PCR

About this book

A thoroughly updated version of the successful first edition with a new chapter on Real-Time PCR, more prokaryotic applications, and more detail in the complex mutagenesis sections. Information on PCR applications in genomics and proteomics will be expanded and integrated throughout the text. There will also be advice on available products and specific pointers to the most appropriate methods. As with the first edition, this will be an ideal practical introduction and invaluable guide to PCR and its applications.

Contents

1: AN INTRODUCTION TO PCR - Introduction; PCR, a 'DNA photocopier'; History of PCR; PCR involved DNA replication; PCR is controlled by heating and cooling; Does PCR replace gene cloning?; PCR applications. 2: UNDERSTANDING PCR - How does PCR work?; PCR: a molecular perspective; The kinetics of PCR; Getting started; Post-PCR analysis 3: REAGENTS AND INSTRUMENTATION - Technical advances in PCR; Reagents; PCR buffers (mention other ions); Nucleotides; PCR premixes; Oligonucleotide primers; Degenerate primers 4: OPTIMIZATION OF PCR - Introduction; Improving specificity pf PCR; Template DNA preparation and inhibitors of PCR; Nested PCR improves PCR sensitivity; Contamination problems; Stress that control tubes should be set up last; Preventing contamination; Trouble-shooting guide 5: ANALYSIS, SEQUENCING AND IN VITRO EXPRESSION OF PCR PRODUCTS - Introduction; Analysis of PCR products; Verification of initial amplification product; Direct DNA sequencing of PCR products; Direct labelling of PCR products and homogenous assays; In vitro expression of PCR product 6: PURIFICATION AND CLONING OF PCR PRODUCTS - Introduction; Purification of PCR products; Cloning of PCR products; PCR cloning vectors; PCR cloning vector systems; Analysis of PCR-cloned DNA fragments 7: PCR MUTAGENESIS - Introduction; Include direct mutagenesis and in vitro methods at the start; Introducing mutations by splicing by overlap extension (SOEing); Deletion mutagenesis; Insertion mutagenesis; Point mutations by SOEing; Inverse PCR mutagenesis; Unique sites elimination; Random mutagenesis; PCR misincorporation procedures; Recombination strategies; Gene synthesis 8: ANALYSIS OF GENE EXPRESSION - Introduction; Reverse transcriptase PCR (RT-PCR); Semi-quantitative and quantitative RT-PCR; Use of internal standard ie. Stably/constitutive expressed genes; How to quantify signals; More info on potential problems and controls; One-tube RT-PCR; Differential display; Advantage compared to RT-PCR 9: REAL-TIME RT-PCR - Introduction and principle; Detection systems (SYBR Green, Taqman, molecular Beacons, Scorpions); Taqman primer and probe design; Polymerases; Thermal cyclers; Software; Multiplex real-time RT-PCR; One-step real-time RT-PCR; Interpreting the results 10: CLONING GENES BY PCR - Cloning genes of known DNA sequence; Using PCR to clone expressed genes; Expressed sequence tags (EST) as cloning tools; Rapid amplification of cDNA ends (RACE); Isolation of unknown DNA sequences; Inverse polymerase chain reaction (IPCR); Multiplex restriction site PCR; Vectorette PCR; Splinkerettes 11: PCR AND GENOMICS - Introduction; Why map genomes?; Single-strand confirmation polymorphism analysis (SSCP); Ligase chain reaction (LCR); Amplification refractory mutation system (ARMS) Appendix - Web directory (must be up-to-date with stable sites)

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