High-speed automated DNA sequencing in ultrathin slab gels.
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Algorithms↗
Biomedical subjects
R L Brumley
Publications and source records attributed to R L Brumley.
An adaptive, object oriented strategy for base calling in DNA sequence analysis.
An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well.
Algorithms↗
High speed automated DNA sequencing in ultrathin slab gels.
We have developed a high speed instrument for automated DNA sequence analysis. The apparatus employs laser excitation and a cooled CCD detector for the parallel detection of up to 18 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (50-100 microns) denaturing polyacrylamide gels. Four hundred and fifty bases of sequence information is obtained from 100 ng of M13 template DNA in less than one hour, corresponding to an overall instrument throughput of over 8000 bases/hr.
Autoanalysis↗
Rapid DNA sequencing by horizontal ultrathin gel electrophoresis.
A horizontal polyacrylamide gel electrophoresis apparatus has been developed that decreases the time required to separate the DNA fragments produced in enzymatic sequencing reactions. The configuration of this apparatus and the use of circulating coolant directly under the glass plates result in heat exchange that is approximately nine times more efficient than passive thermal transfer methods commonly used. Bubble-free gels as thin as 25 microns can be routinely cast on this device. The application to these ultrathin gels of electric fields up to 250 volts/cm permits the rapid separation of multiple DNA sequencing reactions in parallel. When used in conjunction with 32P-based autoradiography, the DNA bands appear substantially sharper than those obtained in conventional electrophoresis. This increased sharpness permits shorter autoradiographic exposure times and longer sequence reads.
Base Sequence↗
An automated instrument for the performance of enzymatic DNA sequencing reactions.
An instrument has been developed for the automation of enzymatic DNA sequencing reactions. Up to 96 DNA templates contained in a microtiter plate can be processed for either radioactive or fluorescence-based sequence analysis in a three-hour period. The quality of the resultant data is comparable to that obtained manually. The system is simple, flexible and is readily adapted to the use of new polymerases or modified experimental protocols.
Automation↗
Fast ELISA for measuring serum antibody responses.
A method which speeds up the enzyme-linked immunosorbent assay (ELISA) is described. The procedure uses a modified Falcon fast assay screening system (Becton Dickinson Labware, Lincoln Park, NJ) and Falcon round-bottom 96-well plates. Antigen is adsorbed onto beads which extend from a lid and fit into 96-well plates. The beads are washed in a trough and reacted to antibody in the round-bottom plate. The labor required to wash the plates after coating with antigen, antibody or conjugate is thereby reduced. Greater flexibility and accuracy result, especially with the use of more than one 96-well plate. In this study, naturally occurring human IgG antibody responses to two isolated bacterial antigens were measured in over 200 subjects. It was found that numerical taxonomy could be used to split out the high IgG responders. The IgM response to one of the antigens was less variable and not significantly related to the IgG response. The fast ELISA is as useful to operate as the standard ELISA, but less stressful on the operator and more rapid.
Actinomyces↗
Separating field strength, temperature, and pulsing effects in pulsed field electrophoresis.
The utility of pulsed field electrophoresis for DNA sequencing is investigated. Previous studies have indicated a beneficial retardation of sequencing fragments when pulsed fields are employed. The interpretation of these results is complicated, however, by concomitant variations in electric field strength and/or temperature. Methods are presented here permitting discrimination of such mobility effects due to pulsing, field strength, and temperature. It is demonstrated that under the conditions employed here, observed mobility effects are due to electric field variations rather than pulsing. These conditions thus correspond to the low frequency/small molecule limit. The effect of temperature is estimated from the steady state solution to the heat conduction equation under appropriate boundary conditions. No temperature effect upon mobility is operative in the thin gel system employed, due to the high efficiency of heat transfer. However, it is shown that in conventional gel systems large temperature-related mobility effects can occur. These methods for dissecting and understanding mobility effects in pulsed field electrophoresis are expected to be of general utility.
Base Sequence↗