REVIEW ARTICLE
Methods in molecular cardiology:N
the polymerase chain reaction
D.G.P. Sonnemans, L.J. de Windt, E.D. de Muinck, P.A. Doevendans
Several polymerase chain reaction (PCR) tech-
niques are described in this review to give insight
into the potential applications for cardiovascular
research. Although PCR can be performed in
several ways, all applications are based on the same
general principle, the amplification of DNA or
RNA by the enzyme polymerase. This amplification
provides the opportunity to detect, identify and
multiply a single copy of DNA or RNA, in or
outside the cell. This powerful technique can be
used in several directions of DNA and RNA
research resulting in the ability to specifically detect
the presence and activity of genes. The use of these
techniques in cardiovascular research is discussed
here. (Neth Heart J 2002;10:412-8.)
Key words: DNA, PCR, RNA
N owadays, molecular biology is commonly used in
clinical cardiology. There are four levels to study
molecular cardiology: DNA (deoxyribonucleic acid),
RNA (ribonucleic acid), protein and protein function.
These are correlated to the transcription of DNA into
RNA, the translation of RNA into proteins and the
protein's function inside or outside the cell. The first
two approaches can give information about genes and
the activation of these genes. Protein and protein
function analysis will provide more information about
gene product and activity. In this review, principles,
D.G.P. Sonnemans.
L.J. de Wlnctt.
E.D. de Mulnck.
P.A. Doevendans.
Department of Cardiology, Cardiovascular Research Institute
Maastricht, University Hospital Maastricht, P0 Box 5800, 6202
AZ Maastricht.
P.A. Doevendans.
Interuniversity Cardiology Institute of the Netherlands,
P0 Box 19258, 3501 DG Utrecht.
Address for correspondence: P.A. Doevendans.
E-mail: p.doevendans@cardio.azm.nl
412
practical approaches and applications of PCR
techniques used for DNA and RNA research will be
discussed. PCRis a basic and essential tool in molecular
cardiology.
The polymerase chain reaction
Historical introduction
The polymerase chain reaction, better known as PCR,
is a basic and essential tool in molecular biology. Kary
Mullis conceived this simple technique in 1983 during
a drive on a moonlit California mountain road, when
he was pondering on new ways to detect specific bases
by DNA sequencing. He developed this technique
with his colleagues into a pervasive and one of the
most widely used techniques of molecular biology." 2
For this Dr Mullis received the Nobel Prize for
chemistry in 1993.
Based on the simple method of PCR, many
variations have been developed. Since the first
publication this has lead to an exponential increase of
references, akin to the DNA produced by PCR. In
1989, Science selected the technique as the 'major
scientific development' and Taq DNA polymerase
'molecule ofthe year'.3 Thanks to PCR, development
of a range of applications in several disciplines was
possible and the amount of starting material no longer
a limitation in research. In medicine, for example, PCR
is used for diagnosis and screening of genetic diseases
and cancer, the characterisation of genes, their cloning
and expression as well as the basic factor in other
techniques, such as sequencing and preparation of
probes for hybridisation. The most impressive result
is, however, the determination of the complete
humane genome.
Basic principals
PCR is a rapid in vitro enzymatic amplification of a
specific DNA or RNA region that lies between two
regions ofknown DNA or RNA sequence. The DNA
molecule, as described in DNA sequencing,4 is a
double helix in its native state. This double helix con-
tains two complementary single strands ofnucleotides
bound to each other by hydrogen bonds. These
Netherlands Heart Journal, Volume 10, Number 10, October 2002

hydrogen bonds are formed between the monomers
of the DNA strands. DNA is a polymer of four
nucleotides, which differ in the sugar base. The sugar
bases are the purines; adenine (A) and guanine (G), and
the pyrimidines; cytosine (C) and thymine (T). They
are bound to a backbone of repeating deoxyribose-
phosphate units.
In DNA C is attached to G on the opposite strand and
Ato T.
The RNA molecule is a single strand of sugar bases
on a ribose-phosphate backbone. RNA, however, has
the pyrimidine base uracil (U) instead of T. Single-
stranded RNA is capable offorming a double-stranded
molecule with either complementary RNA or DNA.
The formation of complementary strands is the basic
principle of PCR.
In vivo the enzyme DNA polymerase carries out the
synthesis of DNA using a single-stranded DNA or
RNA template in the presence of deoxynucleoside
triphosphates (dNTPs). This synthesis is performed in
the 5' to 3' direction starting at a double-strand region
resulting in a complementary strand. This process is
called DNA extension. For in vivo synthesis more
processes are involved in the DNA synthesis, but a
simple scheme of this process is shown in figure 1.
..~~~~~~~~~~~~~~~~S3'
r when the reaction is repeated several times. Starting
liii dAlP
Ct o
1111 P Mg2+
1111 dGTP
-i 1111 amT
5.
3.
Figure 1. Schematic diagram of primer extension by DNA
polymerase using a complementary strand as template.
The start position is determined by the end ofdouble-
strand DNA and can be designed in vitro by using
synthetic oligonudeotide primers. These are 15 to 30
nudeotides in length. Primers can be synthesised by the
investigator or ordered from a biotech company. The
PCRinvolves two oligonudeotide primers, which flank
the target DNA sequence that has to be amplified.
After denaturing the double-stranded template, the
primers can hybridise (anneal) to the opposite strands
of the DNA at the optimal temperature. The orien-
tation ofthese primers is arranged in such way that the
DNA synthesis by the polymerase proceeds covering
the region between the primers. Therefore, the newly
formed strand will become a template for the reaction
itself (figure 2).
Nctherlands Heart Journal, Volume 10, Number 10, October 2002
Methods in molecular cardiology: the polymerase chain reaction
Figure 2. Schematic diagram of the principk of PCR; I Double-
stranded template; II Denaturing and primer annealing (blue
and ellow); III &vtension ofthe prismet;VCyckVs ofdenatunqng,
annealing and extnsion will kad to an eponential amplifieation
of the target sequence.
In figure 2 the second cycle, starting with four primer
sites, will result in eight new templates. Offering these
templates into a third cyde will result in 16 templates
of which two double-stranded molecules are flanked
by the two primers. These molecules, only containing
the target sequence, will accumulate exponentially
with one double-stranded template in the first cycle
will lead to a 270 million-fold amplification of the
discrete product after 30 cycles. The reaction will
continue as long as there is an excess of primers and
oligonudeotides, and the polymerase enzyme maintains
activity.5-9
DNA polymerases
Originally, the Klenow fragment of E. coli DNA poly-
merase I and T4 DNA polymerase were used to
synthesise the oligonucleotide extension. This am-
plification only worked well for the amplification of
short fragments up to 200 base pairs. Longer target
sequences were not amplified. The yield ofthe reaction
was low and the products were not specific. This was
due to the optimal activation temperature of the
enzymes. A non-specific annealing and extension
temperature of 37°C had to be used. Another dis-
advantage of these enzymes is their heat-instability.
After every denaturing step, fresh enzyme had to be
added to the reaction to catalyse the synthesis. A
breakthrough came with the introduction of Taq DNA
polymerase. The DNA polymerase was isolated from
Thermus aquaticus, a bacteria living in hot springs in
Yellowstone National Park. This heat-stable enzyme
pernitted the use ofhigher temperatures for annealing
and extension steps, which improved the stringency
of primer hybridisation to the template and therefore
the specificity of the products. The optimal activity is
found at 720C. The enzyme, however, is not infinitely
413
Methods in molecular cardiology: the polymerase chain reaction

resistant to heat and will lose more ofits efficiency after temperatures balanced for specificity. The primers
denaturing steps. should be made with a GC content. The primer
After the breakthrough with Taqpolymerase, more sequence should not contain any stretches ofpurines
polymerases have been isolated out ofdifferent thermo- or pyrimidines and no repetitive motifs. This will result
philic bacteria, such as other Thermus, Pyrococcus and in secondary structures within the primer itself,
Thermococcus bacteria. Also recombinant polymerases inducing the need for higher annealing temperatures
are commercially available. These DNA polymerases to unroll the structures. The primers should not be
provide an amplification ofmolecules of up to several complementary to each other on the 3' end. This
thousands base pairs of target sequence. The choice would result in annealing of the primers leading to
of polymerase is based on the application ofPCR and primer-dimers. In this case the primers will be the
needs to be taken in account.68"0 template for the polymerase and extension will make
new aspecific primers. These new primers will compete
Deoxynucleoside triphosphates (dNTPs) with the target DNA leading to loss of efficiency.
For DNA synthesis, polymerases need energy and The distance between the primers can be stretched
monomers. This is provided by the addition ofdNTPs to 10,000 bp (10 kb), but most polymerases are
into the reaction mixture. The four different nu- efficient only up to 3 kb. The distance should not be
cleotides need to be added in the same amount for too short either, because the PCRproduct (amplicon)
optimal activity of the polymerase. Optimal concen- needs to be detectable with, for instance, electro-
tration of dNTPs depends on several options. Taq phoresis or probe hybridisation.
polymerase activity, for example, is reduced by the The primers should have more or less the same
addition ofmore than 200 FM each. MgCI and primer annealing temperature. At this temperature the primers
concentration also interact. The length of the target bind specifically to the template DNA and are ready
sequence and the number of cycles influence the for extension. An easy formula is used for calculation
amounts of dNTPs necessary as well. The optimal ofthe primer melting temperature: (number ofA and
concentration of dNTPs is unique and needs to be T) x 2°C + (number of C and G) x 4°C. In general, a
determined for each PCR; a concentration ofaround temperature of 3 to 5°C below this melting point can
100pM each is, however, standard for PCR68 be used as annealing temperature in the PCR. How-
ever, this calculation is just an indication. The most
Buffes and MgCI2 specific annealing temperature has to be determined
All commercially available polymerases are supplied by practical experience with the primer set. The highest
with a specific buffer. Usually they provide a tenfold temperature with the best specific PCR products
concentrated buffer for use with the enzyme. The should be used as the final annealing temperature.
buffer contains several salts in which the enzyme will Another way of improving the specificity of the
be preserved best, although this will be different for each amplification is the use ofnested primers. As the word
enzyme. The buffer also contains non-ionic detergents
such as gelatine, NP40, Tween 20 or Triton X-100.
And finally, to optimise the activity ofthe polymerase, TT P
TCT TAT TOT
the reaction mixture needs to be enriched with MgCI2. TTC TCC TAC J TOC J
Ser
The Mg2+ ions not only interact with the dNTPs for TA LOU
TCA TAA Stop TOA Stop
TTO TCO TAG sop TOO Trp
incorporation in the new strand, they also stimulate CTT 1 CCT CAT COT
the activation ofthe enzyme and the primer annealing CTC lLO eCCC Pr CAC
His
c
1
9 Arg
to the template. The concentration of MgCl2 has an CTA | CCA CAC Gin COA
enormous effect on the specificity and yield of a PCR. CTO J CC<S J CAG coPoJ
So, determination of the optimum concentration is ATT 1 ACT AAT AOT
essential for each unique PCK6-8 ATC II* ACC AAC| AOC
ATA ACA AAA{ LY AGA
ATO
M~~
ATO MA
AOO
ACG
ThrAO AAG A GG
Ar

Primers OTT 1 OCT') OAT 1 GOT1

To obtain a specific PCRproduct an optimal set (two) OTC 0CC OAC sp GCC
of primers must be designed. For most PCR appli- OTA
V

OCA
A

GAA GOAoA
0C0 J O
cations the primers are designed exactly comple- OTC J GAG c

mentary to the template DNA. In other applications

it is essential for them not to be exactly complementary
to, for instance, induce a mutation or obtain fragments
of not completely known template DNA. For primer
design, several computer programmes have now been
developed to assist the investigator. These are useful
but not foolproof. Therefore, the following rules have
to be adhered to. The primers should be between 15
and 30 nucleotides in length and allow high annealing
Figure 3. Genetic code; translation ofamino acidsfrom nucleotide
tripets. Amino acid abbreviations:Ala:Alanine;Arg:Arginine;
Asn: Asparagine; Asp: Aspartic acid; Cys: Cysteine; Gin:
Glutamine; Glu: Glutamicacid; Gly: Glycine; His:Histidine;Ile:
Isoleucine; Leu: Leucine; Lys: Lysine; Met: Methionine; Phe:
Phenylalanine; Pro: Proline; Ser: Serine; Thr: Threonine; Trp:
Tryptophan; Tyr: Tyrosine; Val: Valine. The chain termination
codons are indicated by stop.

414 Netherlands Heart Journal, Volume 10, Number 10, October 2002

already suggests, primers will be used which are
embedded within the amplicon of the first primer
sequence. The products of a first round of PCR are
used in a second PCR. One or both primers in this
second PCRare located internally ofthe primers used
in the first PCR. This will exdude aspecific products
in the first PCRand selectively amplify the target DNA.
Complete matching of the primers is not always
preferable. There are applications which use de-
generated primers. Degenerated primers contain a
mixture ofoligonucleotides with similar sequence but
variations at one or more positions. This is necessary
when only the amino acid sequence ofa gene is known.
A single amino acid can be coded for by several
nucleotide sequence possibilities (redundancy) as
shown in figure 3.
To amplify a piece of this unknown gene all possible
variations in DNA sequence have to be implied in the
reaction. Other applications are to search for novel
members ofknown genes,'2 or for homologous genes
between species.
The latter has the ability to relate species to each
other when primers, based on the gene sequence of one
species, are used in other more or less related species.'3
However, the use ofthese degenerated primers com-
plicates the reaction properties, such as the annealing
temperature. Controlling these reactions is essential
to prevent false positive or negative results.58"'
Instruments
The PCR developers performed the first reactions
manually. Klenow had to be added after each de-
naturing step and the samples were shuffled between
water or oil baths for denaturing, annealing and
extension steps. Automation ofthe procedure became
possible when Taqpolymerase made its entrance. The
enzyme could be added to the reaction mixture once.
A PCR machine could be developed which would
repeatedly and accurately cycle the temperature for a
set number of cydes. Such a procedure can be provided
by a robot arm, fitted with a tube-rack. The rack is
moved between three water baths by the arm. The
tubes are submerged for an incubation step and
transferred to the next step by the arm. This PCR
machine is not practical or effective. The robot and
water baths take a lot of bench space and during
transfer of the tubes temperature fluctuation occurs.
Several companies have developed the programmable
thermo block, as a more efficient machine. These
thermo cyclers should be able to change their
temperature reliably between 4°C and 95°C within
seconds (figure 4). The metal-block-type cyclers are
designed to fit 1.5 ml, 0.5 ml, and 0.2 ml reaction
tubes or even 96-wells plates. Most common is a
combination of the last two versions. Complicated
software is used to control the thermo block for
applying accurate and reproducible uniform tem-
peratures, incubation times and cycle numbers. Tem-
perature controlling, informing the user on progress,
Netherlands Heart Journal, Volume 10, Number 10, October 2002
Methods in molecular cardiology: the polymerase chain reaction
.
lnm (mm)
Figure 4. Temperature profile ofa PCR thermo block, denaturing
of the template DNA at 95°Cfollowed byprimer annealing at i.e.
60°C Primer extension at 720Cends onecyle. Thisprocess (in red)
isrepeatedforapresetnumberofcycles. The extension oftheprimers
in the last cycle is incubatedfor a longer period to ensure that all
the produets arefui length. Most PCR machines have the ability to
store the samples at 40C after the PCR.
elapsed time and the ability to store or print the
reaction is also included in the machines.
Development of this software and more compli-
cated machines even made it possible to perform a PCR
on a temperature gradient thermo block, or control
more thermo blocks simultaneously on one machine.
When heating the samples up to 95°C, evaporation
of the reaction mix has to be prevented. This can be
done by a mineral oil overlay or paraffin wax on the
reaction mix. Thermal cycling devices are now also
available with heated lids. They will heat the tubes and
the air above the reaction mix to about 110°C. When
heating the reaction mix, vapour immediately con-
densates on the colder surface ofthe reaction mix. This
will keep the reaction conditions intact and make the
use of mineral oil unnecessary.68
Detectlon of PCR products
One of the most commonly used techniques is
separation of PCR products by size. The DNA PCR
products are negatively charged. Electrophoresis can
be used to size fractionate the amplicons. Separation
on agarose or polyacrylamide gels, with an appropriate
size marker, makes determination of positive results
possible (figure 5). DNA can be easily visualised by
Figure 5. PCR products on an agarosegel flanked by molecular
weight lahdde,.
415
Methods in molecular cardiology: the polymerase chain reaction
staining with ethidium bromide or another fluorescent
dye. Observing the stained gel on an ultraviolet trans-
illuminator will reveal the DNA bands separated on
the gel.
DNA can also be directly detected with silver
staining. Southern blotting ofthe gel introduces more
detection techniques. The PCR products, now im-
mobilised on a membrane, can be detected with
probes. These probes are small isotope or enzyme or
antigen labelled DNA fragments and complementary
to the PCR product. Hybridisation or annealing to
the denatured PCRproducts will result in a detectable
signal. With these techniques it is not only possible to
detect the presence of products but also to determine
differences in size. Several applications are based on
this principle of size polymorphism, such as DNA se-
quencing and analysis of new disease mutations.47"4
PCR in genetic diagnosis
PCR made it much easier to analyse many genetic
diseases and even to detect genetic variation. One of
the revolutionary advantages of PCRis sample size. In
the early days, patients had to give enormous amounts
of tissue or blood to the laboratory. Analyses of this
material used to take a few days. With PCR, these
results are obtained in only a few hours and only a
pinprick of blood or a cotton swab of the oral mucosa
is needed.
A good example of PCRin genetic diagnosis is sex
determination. PCRcan be used to determine sex from
any tissue. This PCRis based on primers designed on
part of the Y-chromosome. This sequence is not pre-
sent in female samples. In this analysis PCR product
indicates male and a negative result indicates female
gender. To control this PCR for false negative results,
positive controls are usually included. These should
always be positive to confirm the reaction results. This
analysis can, for example, be used for prenatal sexing,
giving families with sex-linked diseases the opportunity
for prenatal care.5-8"'
PCR In labeiling probes
PCR can be used to amplify gene-specific sequences.
When these PCRproducts are labelled in a certain way,
they can be used to detect the genetic variation
through, for instance, Southern blotting (DNA-DNA
hybridisation). The side chains ofmodified nudeotides
do not prevent Taq DNA polymerase from using these
nucleotides for DNA synthesis. Therefore labelling
procedures can be performed radioactively or non-
radioactively during the PCR. The radioactive labelling
is performed in a standard PCR containing dNTPs
with a small part of one of the dNTPs radioactively
labelled with, for instance, 32P. This will result in PCR
products with several incorporated isotope labelled
nucleotides. Examples of non-radioactively labelled
PCR products are the use of digoxigenin-, biotin- or
fluorescent-labelled nucleotides for the same in-
corporation in their products.5"8,"
416
PCR In automated sequencing
One special example of labelling of PCR products is
sequencing. The Sanger method, as described in DNA
sequencing, uses fluorescent-labelled dideoxy-
nucleotides (ddNTPs). Every incorporated ddNTP
will stop the amplification reaction and add a specific
fluorescent label to the product. This base-specific label
can be detected during the electrophoresis by a laser.
Relating size and fluorescent label will reveal the
sequence. Other sequencing applications of PCRwere
described in DNA sequencing.4'6-8
PCR In ampification of RNA
The described methods use DNA as template, but
PCR can also be used for the amplification of RNA.
PCRprovides new ways to analyse RNA, such as gene
expression. Quantities of RNA are very limited and
often insufficient for the standard analysis methods.
The amplification is based on two enzymatic reactions,
which are displayed in figure 6. The first reaction is a
conversion step using RNA as template, resulting in the
translation of the original RNA into copy or com-
plementary DNA (cDNA). This reaction is performed
by the enzyme reverse transcriptase (RT), giving this
method the name of RT-PCR
1I
RT secticn I
If
J A
EE
*_____*____________ 4.
R III
v
-~~~~~~~~~~--
-K Z~~~~~~
Figure 6. Schematic diagram of the principle of RT-PCR using
oligo(d7) primer (leftstion) and a pecific pmer (nht etion);
I Reverse transription ofmRNA into cDN4 II RNaseHactivity
of the Reverse Transcriptase; III Single-stranded cDNA template;
IVPrimer annealing (blue) and exensi VCyclesofdenaturing,
annealing and extension will kad to an exponential amplification
of the target sequence.
This enzyme can be purified from several sources. The
most commonly used are AMV-RT, purified from
avian myeloblastosis virus and MMLV-RT, purified
from Moloney murine leukaemia virus. Some DNA
polymerases, such as the one isolated from T. thermo-
philus (Tth), can also reverse transcribe RNA.
The reverse transcriptase needs a primer to start
the extension ofthe single strand, in the same way as
Netherlands Heart Journal, Volume 10, Number 10, October 2002

DNA polymerase does. This primer can be based on
a gene-specific sequence. Another option is a general
primer based upon the structure of messenger RNA
(mRNA). Most of the mRNAs have a repeat of the
base A in their sequence, a so-called polyadenylated
tail, on the 3' end. This can be used as template for an
oligo(dT) primer. The reverse transcriptase has ribo-
nuclease H activity, which initiates the degradation of
RNA in the RNA-cDNA double strand. The single-
stranded cDNA of this first reaction is used in the
second reaction. This second reaction is a normal PCR,
which uses the cDNA strand as template. In this
reaction the original primer can be used in combination
with a specific primer or two new specific primers can
be used for the amplification. The cDNA product can
be used for several applications such as cloning into a
cDNA library (containing copies of all the transcripts
in a cell or tissue), or proving the presence of a certain
gene transcript in the original RNA isolation.
11~~~~~~~1
IlI
Figure 7. Shematic diagram ofdiscriminatingforgenomic DN4
ISingle-stranded cDNA (left) and double-strandedgenomic DNA
(right) templates in the same PCR; II Annealing and extension
of the primers is prevented in genomic DNA by intron sequences
(qreen/orange);III Cyclesofdenaturing, annealing and ex-tension
will lead to an exponential amplification of the target sequence
and not of thegenomic DNA equivalent.
In RT-PCR it is necessary to prevent false positive
results caused by genomic DNA contamination.
Primers anneal to RNA as well as genomic DNA in
the first reaction leading to cDNA contaminated with
DNA products. This can be prevented in two ways.
First the RNA can be treated with deoxyribonuclease
to destroy any contaminating DNA. Or secondly, the
design of primers can prevent DNA amplification. In
contrast to DNA, RNA only contains coding, or exon
sequence. Choosing primers in a region overlapping
more exons will exclude amplification of genomic
DNA, which will have several kilobases of intron, or
non-coding sequence in between (figure 7).5 8,11
Netherlands Heart Journal, Volume 10, Number 10, October 2002
Methods in molecular cardiology: the polymerase chain reaction
In situ PCR
The RT-PCR is used to detect gene expression. The
template RNA is isolated from a specific tissue and will
give an overall gene expression ofthis tissue. In situ PCR
can be used to perform the amplification steps inside
the cells and thus provides the possibility of defining
the exact location of gene expression. Another appli-
cation of in situ PCR is the detection of pathogens,
such as bacteria and viruses. This technique is per-
formed on tissue or on fixed cells. This introduces
several complications. To perform a PCR the cells need
to be made semi-permeable to allow the PCRreagents
to diffuse inside. The PCRproducts, however, need to
stay on the site of amplification for detection. This
detection can be performed in two ways; detecting
amplified products directly by labelled nucleotides or
indirectly by in situ hybridisation. The latter uses
probes, as shown in section PCR in labelling probes,
which are specific for the amplicons. The in situ PCR
needs to be controlled for false positive and negative
results. A normal PCR is performed with pure DNA
or RNA. This technique uses template material, which
contains several unpredicted circumstances such as
aspecific annealing of the primers and incorporation
of nucleotides. Leakage of the PCR products in and
out of cells can lead to extracellular amplification. These
products can result in false positive cells. False negative
results can occur due to poor permeability, thermo-
conductivity or reaction instabilities. Therefore, ap-
propriate controls are essential for correct interpretation
of the results. This technique and its applications,
however, are too complex and need to be described
separately. 6-8,11
Applications In cardiology
The techniques described above are used in a wide
range of research. An application of PCR within
cardiology is explained in this section.
Angiotensin-converting enzyme gene polymorphism
When Cambien et al. published data about the effect
of a variation in the gene encoding angiotensin-
converting enzyme (ACE) and its possible relation to
myocardial infarction, more genetic factors in cardio-
vascular disease were explored. The role ofACE poly-
morphism in cardiovascular disease is still not exactly
dear. However, the detection technique used in this
research has produced, as the PCR itself, an almost
exponential increase in publications since the first
publication in 1992. The role of PCR in ACE poly-
morphism will therefore be described.
An insertion/deletion DNA polymorphism was
detected in the human ACE gene. The insertion was
localised on intron 16 of the human dipeptidyl
carboxypeptidase 1 (DCPI). The insertion itself
consists of a 287 bp long repetitive sequence. This
polymorphism results in three genotypes, including
the double insertion (II) and double deletion (DD)
homozygotes and the heterozygous genotype with in
417
 Methods in molecular cardiology: the polymerase chain reaction
m ddion (D)
13ifert gen1otpes
Figure 8. ACEpolymorphism withdouble insertion (II) and double
deletion (DD) homozygotes and insertion/dektion heterozygous
genotype (ID).
one allele an insertion and in the other allele a deletion
(ID) (see figure 8). The effect ofdeletion results in a
high ACE activity in the DD homozygote and an
intermediate activity in the heterozygote compared
with the II homozygous genotype.
Using PCR and specific primers flanking the in-
sertion this polymorphism can be detected. Originally,
24-mer primers were used to amplify a part of the
DCPI gene using human genomic DNA as template.
The DNA was amplified for 30 cycles of denaturing,
annealing and extension. The PCR product is a 190
bp fragment in the absence of the insertion and a 477
bp fragment in the presence of the insertion. These
products can be visualised when separated by size using
electrophoresis and compared with a size marker as
shown in figure 9.
Samples with only a product at 190 bp will contain
the double deletion genotype, whereas samples with only
a product at 477 bp will have the double insertion geno-
type. When the PCRresults in both products the hetero-
zygous genotype will be present in the sample.'5-'7
This simple diagnostic test can be performed easily
for ACE genotyping of a patient. With just a cotton
i1D
Figure 9. Gel electrophoresis to determine the ACE genotype by
means ofPCR.
418
swab of the oral mucosa, enough material is provided
to isolate genomic DNA. And an ACE-specific PCR
with reliable positive and negative controls will produce
a genotype within a few hours.
By using PCR and its applications, which can easily be
adapted, more information can be revealed about gene
activity and the effects of polymorphisms on it. Addi-
tionally, there is the possibility to detect pathogens, or
pathological gene expression which prove the power of
these techniques. The knowledge provided by PCRwill
increase our insight in pathophysiological processes. a
Uterature
1 Mullis KB, Faloona F. A Specific synthesis of DNA in vitro via a
polymerase catalysed chain reaction. Methods in Enzymology 1987;
155:335.
2 Mullis KB, Faloona FA, ScharfSJ, Saiki RK, Horn GT, Erlich HA.
Specific enzymatic amplification of DNA in vitro: the polymerase
chainreaction. ColdSpringHarborSympQuantBiol1986;51:263-
73.
3 Guyer R, Koshland DE Jr. The Molecule ofthe Year. Science 1989:
246:1543-6.
4 Theije CC de, Wmdt LJ de, Doevendans PA. DNA sequencing.
Neth HeartJ2002;10:133-41.
5 Old RW, Primrose SB. Principles of Gene manipulation an intro-
duction to genetic engineering 5th edition. 1994 Blackwell Science
Ltd, Oxford.
6 McPherson M, Quinche P, Taylor G. PCR;- A practical approach.
1991 Oxford University Press, New York.
7 Newton CR, Graham A. PCR, Polymerase Chain Reaction 1994
BIOS Scientific Publishers Oxford.
8 Coen DM. The polymerase Chain Reaction in Current Protocols
in molecular biology. Wiley and Sons Inc 1994;pl5.0.1-15.6.8.
9 Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD.
Molecular biology ofthe cell 3rd edition. 1994 Garland Publishing
Inc New York.
10 Saiki RK, Gelfand DH, Stoffel S, ScharfSJ, Higuchi R, Horn GT,
et al. Primer-directed enzymatic amplification ofDNAwith a ther-
mostable DNA polymerase. Science 1988;239:487-91.
11 Boehringer Mannheim PCR Applications Manual 1995
Boehringer Mannheim GmbH, Biochemica.
12 Wilks AF. Two putative protein-tyrosine kinases identified by
application of the polymerase chain reaction. Proc Natl Acad Sci
USA. 1989;86:1603-7.
13 Kaiser P, Sonnemans D, Smith LM. Avian IFN-gamma genes: se-
quence analysis suggests probable cross-species reactivity among
galliforms. JInterferon Cytokine Res 1998;18:711-9.
14 Sonnemans DGP, Wmdt LJ de, Muinck ED de, Doevendans PA.
Methods in Molecular Cardiology: Protein Analysis. Neth Heart
J2002;10:181-8.
15 Cambien F, Poirier 0, Lecerf L, Evans A, Cambou JP, Arveiler
D, et al. Deletion polymorphism in the gene for angiotensin-con-
verting enzyme is a potent risk factor for myocardial infarction.
Nature 1992;359:588-9.
16 Rigat B, Hubert C, Corvol P, Soubrier F. PCRdetection ofthe in-
sertion/deletion polymorphism of the human angiotensin con-
verting enzyme gene (DCPI) (dipeptidyl carboxypeptidase 1).
Nucleic Acids Research 1992;20:1433.
17 Pinto YM, Gilst WH van. The ACE gene polymorphism: the good,
the bad and the ugly. Cardiovascular Research 1999;43:23-4.
Netherlands Heart Journal, Volume 10, Number 10, October 2002