Biomedical subjects

Stephen C Hardies

Publications and source records attributed to Stephen C Hardies.

4 recordsLinked to original sources

PubMed2005

Matrix conditioning for lengthened capillary DNA sequencing.

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

DNA↗

PubMed2005

Temperature-dependence of preconditioning for lengthened capillary DNA sequencing.

A previous study shows that electrophoretic preconditioning of a commercial polymer solution increases the spacing and resolution of DNA fragments fractionated in this solution by CE at 50 degrees C (Griess, G. A. et al., Electrophoresis 2005, 26, 102). The present study shows that this preconditioning effect on peak spacing progressively increases when the temperature of preconditioning increases to 70 degrees C, though fractionation is still performed at 50 degrees C. An increase in peak sharpness accompanies the increase in peak separation for DNA fragments longer than 200 bases. Changing the preconditioning temperature from 50 to 70 degrees C optimally improves resolution of fragment analysis in the range of 600-2000 nucleotides. When DNA sequencing is performed with automated base calling and 70 degrees C preconditioning at 319 V/cm (47 cm long capillary, Applied Biosystems 310 apparatus), the range of high-quality base calls is increased by 25% to 750; the range of low-quality base calls is increased by about 100% to 1200 in comparison to DNA sequencing without preconditioning.

DNA↗

PubMed2004

Improved isolation of undersampled bacteriophages: finding of distant terminase genes.

Isolation and characterization of new environmental bacteriophages are performed for (1) analyzing microbial evolution and ecology and (2) delivering biological therapy. The sampling of environmental bacteriophages appears, however, to be limited by the procedure (usually liquid enrichment culture) used to propagate them. An alternative, less competitive procedure is developed here for the purpose of isolating new bacteriophages. This procedure involves extraction directly into and then propagation in a dilute agarose gel. Adaptations of this procedure are used to avoid repeated isolation of the same bacteriophage. Some newly isolated bacteriophages grow so poorly that they appear inaccessible to liquid enrichment culture. Four comparatively high titer bacteriophages were isolated and characterized by a genomic sequence survey. Some had genomes with extremely distant relationships to those of other bacteriophages, based on a tree built from the large terminase genes. These methods find novel genomes by rapidly isolating and screening diverse bacteriophages.

Bacteriophage lambda↗

PubMed2003

The complete sequence of marine bacteriophage VpV262 infecting vibrio parahaemolyticus indicates that an ancestral component of a T7 viral supergroup is widespread in the marine environment.

The 46,012-bp sequence of the marine bacteriophage VpV262 infecting the bacterium Vibrio parahaemolyticus is reported. The VpV262 sequence reveals that it is a distant relative of marine Roseophage SIO1, and an even more distant relative of coliphage T7. VpV262 and SIO1 appear to represent a widespread marine phage group that lacks an RNA polymerase gene and is ancestral to the T7-like phages. We propose that this group together with the T7-like phages be designated as the T7 supergroup. The ancestral head structure gene module for the T7 supergroup was reconstructed by using sensitive biased Psi-blast searches supplemented by statistical support derived from gene order. In the early and replicative segments, these phages have participated in extensive interchange with the viral gene pool. VpV262 carries a different replicative module than SIO1 and the T7-like phages.

Bacteriophage T7↗