Table Of ContentModern Applications of DNA
Amplification Techniques
Problems and New Tools
Modern Applications of DNA
Amplification Techniques
Problems and New Tools
Edited by
Dirk Lassner
Institute of Clinical Chemistry and Pathobiochemistry
University of Leipzig
Leipzig, Germany
8arbara Pustowoit
Institute of Virology
University of Leipzig
Leipzig, Germany
and
Arndt Rolfs
Clinic and Policlinic of Neurology
University of Rostock
Rostock, Germany
Springer Science+Business Media, LLC
Library of Congress Cataloglng-In-Publlcatlon Data
Modern appllcat10ns of DNA ampilflcatlon techn1Ques problems and nelol
tools edlted by Dlrk ~assner. Barbara PustOWOlt. and Arndt Rolfs.
p. em.
'Praceeo1ngs D~ the- AugustuSburg Conference of Advanced SClence on
Problems of Quantltatlon of NuclelC AClds by Ampllf1cat1on
Technlques. held September 23-26. 1996, 1~ AuguStuSburg. Germany -
-T.p. verso.
Includes blbllographlcal references and lndex.
ISBN 978-1-4613-7455-8 ISBN 978-1-4615-5379-3 (eBook)
DOI 10.1007/978-1-4615-5379-3
1. Polymerase chaln reactlon--Congresses. 2. DNA--Synthesls-
-Congresses. I. Lassner. D1 r k. I I. Pustawo 1t . Barbara.
III. Rolfs. Arndt. 1959- IV. Augustusburg Conference of
Advanced SCience on PrOblems of Quantltatlon of Nucleic AClds by
AmplIficatIon TechnIques (1996)
[DNLM 1. DNA--analysls--congresses. 2. Polymerase Chain
React1on--methods--congresses. 3. Polymerase Chaln Reactlon
-standards--congresses. 4. VIrus Dlseases--dlagnosls--congresses.
QU 58.5 M689 19971
QPSOS.D4SMS3 1997
572.8·S45--dc21 97-41939
CIP
Proceedings of the Augustusburg Conference of Advanced Science on Problems of Quantitalion
of Nucleic Acids by Amplification Techniques, held September 23 - 26, 1996,
in Augustusburg, Germany
ISBN 978-1-4613-7455-8
© 1997 Springer Science+Business Media New York
Originally published by Plenum Press, New York in 1997
Softcover reprint of the hardcover 1s t edition 1997
http://www.plenum.com
10987654321
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form
or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise,
without written permission from the Publisher
v
PREFACE
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 ofa pplication:
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.
Several experimental factors may affect the efficiency of peR amplification: the sequence
being amplified, the length of the target, the sequence of the primers and potential impurities
in the sample. There are additional, as yet unknown, often subtle factors that also affect the
amplification efficiency. Some of the earliest experiments designed to quantify nucleic acid
levels involve the measurement of external standards. Nevertheless, in an attempt to correct
for tube-to-tube variations in amplification efficiency, an internal standard seems to be the
most accurate procedure at the moment.
As a sign for still existing difficulties in the methodological field ofPCR quantification there
are even some highly sophisticated mathematical papers dealing with that problem (e.g.
Nedelman et al. 1992).
To give an actual overview of the state of the art in peR quantification the idea for a
conference on the Augustusburg near Dresden was born during a discussion between the
editors and other scientists. The title of the conference was "Modem application of DNA
amplification -problems and new tools" and the aim was to present a thorough discussion of
problems in that field. However, there is now doubt that due to the vast amount of available
information about peR and the rapid development of new methods, the editors and organizers
may have inadvertently omitted some important and exciting information. The results of all the
investigations in the rapidly growing field of peR technology give a lot of answers to actual
questions. But with each new answer, more questions arise.
VI Preface
The editors would like to thank Joanna Lawrence and Robert Wheeler from Plenum Press
whose infinite patience, encouragement and support have been invaluable. The editors are
also grateful to Ms. Katrin SchmidtIRostock for her patient assistance in all stages of this
undertaking.
We thank all authors for their contributions and for their care in compiling useful practical
accounts of their specialist areas ofPCR technology. The editors hope that the reader will find
that the book represents an informative and useful manual to continue the list of good PCR
books.
Dirk Lassner
Barbara Pustowoit
Arndt Rolfs
VII
CONTENTS
peR Quantification and Technical Aspects
Multiple Competitors for Single-Tube Quantification ofHIV- 1 DNA. . . . . . . . .. 3
Tanya Vener, Malin Stark, Jan Albert, Mathias UhJen, Joakim Lundeberg
Quantitation of p53 Tumor Suppressor Gene Copy Number in Tumor DNA. . . . . II
Samples by Competitive PCR in an ELISA-Format
Meinhard Hahn, Volker Dorsam, Alfred Pingoud
Standardisation of Messenger RNA Quantification Using an RT -PCR
Method Involving Coamplification with a Multi-Specific Internal Control.. 25
David Shire, Pascale Legoux, Adrian J. Minty
Quantitative Analysis of Ruman DNA Sequences by Solid-Phase Mini-
sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Ann-Christine Syviinen
Bioimage Analysers: Application for Ribozyme Kinetics .................... 47
Claus Stefan Vortler, Klara Birikh
First Approaches to Quantitate MDRI-Messenger RNA by in cell PCR . . . . . . .. 55
Dirk Lassner, Joerg Milde, Matthias Ladusch, Karl Droessler,
Harald Remke
Application of in situ-PCR for Detection ofIntracellular mRNAs . . . . . . . . . . . .. 65
Volker Uhlmann, Eilhard Mix, Arndt Rolfs
Psoralen Biotin: A Novel Reagent for Non-Enzymatic and Specific Labeling
ofN ucleic Acid Probes and Oligonucleotides .......... : . . . . . . . . . . . . . 77
Robert L. Burghoff, Jens Beator, Michael A. Harvey
The Effect of Quantitative Ratio between Primer Pairs on PCR Products
in Multi-Target Amplification .................................... 83
Dani Bercov;ch, Zip; Regev, Tal Ratz, Yoram Plotsky
V/lJ Contents
peR Quantification of Infectious Agents
Quantitation of Rubella Virus Genome by QPCR and its Application to
Resolution for Mechanism of Congenital Rubella Syndrome. . . . . . . . . .. 93
Shigetaka Katow, Satoko Arai
Significance ofthe Detection of Rubella Virus RNA by Nested PCR in
Prenatal Diagnostics of Viral Infections. . . . . . . . . . . . . . . . . . . . . . . . . .. 101
Barbara Pus/owoit
Quantitative Detection of Human Cytomegalovirus DNA in Cerebrospinal
Fluid by Polymerase Chain Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 109
Jens-Uwe Vogel, Bernard Weber
Quality Control and External Quality Assessment Schemes for the Diagnostic
Use ofPCR in Microbiological Laboratories -European Trials on
Hepatitis B Virus and Cytomegalovirus ........................... 119
Jurjen Schirm
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 129
Suppliers of Special Items. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 133
Index ........................................................... 139
peR Quantification and Technical Aspects
3
MULTIPLE COMPETITORS FOR SINGLE-TUBE QUANTIFI
CATION OF mV-l DNA
Tanya Venerl, Malin Starkl, Jan Albert2, Mathias Uhlenl and Joakim
Lundebergl
IDepartment ofB iochemistry and Biotechnology, KTH, Royal Institute of
Technology, Teknikringen 30, 10044 Stockholm. 2J)epartment of Clinical
Virology, Swedish Institute for Infectious Disease Control, Karolinska
Institute, 105 21 Stockholm, Sweden
INTRODUCTION
The polymerase chain reaction (PCR) has drastically changed the diagnostic possibilities to
detect and analyse diseases caused by pathogens, such as viruses and parasitesl. Furthermore,
the use of PCR for quantitative analysis has become a key technique in disease prognosis.
However, large scale routine quantification of clinical samples has been hampered by the
amount of labor needed for accurate quantification. Analysis with limiting dilution and
Poisson distribution gives a correct prediction but is labor intensive2. Instead, several methods
based on co-amplification schemes have been described3-7involving mixing varying amounts of
competitor with a constant amount of target. Most of these methods have the disadvantage
that they require multiple tubes per analysis to assess the titration inflection point.
One important clinical area related to PCR quantification systems is the analysis of human
immunodeficiency virus type I (illY-I) during treatment with anti-viral drugs such as AZT,
ddC and ddIl and it has been shown that direct measurement in viral load can be correlated to
the effect of different treatment regimes.
Here, we describe an alternative for viral load analysis ofIDV-l in samples using multiple
competitors in a semi-nested co-amplification procedure. The method has been designed to
allow routine analysis in a non-isotopic format and to yield information on the amount of viral
mV-l DNA in peripheral blood mononuclear cells (PBMC). The competitor DNAs have
been designed to contain the same LTR primer binding sequences as the wild type DNA, but
with different internal sequences and length. Discrimination between the wild type DNA and
the four competitors has been performed by using one fluorescently labelled inner PCR primer
followed by fragment analysis using standard automated sequencers. A calibration curve can
be established using the peak area ofthe four competitors for accurate determination of target
Modem Applications ofD NA Amplification Techniques
Edited by Lassner el al., Plenum Press. New York. 1997
