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Biomedical subjects

D T Burke

Publications and source records attributed to D T Burke.

At least 19 recordsLinked to original sources
PubMed1996

Directed mutagenesis of YAC-cloned DNA using a rapid, PCR-based screening protocol.

We have developed a system which facilitates the rapid modification of yeast artificial chromosome (YAC) insert DNA. Specific modifications, such as deletions, insertions and point mutations, can be generated by a two-step allele replacement method using the yeast translational suppressor, SUP4-o, as both a positive and negative selection. The introduction of the SUP4-o gene was successful in 4 out of 24 selected transformant colonies, while the subsequent homologous elimination occurred in 2 out of 30 colonies. The use of a simple, short-range PCR assay rapidly identified the correct events among the genetically selected isolates and should be generally applicable to YAC modifications.

Animals↗
PubMed1996

Microfabricated structures for integrated DNA analysis.

Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration.

Automation↗
PubMed1996

Single nucleotide primer extension: quantitative range, variability, and multiplex analysis.

The quantitative measurement of transcription products from homologous alleles at a diploid locus has broad application for the study of mammalian gene expression. Single nucleotide primer extension (SNuPE) analysis is a simple and sensitive method for allelic transcript discrimination requiring only 1 bp difference between alleles. In this study the effective range, experimental variation, and the influences of poly(dT)-primed and gene-specific reverse transcriptions are characterized. The ability to analyze several genes from a single reverse transcription reaction is assessed as well. For the genes examined, the maximum range of detection is reached when the minor transcript represents 1/250 of the major allele. Relatively little error is seen within or between assays and linearity of response is maintained over an approximately thousandfold range.

Alleles↗
PubMed1994

Splinting for carpal tunnel syndrome: in search of the optimal angle.

Carpal tunnel syndrome (CTS) is the most common of the compression neuropathies. Several studies have demonstrated the efficacy of wrist splinting in relieving the symptoms of CTS; however, the chosen angle of immobilization has varied. Wick catheter measurements of carpal tunnel pressures suggest that the neural position has less pressure and, therefore, greater potential to provide relief from symptoms. This study is a prospectively gathered, blind trial comparing the symptom relief experienced by wearers of splints immobilized at 20 degrees extension and at neutral. The results indicate that the neutral angle provided superior symptom relief, and that the relief did not often improve between 2 weeks and 2 months of wear. Relief of symptoms was not related to the length of time that the patient had experienced of CTS symptoms. The results also indicate that the results of the electromyography/nerve conduction study (EMG/NCS) do not provide information about the subjects' likely response to splinting.

Carpal Tunnel Syndrome↗
PubMed1992

Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts.

A yeast artificial chromosome library with mouse genomic DNA inserts has been constructed. The library encompasses a 2.5-fold coverage of the mouse genome, with an average insert size of 250 kilobases. The screening strategy uses the polymerase chain reaction on pooled DNAs prepared from individually stored clones. The usefulness of the library for chromosome walking was illustrated by constructing a 600-kilobase-long contig of DNA surrounding Hba-ps4, a DNA marker that is tightly linked to the fused (Fu) locus on chromosome 17.

Animals↗
PubMed1991

The role of yeast artificial chromosome clones in generating genome maps.

The cloning of large DNAs as yeast artificial chromosomes (YACs) holds the promise of greatly expanding the scope of physical genomic mapping. Recent improvements in YAC clone libraries and their screening, as well as in the analysis of YAC-cloned DNA, are beginning to fulfill the potential of this system.

Animals↗
PubMed1989

Mapping the Drosophila genome with yeast artificial chromosomes.

The ability to clone large fragments of DNA in yeast artificial chromosomes (YAC's) has created the possibility of obtaining global physical maps of complex genomes. For this application to be feasible, most sequences in complex genomes must be able to be cloned in YAC's, and most clones must be genetically stable and colinear with the genomic sequences from which they originated (that is, not liable to undergo rearrangement). These requirements have been met with a YAC library containing DNA fragments from Drosophila melanogaster ranging in size up to several hundred kilobase pairs. Preliminary characterization of the Drosophila YAC library was carried out by in situ hybridization of random clones and analysis of clones containing known sequences. The results suggest that most euchromatic sequences can be cloned. The library also contains clones in which the inserted DNA is derived from the centromeric heterochromatin. The locations of 58 clones collectively representing about 8 percent of the euchromatic genome are presented.

Animals↗
PubMed1989

Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

Chromosomes, Fungal↗
PubMed1989

Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.

Base Sequence↗
PubMed1987

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.

Chromosomes↗
PubMed1986

Oligodeoxynucleotide-directed mutagenesis of Escherichia coli and yeast by simple cotransformation of the primer and template.

A method of oligodeoxynucleotide-directed mutagenesis is presented which requires no in vitro DNA synthesis. Cotransformation of the synthetic primer and single-stranded template into competent spheroplasts of Escherichia coli or Saccharomyces cerevisiae generates the directed mutation. The desired event is detected genetically or by hybridization screening using the mutagenic oligodeoxynucleotide as a probe. The targeted mutation arises at a frequency of approximately 0.1%. In one extensively studied case in E. coli, involving creation of a 99-bp deletion, this procedure produced many fewer untargeted mutation events than did conventional protocols.

Escherichia coli↗