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The primary purpose of this volume is to demonstrate the range of applications of the Pyrosequencing technology in research and diagnostics and to provide detailed protocols. Beginning with an up-to-date overview of the biochemistry, the volume continues with quantitative analysis of genetic variation, ratio of expressed alleles at the RNA level, analysis of DNA methylation, global DNA methylation assays, specialized applications for DNA methylation analysis including loss of imprinting, single blastocyst analysis, allele-specific DNA methylation patterns, DNA methylation patterns associated with specific histone modifications. The volume further details tools and protocols for the detection of viruses and bacteria, and genetic and epigenetic analyses for forensics using Pyrosequencing. As a volume in the highly successful Methods in Molecular Biology series, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and adaptable, Pyrosequencing: Methods and Protocols, Second Edition will greatly aid doctorial students, postdoctoral investigators and research scientists studying different aspects of genetics and cellular and molecular biology.
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Table of Contents

Part I: Introduction1. The History of PyrosequencingOPal Nyren 2. PyroMark (R) Instruments, Chemistry, and Software for Pyrosequencing (R) Analysis Martin Kreutz, Gerald Schock, Julia Kaiser, Norbert Hochstein, and Ralf Peist 3. Software-Based Pyrogram (R) Evaluation Guoli Chen, Matthew T. Olson, and James R. Eshleman 4. Quantitative Validation and Quality Control of Pyrosequencing (R) Assays Ulrich Lehmann Part II: Analysis of Genetic Variation 5. Extended KRAS and NRAS Mutation Profiling by Pyrosequencing (R) Andreas Jung 6. Universal BRAF State Detection by the Pyrosequencing (R)-based U-BRAFV600 Assay Alexander Skorokhod 7. Pyrosequencing (R)-based Identification of Low Frequency Mutations Enriched through Enhanced-ice-COLD-PCR Alexandre How-Kit and Joerg Tos 8. Analysis of Mutational Hotspots in Routinely Processed Bone Marrow Trephines by Pyrosequencing (R) Stephan Bartels and Ulrich Lehmann 9. Analysis of Copy Number Variation By Pyrosequencing (R) using Paralogous Sequences Marianne Kristiansen Kringen 10. Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing (R) Hui Ye, Haiping Wu, Yunlong Liu, Bingjie Zou, Tomoharu Kajiyama, Hideki Kambara, and Guohua Zhou 11. HLA-B and HLA-C Supratyping by Pyrosequencing (R) Irene Vanni, Elisabetta Ugolotti, Patrizia Larghero, and Roberto Biassoni 12. Allele Quantification Pyrosequencing (R) at Designated SNP sites to Detect Allelic Expression Imbalance and Loss-of-heterozygosity Chau-To Kwok and Megan P. Hitchins Part III: Analysis of DNA Methylation Patterns 13. Quantitative DNA Methylation Analysis by Pyrosequencing (R) Jessica Roessler and Ulrich Lehmann 14. -Quantitative Methylation Analysis of the PCDHB Gene Cluster Barbara Banelli and Massimo Romani 15. Assessment of Changes in global DNA Methylation Levels by Pyrosequencing (R) of Repetitive Elements Ali M. Tabish, Andrea A. Baccarelli, Lode Godderis, Timothy M. Barrow, Peter Hoet1 and Hyang-Min Byun 16. Global Analysis of DNA 5-methylcytosine using the Luminometric Methylation Assay, LUMA Karin Luttropp, Louise K Sjoeholm, and Tomas J Ekstroem 17. Limiting Dilution Bisulfite Pyrosequencing (R): a Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells Nady El Hajj, Juliane Kuhtz, and Thomas Haaf 18. Detection of Loss of Imprinting by Pyrosequencing (R) Silvia Tabano, Eleonora Bonaparte, and Monica Miozzo 19. Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing (R) John Huntriss, Kathryn Woodfine, Joanna E Huddleston, Adele Murrell, and Helen Mary Picton 20. Allele-specific DNA methylation detection by Pyrosequencing (R) Lasse Sommer Kristensen, Jens Vilstrup Johansen, and Kirsten Gronbaek 21. SNP-based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing (R) Florence Busato and Joerg Tost 22. DNA Methylation Analysis of ChIP Products at Single Nucleotide Resolution byPyrosequencing (R) Celine Moison, Fanny Assemat, Antoine Daunay, Paola B Arimondo, and Joerg Tost Part IV: Bacterial Typing and Identification 23. Multiplex Pyrosequencing (R): Simultaneous Genotyping Based on SNPs from Distant Genomic Regions Piotr Wojciech Dabrowski, Kati Bourquain, and Andreas Nitsche 24. Detection of Drug-Resistant Mycobacterium tuberculosis Anna Engstroem and Pontus Jureen 25. Application of Pyrosequencing (R) in Food Biodefense Kingsley Kwaku Amoako Part V: Forensics 26. Forensic Analysis of Mitochondrial and Autosomal Markers using Pyrosequencing (R) Magdalena M. Bus, Hanna Edlund, and Marie Allen 27. Tissue-specific DNA Methylation Patterns in Forensic Samples Detected By Pyrosequencing (R) Joana Antunes, Kuppareddi Balamurugan, George Duncan, and Bruce McCord

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