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The human genome project is set to revolutionize health care and medicine. The genetic make up of each individual gives clues as to the genetic factors that predispose one to a particular genetic disease. Single Nucleotide Polymorphisms (SNPs), single base changes in the nucleotide DNA sequence of individuals, are thought to be the main cause of genetic variation. By comparing patterns of SNP allele frequencies between disease affected and control populations, disease associated SNPs can be identified and potential disease gene(s) located. These types of study necessitate genotyping of thousands of SNPs which requires the use of powerful, high throughput, systems of analysis. Mass spectrometry is fast becoming the preferred technology for this type of high throughput analysis. This book contains a collection of descriptions of some of the most outstanding advances in the field of mass spectrometry from which, it is hoped, the reader will be able to learn both the principles and the most up-to-date methods for its use in genomic analysis. It covers the general principles of the technologies and more in depth detailed descriptions for more academic reading and information.
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76 2. Short Oligonucleotide Mass Analysis 76 2. 1. Method Outline 76 2. 2. Design of PCR Primers and Fragments for Analysis 78 2. 3. Typical PCR Reaction Conditions 79 3. Electrospray Ionisation Mass Spectrometry 79 Formation of Ions 3. 1. 79 3. 2. Tandem Mass Spectrometry 79 3. 3. Typical ESI-MS Settings for SOMA 80 4. Purification Procedures 80 4. 1. Phenol/Chloroform Extraction and Ethanol Precipitation 80 4. 2. In-line HPLC Purification 81 5. Genotyping Using SOMA 81 5. 1. APC Genotyping in Human Subjects 81 5. 2. APC Genotyping in Min Mice 85 5. Mutation Detection Using SOMA 86 6. 1. Analysis of p53 Mutations in Liver Cancer Patients 86 6. 1. 1. p53 Mutations in Liver Tumours 87 6. 1. 2. p53 Mutations in Plasma Samples 88 7. Advantages and Disadvantages of SOMA 89 8. Future Perspectives 90 9. Acknowledgements 91 10. References 91 CHAPTER 7 WV. Bienvenut, M. Müller, PM. Palagi, E. Gasteiger, M. Heller, E. Jung, M. Giron, R. Gras, S. Gay, PA. Binz, G J. Hughes, JC. Sanchez, RD. Appel, DF. Hochstrasser Proteomics and Mass Spectrometry: Some Aspects and Recent Developments 1. Introduction to Proteomics 93 2. Protein Biochemical and Chemical Processing Followed by Mass Spectrometric Analysis 94 2. 1. 2-DE Gel Protein Separation 95 Protein Identification Using Peptide Mass Fingerprinting and Robots 96 2. 2. 2. 2. 1. MALDI-MS Analysis 98 2. 2. 2. MS/MS Analysis 102 Improvement of the Identification by Chemical Modification of Peptides 106 2. 2. 3.