Pyrosequencing

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.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
The History of Pyrosequencing Ò.- Pyro Mark® Instruments, Chemistry and Software for Pyrosequencing® Analysis.- Software-Based Pyrogram ® Evaluation.- Quantitative Validation and Quality Control of Pyrosequencing ® Assays.- Extended KRAS and NRAS Mutation Profiling by Pyrosequencing®.- Universal BRAF State Detection by the Pyrosequencing®-based UBRAF V600 Assay.- Pyrosequencing ® -based Identification of Low Frequency Mutations Enriched through Enhanced- ice -COLD-PCR.- Analysis of Mutational Hotspots in Routinely Processed Bone Marrow Trephines by Pyrosequencing ®.- Analysis of Copy Number Variation By Pyrosequencing ® using Paralogous Sequences.- Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing ®.- HLA-B and HLA-C Supratyping by Pyrosequencing ®.- Allele Quantification Pyrosequencing® at Designated SNP sites to Detect Allelic Expression Imbalance and Loss-of-heterozygosity.- Quantitative DNA Methylation Analysis by Pyrosequencing®.- Quantitative Methylation Analysis of the PCDHB Gene Cluster.- Assessment of Changes in global DNA Methylation Levels by Global Analysis of DNA 5-methylcytosine using the Luminometric Methylation Assay, LUMA.- Limiting Dilution Bisulfite Pyrosequencing ® : a Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.- Detection of Loss of Imprinting by Pyrosequencing ®.- Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing ®.- Allele-specific DNA methylation detection by Pyrosequencing ®.- SNP-based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing ®.- DNA Methylation Analysis of ChIP Products at Single Nucleotide Resolution byPyrosequencing ®.- Multiplex Pyrosequencing®: Simultaneous Genotyping Based on SNPs from Distant Genomic Regions.-Detection of Drug-Resistant Mycobacterium tuberculosis.- Application of Pyrosequencing ® in Food Biodefense.- Forensic Analysis of Mitochondrial and Autosomal Markers using Pyrosequencing ®.- Tissue-specific DNA Methylation Patterns in Forensic Samples Detected By Pyrosequencing ®.