Advances and Clinical Practice in Pyrosequencing

The aim of this book is to improve pyrosequencing protocols as well as instrumentation for better clinical use by describing improvements and novel applications of pyrosequencing technology. Divided into five parts, the book's thirty chapters explore advances in pyrosequencing template preparation, pyrosequencing technology innovations, multiplex pyrosequencing based on barcodes, the miniaturization of pyrosequencing equipment, as well as various applications. As part of the Springer Protocols program, chapters contain the kind of detail and practical implementation advice to guarantee successful results in the lab.

Comprehensive and thorough, Advances and Clinical Practice in Pyrosequencing serves as a valuable reference for researchers who are engaged in personalized medicine, disease control, and DNA diagnosis in numerous other fields.

Provides a comprehensive description of current techniques and clinical applications of pyrosequencing Addresses both the technical and clinical aspects of pyrosequencing uses Describes the current applications of pyrosequencing in different fields, emphasizing their practicality and utility in clinical treatment, especially gene-guided personalized medicine Provides technical protocols to measure SNPs or mutations, mRNA and microRNA quantification, as well as resequencing by pyrosequencing Includes supplementary material: sn.pub/extras

Inhalt
Preparation of single-stranded DNA for pyrosequencing by LATE-PCR.- A simplified protocol for preparing pyrosequencing templates based on LATE-PCR using whole blood as starting material directly.- Improvement of LATE-PCR to prepare pyrosequencing template.- Pyrosequencing-templates generated by nicking PCR products with nicking endonuclease.- Pyrosequencing-templates generated by asymmetric Nucleic Acid Sequence-Based Amplification (asymmetric-NASBA).- Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing.- Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.- A novel pyrosequencing principle based on AMP-PPDK reaction for improving the detection limit.- Pyrosequencing chemistry coupled with modified primer extension reactions for quantitative detection of allele frequency in a pooled sample.- A gel-free SNP genotyping method based on pyrosequencing chemistry coupled with modified primer extension reactionsdirectly from double-stranded PCR products.- Improvement of pyrosequencing to allow multiplex SNP typing in a program.- Improvement of pyrosequencing-sensitivity based on the capture of free adenosine 5'-phosphosulfate with adenosine triphosphate sulfurylase.- Using polymerase preference index to design imLATE-PCR primers for an efficient pyrosequencing.- Preparation of thermalstable biotinlyted firefly luciferase and its application in pyrosequencing.- Expression of thermostable recombinant luciola lateralis luciferase and its function on setting up of heat-stable pyrosequencing system.- Expression and purification of ATP sulfurylase from saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing.- Characterization of recombinant Escherichia coli single-strand binding protein and its application in pyrosequencing.- Expression of PPDK from Microbispora Rosea Subsp.aerata in E.coli and its application in pyrosequencing.- Construction of 3-plex barcodes for differential gene-expression analysis with pyrosequencing.- Development of pyrosequencing-based multiplex bioassay by designing barcodes encoded with artificially designed sequences.- Quantitatively discriminating multiplexed LAMP products with pyrosequencing-based bio-barcodes.- Multiplex ligation-dependent sequence-tag amplification coupled with pyrosequencing for CNV analysis.- Pyrosequencer miniaturized with capillaries to deliver deoxynucleotides.- Pyrosequencer with a senor of a photo-diode array.- Pyrosequencing on acryl-modified glass chip.- Pyrosequencing on-chip based on a gel-based solid-phase amplification.- Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR.- Comparative gene expression analysis of breast-cancer related genes by multiplex pyrosequencing coupled with sequence-barcodes.- MicroRNA quantification by pyrosequencing with a sequence-tagged stem-loop RT primer.- Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.- Genotyping of pathogenic serotypes of S. suis with pyrosequencing.- Differential gene expression analysis of breast cancer by combining sequence-tagged reverse-transcription PCR with pyrosequencing.- Detection of avian influenza A virus by pyrosequencing.- Genotyping of the alcohol dehydrogenase gene by pyrosequencing coupled with improved LATE-PCR using human whole blood as starting material.- Pyrosequencing on a single cell. <p