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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.
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