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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.
PCR Quantification and Technical Aspects: Multiple Competitors for
Single-Tube Quantification of HIV1 DNA; T. Vener, et al.
Quantitation of p53 Tumor Suppressor Gene Copy Number in Tumor DNA
Samples by Competitive PCR in an ELISA-Format; M. Hahn, et al.
Standardisation of Messenger RNA Quantification Using an RTPCR
Method Involving Coamplification with a Multi-Specific Internal
Control; D. Shire, et al. Quantitative Analysis of Human DNA
Sequences by Solid-Phase Minisequencing; A.C. Syvanen. Bioimage
Analysers: Application for Ribozyme Kinetics; C.S. Voertler, K.
Birikh. First Approaches to Quantitate MDR1Messenger RNA by in cell
PCR; D. Lassner, et al. Application of in situ-PCR for Detection of
Intracellular mRNAs; V. Uhlmann, et al. Psoralen Biotin: A Novel
Reagent for Non-Enzymatic and Specific Labeling of Nucleic Acid
Probes and Oligonucleotides; R.L. Burghoff, et al. The Effect of
Quantitative Ratio between Primer Pairs on PCR Products in
Multi-Target Amplification; D. Bercovich, et al. PCR Quantification
of Infectious Agents: Quantitation of Rubella Virus Genome by QPCR
and Its Application to Resolution for Mechanism of Congenital
Rubella Syndrome; S. Katow, S. Arai. Significance of the Detection
of Rubella Virus RNA by Nested PCR in Prenatal Diagnostics of Viral
Infections; B. Pustowoit. Quantitative Detection of Human
Cytomegalovirus DNA in Cerebrospinal Fluid by Polymerase Chain
Reaction; J.U. Vogel, B. Weber. Quality Control and External
Quality Assessment Schemes for the Diagnostic Use of PCR in
Microbiological Laboratories-European Trials on Hepatitis B Virus
and Cytomegalovirus; J. Schirm. Suppliers of Specialist Items.
Index.
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