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R H Tullis

Publications and source records attributed to R H Tullis.

10 recordsLinked to original sources
PubMed1992

Microelectrophoresis for the separation of DNA fragments.

A methodology has been developed which significantly reduces the linear dimension necessary for the electrophoretic separation of DNA fragments and oligonucleotides. DNA fragments are rapidly separated into compact, resolvable microscopic banding patterns which can be detected using a high-resolution electronic imaging system. Separations can be carried out in either capillary tube or thin-layer (slab) microgel formats of one centimeter or less in length. The complete separation of all eleven fragments (1353 to 72 base pairs) of the pi X174 DNA/HaeIII restriction ladder was achieved in a total running distance of less than 2 mm and in less than 2 min. The observed band widths for the larger fragments (1353-603 bp) ranged from 18 to 25 microns, with the intermediate and smaller fragments (310 to 72 bp) ranging from 30 microns to 60 microns. The ethidium bromide-stained microgels were analyzed using an epifluorescent microscope combined with an intensified charged coupled device imaging system. In other experiments, single-base resolution of fluoresceinated oligonucleotides in the 20-30 nucleotide range was demonstrated. DNA sequencing may be possible with further optimization. This new methodology departs from the conventional gel formulations and electrophoretic procedures used for the separation DNA fragments. High voltage gradients and the use of highly concentrated and crosslinked homogeneous polyacrylamide gels effects the rapid separation of DNA fragments in very short distances. Analysis of the microgels with proteins of known size (Stokes radius) indicates that separations are occurring in gels with pore sizes close to the diameter of double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage phi X 174↗
PubMed1990

Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Animals↗
PubMed1986

Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.

Alkaline Phosphatase↗
PubMed1981

Fluorescence and kinetic studies on the divalent metal ion induced conformational changes in DNase a.

DNase a has a broad, asymmetric fluorescence emission peak centered at 341 nm. Binding of Ca2+ of Mg2+ to DNase shifted the peak to 342 nm and caused a 10% fluorescence enhancement. Half of the maximum change occurred at 6 x 10(-5) M Ca2+ or at 6 x 10(-4) M Mg2+. The change in fluorescence is most likely due to a conformational change in DNase which occurs when Ca2+ or Mg2+ is bound to the nonspecific tight Ca2+ binding site on DNase. The kinetics of the fluorescence change, followed by stopped flow techniques, show a fast phase (65%) and a slow phase (35%). At 1 mM Ca2+, the half-time for the fast phase is 17 ms, and for the slow phase, 3.5 min. Both phases of the reaction are first order in DNase and independent of Ca2+ at concentrations above 1 mM. DNase incubated with Ca2+ undergoes a slow (t 1/2 approximately equal to 6 min) 1.5-fold increase in activity. This activation follows pseudo-first order kinetics and is not due to the presence of additional Ca2+ in the substrate. The simplest hypothesis which accounts for these data, and previously reported studies on the effect of Ca2+ on DNase, is that DNase exists in 3 conformational states at 25 degrees C and pH 7.5. The kinetics are consistent with a mechanism which can be diagrammed as: (formula: see text) where A, A, and B represent three conformational states of DNase.

Calcium↗
PubMed1972

Adenoid cystic carcinoma of the breast.

Adenoid cystic carcinomas of breast are rarely recorded tumors affording an excellent prognosis. This present report documents three new cases and reviews the literature. Histologic features distinguishing it from cribriform intraductal carcinoma are emphasized.

Adult↗