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C W Fuller

Publications and source records attributed to C W Fuller.

At least 37 records · Page 2Linked to original sources
PubMed1995

Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis.

We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5' end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (448 nm) and fluorescence emission maxima of 525, 555, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and acceptors is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and mobilities of the single base extension DNA fragments generated with primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.

Base Sequence↗
PubMed1995

A novel thermostable polymerase for DNA sequencing.

Using recent findings by Tabor and Richardson, a novel thermostable DNA polymerase that generates sequence data comparable with T7 Sequenase DNA polymerase has been developed for improved base calling and longer reads.

Amino Acid Sequence↗
PubMed1995

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Bacteriophage M13↗
PubMed1995

Implications of health and safety legislation for the professional sportsperson.

There are a large number of injuries in most professional sports every year, and research interest has usually centred around the medical aspects of these injuries. However, health and safety legislation also applies to professional sport, and it is important, as with any other occupation, to understand the underlying causes of the incidents leading to the injuries and to take steps to minimize future injuries. The introduction of the Management of Health and Safety at Work Regulations 1992 formalized a risk assessment approach to health and safety issues; an approach that has always been implicit in the Health and Safety at Work etc. Act 1974. This review assesses how the relevant UK health and safety legislation impacts on the professional sportsperson.

Accidents, Occupational↗
PubMed1992

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Base Sequence↗
PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis.

In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.

Base Sequence↗
PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis.

Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome. Bidirectional DNA synthesis from this site results in complete replication of the chromosome. The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C. W., and Richardson, C. C. (1985) J. Biol. Chem. 260: 3185-3196). DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules. The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses. These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template. The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis. Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template. Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein. Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.

Centrifugation, Density Gradient↗
PubMed1980

A simple procedure for covalent immobilization of NADH in a soluble and enzymically active form.

Using a new and simplified technique for covalent immobilization of adenine nucleotides [Fuller, C. W. and Bright, H. J., J. Biol. Chem. 252, 6631 (1977)], we have prepared immobilized NADH using two water-soluble polymers and tested these preparations for activity with several dehydrogenases. The first polymer used, having a weight-average molecular weight of about 350000, is a copolymer of methacrylyl choline and the epoxide-containing monomer 3-[4-(2,3-epoxypropoxy)butoxy]-2-hydroxypropyl acrylate. Immobilization of NADH onto this copolymer was accomplished in three steps, namely, alkylation of NAD at N-1, reduction of the nicotinamide moiety with dithionite and Dimroth rearrangement of the alkyl linkage from the N-1 to the C-6 amino position. The second copolymer tested was a copolymer of the same epoxide-containing monomer and N-methacrylyl-2-glucosamine. Using this copolymer, immobilization of NADH through the adenine C-6 amino position was accomplished in a single step. Measurements of the steady-state kinetics of five dehydrogenases at pH 7 and pH 9 showed that, on the average, V and Km values obtained with the immobilized NADH were, respectively, about one third and twice those found for the free coenzyme. In general, when compared with the free coenzyme, the immobilized NADH had greater relative enzymic reactivity at pH 9 than at pH 7. The simplicity of this method, the general enzymic reactivity, and the ability to be recycled enzymically suggest that this immobilized NADH may be useful in creating enzyme reactors for synthetic, analytical and other purposes.

Acrylates↗
PubMedDate not supplied

Uniform band intensities in fluorescent dye terminator sequencing.

The use of Cyanine dye (Cy5 and Cy5.5) labeled dideoxy terminators with Thermo Sequenase DNA polymerase in DNA sequencing provides uniform band intensity, improved sequence read-length, and accuracy. It also greatly improves the ability to detect single base heterozygotes with dye-terminator sequencing method.

Base Sequence↗
PubMedDate not supplied

Fluorescence resonance energy transfer dye nucleotide terminators: a new synthetic approach for high-throughout DNA sequencing.

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.

Base Sequence↗
PubMedDate not supplied

Analogs of guanine nucleoside triphosphates for sequencing applications.

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2'-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase T7 DNA polymerase or Thermo Sequenase DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza-dGTP meet our requirements as better sequencing reagents.

Guanine Nucleotides↗
PubMedDate not supplied

An assessment of player error as an injury causation factor in international football.

OBJECTIVE: To quantify the frequency of injury in football as a function of tackle parameters. METHOD: Video recordings of 123 international matches in three Federation Internationale de Football Association tournaments were used to analyze tackle parameters, and team physicians provided reports of postmatch medical attention to players. RESULTS: A total of 8572 tackles were assessed, of which 3464 (40.4%) were fouls. There were 299 incidents of on-pitch medical attention, of which 131 (44%) resulted from foul tackles, and 200 postmatch team physicians' reports, of which 96 (48%) resulted from foul tackles. The cases of on-pitch medical attention resulted in 76 (25%) postmatch physicians' reports, but 124 (62%) of the postmatch physicians' reports were not associated with on-pitch medical attention. Tackled players received 74% of the postmatch medical reports. Tackle types with the greatest probability of requiring medical attention were from the side in terms of tackle direction, jumping vertically in terms of tackle mode, and a clash of heads in terms of tackle action. CONCLUSION: Human error on the part of players during the process of tackling and inadequacies in the laws of football and/or their application by match referees were equally responsible for the high levels of injury observed.

Adolescent↗
PubMedDate not supplied

The influence of tackle parameters on the propensity for injury in international football.

PURPOSE: To understand how tackling leads to injury in football, to develop a framework for classifying tackles, and to identify tackles with the greatest propensity to cause injury. METHOD: Video recordings of 123 matches in three FIFA tournaments were used to identify tackling parameters. Team physicians prepared reports of postmatch medical attention to players. RESULTS: Tackles from the side were twice as likely to require postmatch medical attention as tackles from behind. Injuries to the head/neck of tackled and tackling players and the torso of tackling players were more likely to receive on-pitch medical attention than other injuries. Injuries to the foot for tackled and tackling players and the lower leg and thigh for tackling players were less likely to receive on-pitch medical attention than other injuries. Tackles with the greatest propensity for causing injury involved clash of heads and two-footed tackles for tackled players and clash of heads, two-footed tackles, jumping vertically, and tackles from the side for tackling players. CONCLUSION: The laws of football relating to tackling should be reviewed to provide greater protection from injury by reducing the overall level of risk and, in particular, by protecting players from tackles with the highest propensity for causing injury.

Athletic Injuries↗