by Kwok Fung, Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 89-97). / Acknowledgement --- p.i / Table of Content --- p.ii / Abstract --- p.vi / List of Abbreviation --- p.viii / List of Tables and figures --- p.ix / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The immunoglobulin --- p.1 / Chapter 1.1.1 --- Immunoglobulin structure --- p.1 / Chapter 1.1.2 --- Immunogloblulin genes --- p.1 / Chapter 1.1.3 --- Immunogloblulin gene recombination --- p.3 / Chapter 1.1.4 --- Antibody diversity --- p.4 / Chapter 1.1.4.1 --- Imprecise joining --- p.5 / Chapter 1.1.4.2 --- N region addition --- p.5 / Chapter 1.1.4.3 --- Somatic mutation --- p.6 / Chapter 1.2 --- Hypermutation --- p.6 / Chapter 1.2.1 --- Features of hypermutation --- p.6 / Chapter 1.2.2 --- Germinal centre & Affinity maturation --- p.8 / Chapter 1.2.3 --- Mutational Hotspots --- p.9 / Chapter 1.2.4 --- Intrinsic characteristics of hypermutation --- p.10 / Chapter 1.2.5 --- Models for the mechanism of hypermutation --- p.11 / Chapter 1.2.5.1 --- DNA replication --- p.11 / Chapter 1.2.5.2 --- DNA repair --- p.12 / Chapter 1.2.5.3 --- Gene conversion --- p.13 / Chapter 1.2.5.4 --- Transcription --- p.15 / Chapter 1.2.5.5 --- Homologous recombination of reverse transcribed mRNA --- p.16 / Chapter 1.2.5.6 --- Transcription-coupled repair --- p.17 / Chapter 1.3 --- Scope of investigation --- p.17 / Chapter Chapter 2 --- Material and Method --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Methods (first generation of LMPCR) --- p.21 / Chapter 2.2.1 --- Animal and cell lines --- p.21 / Chapter 2.2.2 --- Oxazolone antigen immunization --- p.21 / Chapter 2.2.2.1 --- Preparation of Bordetella pertussis --- p.21 / Chapter 2.2.2.2 --- Coupling of phenyloxazolone with CSA or BSA --- p.22 / Chapter 2.2.2.3 --- Preparation of aluminium hydroxide adjuvant --- p.23 / Chapter 2.2.2.4 --- Mice immunization --- p.23 / Chapter 2.2.3 --- Detection of anti-phOx antibody by enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.3.1 --- Reagents --- p.24 / Chapter 2.2.3.2 --- Assay procedure --- p.24 / Chapter 2.2.4 --- Extraction of genomic DNA (mice/cell line) --- p.25 / Chapter 2.2.4.1 --- Reagents --- p.25 / Chapter 2.2.4.2 --- Isolation of DNA from cell line (NQ2.12.4 & NQ5.4.3) --- p.25 / Chapter 2.2.4.3. --- DNA extraction from mice --- p.26 / Chapter 2.2.5 --- Ligation-mediated polymerase chain reaction (LMPCR) --- p.26 / Chapter 2.2.5.1 --- Procedure --- p.26 / Chapter 2.2.5.1.1 --- First primer extension --- p.29 / Chapter 2.2.5.1.2 --- Ligation --- p.29 / Chapter 2.2.5.1.3 --- PCR amplification --- p.30 / Chapter 2.2.5.1.4 --- Labelling of LMPCR product --- p.30 / Chapter 2.2.6 --- Marker preparation --- p.31 / Chapter 2.2.7 --- Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.7.1 --- Reagents --- p.32 / Chapter 2.2.7.2 --- Procedure --- p.32 / Chapter 2.2.8 --- Southern blot hybridization --- p.33 / Chapter 2.2.8.1 --- Reagents --- p.33 / Chapter 2.2.8.2 --- DNA blotting --- p.34 / Chapter 2.2.8.3 --- Preparation of 32P labelling DNA probe --- p.34 / Chapter 2.2.8.4 --- Prehybridization and Hybridization --- p.35 / Chapter 2.2.9 --- Simplified protocol for the first generation of LMPCR --- p.36 / Chapter 2.3 --- Method (second generation of LMPCR) --- p.37 / Chapter 2.3.1 --- Excess linker removal --- p.37 / Chapter 2.3.1.1 --- Exonuclease III Treatment --- p.37 / Chapter 2.3.1.2 --- Mung bean nuclease Treatment --- p.37 / Chapter 2.3.1.3 --- Chroma Spin Treatment --- p.37 / Chapter 2.3.2 --- HindIII digestion after LMPCR --- p.38 / Chapter 2.3.3 --- DNA sequencing --- p.38 / Chapter 2.3.3.1 --- Cloning of amplified sequences to M13mpl9 plasmid --- p.38 / Chapter 2.3.3.2 --- Plaque hybridization --- p.39 / Chapter 2.3.3.3 --- Preparation of single-stranded templates --- p.39 / Chapter 2.3.3.4 --- Sanger dideoxy sequencing of single-stranded DNA --- p.40 / Chapter 2.3.4 --- Simplified protocol for second generation of LMPCR --- p.41 / Chapter Chapter 3 --- First generation of LMPCR --- p.42 / Chapter 3.1 --- General design --- p.42 / Chapter 3.1.1 --- LMPCR protocol and its modification --- p.42 / Chapter 3.1.2 --- Oligonucleotide design --- p.44 / Chapter 3.1.3 --- Experimental design --- p.48 / Chapter 3.2 --- Result --- p.50 / Chapter 3.2.1 --- Anti-phOx Ig level in normal and immunized mice --- p.50 / Chapter 3.2.2 --- LMPCR analysis of the sense strand of VkOxl --- p.50 / Chapter 3.2.2.1 --- Overall patterns of the LMPCR signals --- p.50 / Chapter 3.2.2.2 --- Southern hybridization --- p.50 / Chapter 3.2.2.3 --- Distribution of signals --- p.57 / Chapter 3.2.2.4 --- LMPCR analysis of the VkOxl-Jk5 anti-phOx transgene --- p.61 / Chapter 3.2.2.5 --- Effect of the number of cells carrying the VkOxl-Jk5 gene on LMPCR --- p.61 / Chapter 3.2.3 --- LMPCR analysis of the antisense strand of VkOxl --- p.64 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Second generation of LMPCR --- p.72 / Chapter 4.1 --- Introduction(experi mental modification) --- p.72 / Chapter 4.1.1 --- Tagging the specific LMPCR products by addition of a Hin dIII site in the linker --- p.72 / Chapter 4.1.2 --- "Removal of excess linker, OXUH" --- p.72 / Chapter 4.1.2.1 --- Exonuclease III treatment --- p.73 / Chapter 4.1.2.2 --- Chroma spin treatment --- p.73 / Chapter 4.1.2.3 --- Mung Bean Nuclease treatment --- p.75 / Chapter 4.1.3 --- Other modifications in LMPCR --- p.75 / Chapter 4.2 --- Results --- p.75 / Chapter 4.2.1 --- Effect of including Exonuclease III treatment --- p.75 / Chapter 4.2.2 --- Effect of including Mung Bean Nuclease treatment --- p.76 / Chapter 4.2.3 --- Effect of including Chroma spin treatment --- p.76 / Chapter 4.2.4 --- Strand break positions detected at the sense strand --- p.76 / Chapter 4.2.5 --- DNA sequence analysis of the antisense strand LMPCR products --- p.82 / Chapter 4.3 --- Discussion --- p.84 / References --- p.89 / Appendix I --- p.98
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321916 |
| Date | January 1997 |
| Contributors | Lo, Kwok Fung., Chinese University of Hong Kong Graduate School. Division of Microbiology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, x, 99 leaves : ill. (some mounted) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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