Ralph Rapley

Molecular Biology and Biotechnology

Molecular biology and biotechnology , Molecular biology and biotechnology , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Molecular biomethods handbook

Ralph Rapley and John Walker, Molecular Biomethods Handbook (Totowa: Humana Press, 1998), ISBN:096035018

Enhancing PCR amplification and sequencing using DNA-binding proteins.

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.

RNA isolation and characterization protocols

Ralph Rapley and David Manning, RNA Isolation & Characterization Protocols (Totowa: Humana Press, 1998), ISBN: 0896034941

The biotechnology and applications of antibody engineering.

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.

Medical biomethods handbook

Ralph Rapley and John Walker, Medical BioMethods Handbook (Totowa, Humana Press, 2005), ISBN 1-58829-334-3

PCR Sequencing Protocols

Preparation and Analysis of DNA Sequencing Gels Purification of PCR Products from Agarose Gels for Direct Sequencing Enzymatic Fluorescence and Biotin Labeling of Primers for PCR Sequencing Direct Sequencing of Double-Stranded PCR Products with the Sequencase Kit and [a-35S] dATP Direct Sequencing by Thermal Asymmetric PCR Rapid Sequencing of cDNA Clones: Direct Sequencing Using Sequential Linear/Asymmetric PCR Direct Sequencing of PCR Products Using Chemiluminescent Detection Direct DNA Sequencing of PCR Products Using Magnetic Beads Affinity Capture and Solid-Phase Sequencing Biotinylated PCR Products Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing. Nonradioactive PCR Sequencing Using Digoxygenin Silver SequencingTM: Nonradioactive Cycle Sequencing of dsDNA Direct Sequencing of PCR Products with DNA-Binding Proteins PCR Sequencing with the Aid of Detergents Direct Sequencing with Highly Degenerate and Inosine-Containing Primers Determination of Unknown Genomic Sequences Without Cloning DNA Sequencing by the Chemical Method Direct PCR Sequencing with Denaturants (Formamide) Efficient PCR Production of Single-Stranded DNA Sequencing Templates Preparation and Direct Automated Cycle Sequencing of PCR Products Solid-Phase Automated Sequencing of PCR-Amplified Genomic DNA Cloning PCR Products for Sequencing in M13 Vectors Sequencing PCR Products Cloned into M13 Vectors Genome Amplification with Transcript Sequencing (GAWTS) DNA Rescue by Vectorette Method Sequencing (dAidT) of Cloned Mixed PCR Products from Microbial Populations Index

Route maps in gene technology

Preface About the Route Maps format The Concept Of Genes Is DevelopedGenes Are Located To ChromosomesGenes Are Composed Of DNAThe Chemical Building Blocks Of Nucelic AcidsFormation Of The DNA Double HelixPackaging Of DNA Within CellsChromatin Structure And The Functional Activity Of GenesTypes And Functions Of DNA-Protein InteractionsOrganization Of Genomes Into Multiple ChromosomesDistribution Of Nucleic Acids Within Eukaryotic CellsTypes Of RNA MoleculesThe Anatomy Of Eukaryotic ChromosomesThe Organisation Of Genes Within ChromosomesThe Molecular Anatomy Of Eukaryotic GenesChromosome Aberrations And Human DiseaseTypes Of Mutations And Their EffectsForms Of Chemically Altered DNA DNA Repair MechanismsLinkage AnalysisPedigree Analysis And Modes Of InheritanceGenes Dictate The Nature Of ProteinsThe Nature Of The Genetic CodeTranscription: Forming Genetic MessagesPost-Transcriptional Processing Of Messenger RNATransfer And Ribosomal RNA Processing/ModificationMechanisms Regulating Gene ExpressionTranscriptional Regulatory SequencesOperons And Prokaryotic Control Of Gene ExpressionTranscription Factors And Gene ExpressionIn Vivo Translation: Decoding Genetic MessagesSequences Involved In Cellular Protein TargetingEukaryotic Cell Division: Mitosis And MeiosisMolecular Mechanisms Of Cell Cycle ControlGenetic Recombination MechanismsGene Transfer During Bacterial ReproductionTransposable Genetic Elements: TransposonsIn Vivo DNA ReplicationGenetic Control Of DevelopmentThe Natural Biology Of BacteriophagesBacteriophage GeneticsRecombinant DNA TechnologyEnzymes Commonly Used In Molecular Biology MethodsRestriction EndonucleasesRestriction Fragment Length PolymorphismsIsolation Of Nucleic Acids From Cells And TissuesVisualising Mucleic AcidsElectrophoresis Of Nucleic AcidsIn Vitro HybridisationTypes Of Hybridisation Assay FormatsSouthern Blotting In Situ HybridisationMeasuring Transcriptional Activity Via Messenger RNAConverting Messenger RNA Into Complementary DNA (Cdna)Methods For Determining DNA Nucleotide SequencesThe Polymerase Chain ReactionAlternatives To PCR-Based In Vitro DNA/RNA AmplificationIn Vitro Translation MethodsTypes And Methods Of Gene Probe GenerationChemical Synthesis Of OligonucleotidesTypes And Applications Of Nucleotide AnaloguesMethods For Labelling Gene ProbesFundamental Principles Of CloningThe Nature Of Cloning VectorsInserting Foreign DNA Into VectorsThe Development Of Bacteriophage VectorsPlasmids: Development As Clonign VectorsYeast-Derived Plasmid VectorsPhagemids: Hybrid Phage And Plasmid VectorsVectors For Use In Plant And Animal CellsDelivering DNA Into Cells Principal Genomic And Cdna Cloning StrategiesStrategies For Identifying Desirable Recombinant ClonesGene Mapping TechniquesDetecting DNA-Protein Interaction SitesDetecting Promoter And Enhancer SequencesMethods For Identifying Protein Encoding SequencesGenetic FingerprintingAnalysing Ancient DnasIn Vitro Mutagenesis MethodsGenetically Modified Micro-OrganismsGenetically Engineered PlantsGenetically Engineered AnimalsMolecular Techniques In Prenatal DiagnosisThe Genetics Of Cystic FibrosisThe Dystrophin Gene And Muscular DystrophiesIdentifying The Gene For Huntingdons DiseaseLipoprotein Genes And Heart DiseaseThe Detection Of Microbial InfectionsMolecular Biology Of Human Immunodeficiency Virus And AIDSEngineering Microbial BioluminescenceRecombinant DNA Techniques In Vaccine DevelopmentReceptors And Cellular Signalling MechanismsOncogenes And The Molecular Basis Of CancerMolecular Diagnosis And Therapy Of CancersDrug Development Using Recombinant DNA ApproachesProtein EngineeringImmunoglobulin GeneticsGenetic Engineering Of Recombinant AntibodiesCurrent Approaches To Gene TherapyThe Human Genome Mapping ProjectBibliography Index

PCR mutation detection protocols

Theophilus, Bimal D.M., Rapley, Ralph (Eds.), PCR Mutation Detection Protocols, 2nd Edition, Humana Press. eBOOK ISBN: 978-1-60761-947-5, Hardcover ISBN: 978-1-60761-946-8, DOI: 10.1007/978-1-60761-947-5.

Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes

In order to establish the feasibility of applying recombinatorial library technologies to investigate human in vivo IgE responses, and as a pre-requisite of recombinatorial library construction, we have attempted to determine workable peripheral blood sample volumes required for isolation of mRNA for polymerase chain reaction (PCR) amplification of human IgE Fd encoding sequences. Cells secreting chimeric human IgE monoclonal antibody specific for the hapten NIP were used to establish the conditions for specific amplification of C epsilon 1 domain and Fd encoding sequences, as determined by Southern hybridisation. Amplification of C epsilon 1 domain sequences could be achieved using as few as ten cultured cells as the source of RNA. Specific IgE+ B cell enrichment using immuno-magnetic particles prior to RNA extraction was, however, required to obtain amplification of IgE C epsilon 1 and Fd fragments from lymphocytes prepared from 40 ml human peripheral blood. IgG1+ B cell enrichment from similar samples was not required for detectable amplification of human C gamma 1 cDNA sequences. However, this procedure improved amplification efficiency. Optimisation of methods to separate specific B cell populations, or specific RNA/cDNA sequences, will facilitate in vitro generation of human IgE Fab fragments from peripheral blood.