Barbara Pustowoit – författare

In the ten years since the first publication on PCR (Saiki et al. , 1985), this in vitro method of nucleic acid replication and modification has grown to rival in popularity traditional microbiological, genetical und technical procedures for cloning, sequencing, gene detecting and related procedures. To date the PCR literature has emphasized six main areas of application: genetic mapping, detection of mutations, genetic polymorphism, transcriptional splicing and regulation, molecular virology and quantitative procedures. The overwhelming focus of quantification of DNA or RNA by PCR has been on human microbiology and oncological problems. The exquisite sensitivity of PCR gives this method the ability to detect extremely rare DNAs, mRNAs, mRNAs in small numbers of cells or in small amounts of tissue, and mRNAs expressed in mixed-cell populations. However, the exact and accurate quantification of specific nucleic acids in biological samples is in spite of numerous publications in that field still a general problem: during the peR process, an unknown initial number of target sequences are used as a template from which a large quantity of specific product can be obtained. Although the amount of product formed is easy to determine, it is difficult to deduce the initial copy number of the target molecule because the efficiency of the peR is largely unknown.

In the ten years since the first publication on PCR (Saiki et al. , 1985), this in vitro method of nucleic acid replication and modification has grown to rival in popularity traditional microbiological, genetical und technical procedures for cloning, sequencing, gene detecting and related procedures. To date the PCR literature has emphasized six main areas of application: genetic mapping, detection of mutations, genetic polymorphism, transcriptional splicing and regulation, molecular virology and quantitative procedures. The overwhelming focus of quantification of DNA or RNA by PCR has been on human microbiology and oncological problems. The exquisite sensitivity of PCR gives this method the ability to detect extremely rare DNAs, mRNAs, mRNAs in small numbers of cells or in small amounts of tissue, and mRNAs expressed in mixed-cell populations. However, the exact and accurate quantification of specific nucleic acids in biological samples is in spite of numerous publications in that field still a general problem: during the peR process, an unknown initial number of target sequences are used as a template from which a large quantity of specific product can be obtained. Although the amount of product formed is easy to determine, it is difficult to deduce the initial copy number of the target molecule because the efficiency of the peR is largely unknown.

In this laboratory "cook-book", the authors provide a concise guide to PCR-based techniques to quantify nucleic acids in biological and clinical samples using exclusively nonradioactive detection methods, e.g. HPLC, biotin and digoxigenin based protocols. Each method presentation also includes sections on theory, reagents, standards, applicability, limitations, and trouble shooting. In addition to the protocols, the authors also provide the necessary information on: general aspects of nucleic acid quantitation; design of PCR standards; mRNA purification; cDNA synthesis; solution hybridization; DNA sequencing. This laboratory guide enables professionals as well as beginners to adopt easily quantitative PCR protocols into their own clinical or biomedical research.