Real-time PCR applications in haemostasis and transfusion medicine [Elektronische Ressource] / vorgelegt von Jens Müller

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

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Real-time PCR Applications in Haemostasis and Transfusion Medicine





Inauguraldissertation
zur Erlangung des Grades eines Doktors der Humanbiologie
des Fachbereichs Medizin
der Justus-Liebig-Universität Gießen













vorgelegt von Jens Müller
aus Gießen



Gießen 2007


Aus dem Biochemischen Institut der Justus-Liebig-Universität Gießen
Direktor: Prof. Dr. Klaus T. Preissner

und

dem Institut für Experimentelle Hämatologie und Transfusionsmedizin
der Rheinische Friedrich-Wilhelms-Universität Bonn
Direktor: Prof. Dr. Johannes Oldenburg















Gutachter: Herr Prof. Dr. Pötzsch

Gutachter: Herr Prof. Dr. Domann


Tag der Disputation: 13.03.2007 Contents


Chapter 1 Introduction 5

Chapter 2 Development and Validation of a Real-Time PCR Assay for
Routine Testing of Blood Donations for Parvovirus B19 DNA
Infus Ther Transfus Med 2002;29:254-258 37

Chapter 3 Quantitative Tissue Factor Gene Expression Analysis in Whole
Blood: Development and Evaluation of a Real-Time PCR Platform
Clin Chem 2004;50:245-247 50

Chapter 4 Desmopressin Acetate (DDAVP) Administration Induces Tissue
Factor-dependent Procoagulant Activity in Monocytes
Submited 58

Chapter 5 Gene Expression Analysis in Platelets from a Single Donor:
Evaluation of a PCR-based amplification technique
Clin Chem 2004;50:2271-2287 74

Chapter 6 Molecular analysis of thrombophilic risk factors in patients with
dural arteriovenous fistuals
J Neurol 2002;249:680-682 95

Chapter 7 Discusion 102

Chapter 8 Summary 110

Zusammenfassung 112

Erklärung 116

Danksagung 117

Curiculm vitae 118 5
Chapter 1

Introduction

THE HISTORICAL BACKGROUND OF THE POLYMERASE CHAIN REACTION
The Polymerase Chain Reaction (PCR) was pioneered by Kary B. Mullis, born in 1944 in
Lenoir, North Carolina, USA [1]. Charged with making oligonucleotides for DNA
sequence analysis, Mullis worked as a scientist for the Cetus Corporation of Emeryville,
California. In 1983, whilst driving along a mountain road into northern California’s
redwood country, Mullis was thinking about a means of determining the nucleotide
present at a specific position of the beta-globin gene for diagnosis of sickle cell anaemia
[2]. His thoughts were based on the techniques of dideoxy sequencing, a method first
described by Sanger and Coulson in 1975 [2,3]. He then had a sudden intuition showing
him a way of amplifying a DNA sequence in a single test tube [1,2]. Though we know
today that the PCR uses multiple cycles at different temperature conditions to generate
double-stranded DNA from a single-stranded DNA template (see ‘Basic principles of
PCR’, page 6), Mullis was not certain during the early phase of his research that the
reaction would not cycle itself. Assuming any chemical equilibrium to have some finite
value, he believed that a portion of any nominally double-stranded DNA would be single-
stranded, meaning that single-stranded DNA templates would be permanently present
during the amplification process [1,4]. Thus, the first Polymerase Chain Reaction,
labelled ‘PCR01’ (see figure 1, page 6), was an isothermal process that did not lead to
the expected DNA band on ethidium bromide stained agarose gel (see ‘Post-PCR
Detection of Amplification Products’, page 9). It took Mullis and his colleagues from
Cetus several months to achieve conditions producing convincing results [4]. Now
familiar with exponential amplification of DNA sequences, they were able to present a
method for the diagnosis of sickle cell anaemia starting from minimum amounts of
genomic DNA [5].
As PCR requires denaturation of double-stranded DNA through application of a high
temperature to the reaction tube, a problem in the early phase of PCR was that the
enzyme used for DNA amplification (the Klenow fragment of Escheria coli DNA
polymerase) had to be replenished after each denaturation step [5,6]. This problem was 6
overcome by replacing the initially used enzyme with a heat stable protein isolated from
an organism native in hot springs (Thermus aquaticus [Taq] DNA polymerase) [7,8,9].
Kary B. Mullis was awarded the Nobel Prize in chemistry in 1993 for his discovery of the
PCR method [10,11].



















Figure 1.: The first Polymerase Chain Reaction. Page from the notebook of Kary B. Mullis
thshowing the reagents put together for ‘PCR01‘ in a purple-capped tube on September 8 , 1983 [4].

BASIC PRINCIPLES OF PCR
Various reaction components play a role in PCR. The DNA template sequence to be
amplified can include purified DNA or RNA sequences converted to cDNA by reverse
transcription [12,13]. A large molar excess of small pieces of synthetic single stranded
DNA (so-called ‘primers’) determine the length and sequence of the amplification
product [5]. The most frequently used thermostable polymerase is Taq DNA polymerase 7
[7,8]. Amplification reactions also include PCR buffer, deoxynucleotide triphosphates
(dNTPs, an equal mixture of dATP, dTTP, dGTP and dCTP) and magnesium chloride
(MgCl ) [7,9,14,15]. The sensitivity and specificity of a PCR reaction depend on the 2
interaction of these reaction components, but the most important aspect is the primer
design [16,17]. The ideal primer pair anneals to unique sequences that flank the target
and not to other sequences in the sample [18]. A primer needs to be long enough to
reduce the probability of the sequence being found at non-target sites. Typical primers
are 18 to 24 nucleotides in length [5,6,19]. Despite some general desirable
characteristics of primer sequences, the melting temperature (T ) is another important m
parameter [20]. T is the temperature at which 50% of an oligonucleotide and its m
complementary sequence are present in a duplex DNA molecule [21]. Ideally, the
annealing temperature of a primer is low enough to guarantee efficient annealing but
high enough to minimise nonspecific binding [22]. Temperatures for primer annealing
are initially set about 5°C below the T of the primers. Higher annealing temperatures m
are then tested to reduce the formation of any formed primer-dimers or nonspecific
products [15].
The principle underlying PCR is shown in detail in figure 2 (page 8). In the first stage of
the PCR reaction, double-stranded DNA molecules are separated at high temperature
into their two component strands. The reaction tubes are then cooled down to a
temperature that allows the primers to anneal at specific locations on the opposite
strands of the target sequence. While the so-called “forward” (or “sense”) primers anneal
to the anti-sense strands of the separated DNA template, the “reverse” (or “anti-sense”)
primers anneal to the sense strand downstream from the sequence chosen for the
forward primers. Taq DNA polymerases then "extend" the primers by attaching a
complementary nucleotide to each nucleotide in the template strand. In this manner, the
polymerase creates two identical double-stranded DNA helices from the two separated
single strands of the original template molecule. The cycle of denaturation, primer
annealing and strand synthesis is then repeated several times. As the products from one
round serve as a template for the next amplification cycle, each additional cycle
theoretically doubles the amount of accumulated PCR products [2,5,6,19].

8
1
2
3



Figure 2. Basic principle of PCR. First, double stranded DNA sequences are separated into single
strands by applying high temperatures (usually 95°C) to the reaction tubes. 2. During the subsequent
annealing step at an optimised temperature, the forward and reverse primers hybridise to their target
sequences located on the opposite strands of the target DNA molecule. 3. Taking the resulting
primer:template duplexes as a starting point, Taq DNA polymerases then synthesise a
complementary strand to the single-stranded DNA matrices. The process of strand separation, primer
annealing and extension is then performed repeatedly, resulting in the production of a large number
of identical DNA strands. Since amplified products from the previous cycle serve as a template for
the next amplification cycle, PCR is an exponential process and a highly sensitive technique for
nucleic acid detection. 9
KINETICS OF PCR REACTIONS
The amplification of nucleic acids is an exponential process that can be described by the
equation

n P = I * (1+E)

where P is the copy number of accumulated PCR product, I is the initial copy number of
the nucleic acid template of interest, E is the efficiency of the PCR reaction and n is the
number of perfor