and at an electric field of 150 V/cm. The sieving medium was 5%T, non-cross-linked polyacrylamide in 7 M urea. The use of high temperature for the separation reduces formation of secondary structures in the sequencing fragments, generating a sequence that is free of compressions without the use of strongly denaturing gels. The use of high temperatures also increases the separation rate compared with room-temperature operation. Fragments up to 640 bases are separated in less than 2 h.

Selective capture of transcribed sequences (SCOTS) has been employed to identify 54 cDNA molecules that represent 46 genes that are expressed by Mycobacterium avium during growth in human macrophages. Some cDNA molecules correspond to genes that are apparently expressed 48 h after infection of macrophages, while others correspond to genes expressed 110 h after infection, and still others correspond to genes expressed throughout the course of infection in our model system. Genes expressed by M. avium during growth in macrophages include genes encoding enzymes of several biosynthetic pathways (pyrimidines, mycobactin, and polyketides); genes that encode enzymes involved in intermediary metabolism, energy metabolism (tricarboxylic acid cycle, glyoxalate shunt), and nitrogen metabolism; and genes that encode regulatory proteins. A number of genes of unknown function were also identified, including genes that code for proteins similar to members of the PPE family of proteins of Mycobacterium tuberculosis and proteins similar to those encoded by the M. tuberculosis mce genes, which have been previously associated with mycobacterial virulence. The SCOTS technique, followed by enrichment for cDNA molecules that are up-regulated or are uniquely expressed by M. avium during growth in human macrophages (compared to growth in laboratory broth culture), allows recovery and identification of a greater diversity of cDNA molecules than does subtractive hybridization between cDNA mixtures from macrophage-grown and broth-grown M. avium. Data are presented demonstrating the reproducibility of recovery of a subset of cDNA molecules from cDNA mixtures purified by SCOTS on several different occasions. These results further demonstrate the beneficial utility of the SCOTS technique for identifying genes whose products are needed for successful survival and growth by an organism in a specific environment.Mycobacterium avium is a ubiquitous, saprophytic mycobacterium commonly found in soil and water (13,20,27). Prior to the AIDS epidemic, M. avium was rarely identified as a pathogen in humans. However, during the early years of the AIDS epidemic, M. avium became one of the most significant opportunistic pathogens of severely immunocompromised human immunodeficiency virus (HIV)-infected individuals (those whose CD4 ϩ T-cell counts were Յ100/ml of blood; 11, 26, 29). Implementation of highly active antiretroviral therapy (HAART) in the treatment of HIV-infected individuals has significantly improved the degree of immunocompetency of AIDS patients and has resulted in a concomitant decrease in the incidence of opportunistic infections in these individuals (1, 30, 38). However, newly diagnosed AIDS patients, those for whom HAART has been ineffective, and those who are unable to afford HAART remain at risk for opportunistic infections. Thus, we must not become complacent in thinking that M. avium is no longer a significant pathogen. Moreover, increasing numbers of non-HIV-infected individuals are being diagnosed with M. avium infections of the...

We present the mobility of DNA sequencing fragments as a function of temperature in 5%T, 0%C noncross-linked polyacrylamide and at an electric field of 150 V/cm. The mobility of the fragments follows the Arrhenius equation. The activation energy for migration of a fragment through the polymer decreases monotonically with fragment length. The data were also analyzed in terms of the biased reptation model; the onset of biased reptation with stretching occurs for longer fragments as the temperature increase. These results are quite different from those observed for separation of DNA in cross-linked gels and represent a fundamental difference in the physics of the cross-linked and noncross-linked polyacrylamide.

No abstract

The mobility of DNA sequencing fragments was measured in Long-Ranger gels at an electric field ranging from 200 to 1200 V cm-1 and in noncross-linked polyacrylamide at electric fields ranging from 100 to 300 V cm-1. In both cases, N*, the fragment length that denotes the onset of biased reptation with orientation, is inversely proportional to electric field. The inverse dependence of N* is inconsistent with the original biased reptation model but is consistent with modern models of DNA migration. While separation speed increases dramatically with electric field, the number of bases determined in a separation decreases in proportion to field strength. We present a DNA sequencing run at an electric field of 1200 V cm-1. Roughly 200 bases of sequence are determined in 3.5 min.