Polymerase chain reaction (PCR) methodology (1) has become a routine method for selectively ampli... more Polymerase chain reaction (PCR) methodology (1) has become a routine method for selectively amplifying segments of DNA from a wide variety of sources. Amplification of specific sequences is dependent upon an exact match between the template DNA and the oligonucleotide primers. Mismatches at the 3' terminus lead to greatly reduced amplification, with no detectable product when amplified under the appropriate conditions (2,3). We demonstrate here that an oligonucleotide which can be extended by Taq DNA polymerase is able to block the amplification of a PCR product when situated between two flanking PCR primers. An oligonucleotide mismatched at its 3' terminus, however, does not demonstrate this ability. This allows the development of a method for the differential amplification of molecules with identical 5' and 3' ends. Unless otherwise indicated, PCRs were performed in a programmable heating block using 20 rounds of temperature cycling (94°C for 1 min, 55°C for 2 min and 72°C for 3 min) followed by a final 10 min step at 72 °C. One microgram of each primer, 5 ng of 'blocking' oligonucleotide, 10 ng of template and 2.5 units of Taq DNA polymerase (Perkin-Elmer Cetus) were used in a final volume of 100 /il with the reaction buffer as recommended by the manufacturer. The 'blocking' oligonucleotides, designed to block PCR amplification of the DX48 template, are indicated in Table . The templates used were a CAMPATH-1H humanised monoclonal antibody (mAb) heavy (H) chain (4) and DX48 humanised mAb H chain (unpublished), both in cDNA context in the plasmid vector pUC18 (GffiCO/BRL). The 5' PCR primer, X (5'-GATCAA-GCTTTACAGTTACTCAGCACACAG), was designed to anneal to both templates such that the 3' end was at a position 12 nucleotides (nt) upstream of the ATG initiation codon in each case, and the 3' primer, Y (5'-GATCAAGCTTTCATTTA-CCCGGAGACAGGGAGA), was designed to anneal with the 3' end 20 nt upstream of the termination codon. The products of PCRs carried out on both templates using these primers would be expected to be 1462 base pairs (bp) in length.
Network analysis of Escherichia coli metabolic models
We are currently in the process of integrating the metabolic, genetic and product-interaction net... more We are currently in the process of integrating the metabolic, genetic and product-interaction networks of Escherichia coli, with a view to creating a whole cell model [1]. In an effort to assess the physiological accuracy of our current model, we undertook extensive analysis of the metabolic component of the model, which consists of over 850 enzymes. Conversion of this network into a stoichiometric matrix allowed interpretation through the use of graph theory and its associated algorithms. Our strategy, and subsequent results, differed from that previously described [3]. Advantageously, our strategy allows consideration of either metabolites or enzymes as nodes and ameliorates the need to introduce temporary complexes. Various analyses were performed to describe the topology and other properties of the metabolic network. The ‘small-world’ characteristic described as being inherent in metabolic systems was investigated by determining node interconnectivity . To determine the metaboli...
D., Hotz, HR, Tang, H., Goryanin, I. and Hodgman
Simultaneous modelling of metabolic, genetic and product-interaction networks
Briefings in Bioinformatics, 2001
Constructing an enzyme-centric view of metabolism
Bioinformatics, 2004
Simultaneous modelling of metabolic, genetic and product-interaction networks
Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patie... more Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used. polymerase chain reaction and highly degenerate, inosine cont~~ng oligonucleot~des derived from sequence ali~men~ of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the Iutropin/choriogonadotropin (LH/CG} receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GTl) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GTI clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH,CG receptors it is possible to amplify the TSH-R receptor sequence.
The creation of cell models from annotated genome information, as well as additional data from ot... more The creation of cell models from annotated genome information, as well as additional data from other databases, requires both a format and medium for its distribution. Standards are described for the representation of the data in the form of Document Type Definitions (DTDs) for XML files. Separate DTDs are detailed for genetic, metabolic and gene product- interaction networks, which can
Most of the published quantitative models in biology are lost for the community because they are ... more Most of the published quantitative models in biology are lost for the community because they are either not made available or they are insufficiently characterized to allow them to be reused. The lack of a standard description format, lack of stringent reviewing and authors' carelessness are the main causes for incomplete model descriptions. With today's increased interest in detailed biochemical models, it is necessary to define a minimum quality standard for the encoding of those models. We propose a set of rules for curating quantitative models of biological systems. These rules define procedures for encoding and annotating models represented in machine-readable form. We believe their application will enable users to (i) have confidence that curated models are an accurate reflection of their associated reference descriptions, (ii) search collections of curated models with precision, (iii) quickly identify the biological phenomena that a given curated model or model constituent represents and (iv) facilitate model reuse and composition into large subcellular models.
Motivation: Molecular biotechnology now makes it possible to build elaborate systems models, but ... more Motivation: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. Results: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others.
Taq DNA polymerase extension of internal primers blocks polymerase chain reactions allowing differential amplification of molecules with identical 5′ and 3′ ends
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Papers by Hugh Spence