Review
Technical Aspects of Quantitative
Competitive PCR
BioTechniques 21:268-279 (August 1996)
Klaus Zimmermann and Josef
W. Mannhalter
Immuno AG
Vienna, Austria
INTRODUCTION
The polymerase chain reaction
(PCR), first described by Saiki et al.
(79), is a highly sensitive and specific
methodology for detection of nucleic
acids and a useful tool for quantitation
of the amount of specific nucleic acids
present in a sample. The simplest ap-
proach to quantitation of PCR and re-
verse transcription PCR (RT-PCR)
products (reviewed by References 17,
21, 31 and 32) is measurement of the
amount of amplification product in the
exponential phase by reference to the
dilution series of an external standard.
However, accurate quantitation with
this type of PCR is hampered by a
number of variabilities that can occur
during sample preparation or in the
course of the reaction, and minor
varia- tions in reaction conditions are
greatly magnified during the
amplification process. These
variabilities may partly be overcome
by normalizing the amount of PCR
products of the specific template with
respect to an internal ref- erence
template such as the cellular gene -
globin (20) amplified in the same
reaction tube.
Alternatively, limiting dilution
using a nested primer methodology
(57,90) can be used in combination
with Pois- son statistics for evaluation
of the results.
The most precise quantitation of
DNA and RNA can, however, be ob-
tained by competitive PCR and com-
petitive RT-PCR (reviewed in Refer-
ences 16–18, 21, 31, 32 and 84). This
methodolo- gy, concentrating in particular on tech-
nical aspects of competitive PCR, such as the
assay is based on competitive co-
construction of competitive tem-
ampli- fication of a specific target
sequence to- gether with known
concentrations of an internal standard
in one reaction tube. The internal
standard has to share primer
recognition sites with the spe- cific
template, both specific template and
internal standard must be PCR-am-
plified with the same efficiency and it
must be possible to analyze the PCR-
amplified products of specific
template and internal standard
separately. Quan- titation is then
performed by comparing the PCR
signal of the specific template with the
PCR signals obtained with known
concentrations of the competitor (the
internal standard). Ever since this
method was first described (6,38,99),
it has been widely used for
quantitation of cellular RNA and DNA
as well as vi- ral and bacterial nucleic
acids. Exam- ples reported on include
the quantita- tion of cytokine
expression (6,38,52,91, 99,103) and of
viral nucleic acids such as hepatitis B
(46), hepatitis C (9,40,53,
58,75,78,106), human
cytomegalovirus (35), herpes simplex
virus (74) or hu- man
immunodeficiency virus type 1 (HIV-
1) (3,5,36,51,62,70,71,81,88,92)
and quantitation of bacteria, especially
of slow-growing species such as my-
cobacteria (55). Furthermore, the utili-
ty of competitive PCR or RT-PCR has
been demonstrated in the quantitation
of mitochondrial DNA (100) or
mRNA expression (29,69) and
assessment of hereditary deficiencies
(e.g., Reference
43) or leukemias (22,93).
Considering the hundreds of pub-
lished papers on the use of competitive
PCR, it is not surprising that a great
va- riety of protocols exist. In the
present article, we shall review this
plates, different PCR
strategies and modes of
detecting PCR products.
CONSTRUCTION OF
INTERNAL
STANDARDS FOR
COMPETITIVE PCR
Generation and
testing of suitable
internal standards and
the choice of primer
pairs are among the most
crucial and time-
consuming aspects of
setting up a competitive
PCR protocol. The
simplest way to choose
suitable pri- mers is to
use, as far as possible,
268 BioTechniques
primers already widely
used and/or well
described. If new
primers have to be
designed, computer
programs in combination
with sequence data bases
may be helpful tools.
General concepts of
PCR primer de- sign
have been reviewed by
Dieffen- bach et al. (26).
Since different primer
pairs for the same gene
can exhibit up to 1000-
fold differences in
sensitivity (44), special
emphasis should be
placed on testing the
specificity and efficiency
of primer pairs in
advance before inter- nal standards are
generated. Once the appropriate
primer pair has been select- ed, various
strategies for the construc- tion of
internal standards may be used.
Internal standards for competitive
PCR or RT-PCR are DNA or RNA
fragments sharing the primer recogni-
tion sequences with the specific target
yet yielding PCR products that are dis-
tinguishable from the wild-type tem-
plate. The easiest way to distinguish
between wild-type template and inter-
nal standard is by differences in the
size of the two products. This can be
achieved, for example, by constructing
standards having the same sequence as
the specific target but containing a dele-
tion or an insertion. The simplest con-
struction procedure is to use a
compos-
Vol. 21, No. 2 (1996)
ite primer containing two specific tar- and competitor
get sequences at a predetermined dis-
tance from each other (thus resulting
in a deletion) and a second primer
specific for the opposite strand
(14,33,48, 61,77). The amplification
of wild-type templates with such
primers results in a PCR product that
is shorter than the wild-type template
and can thus easily be identified (e.g.,
after electrophoretic or HPLC
separation). Alternatively, elongated
internal standards can be constructed
using a “looped oligo” method by
amplifying cDNA with a primer
containing a non-templated nu-
cleotide insertion between template se-
quences (80).
When deletions or insertions in the
center of the internal standard are de-
sired, a splice overlap extension PCR
can be used (39,72,82,92). First, two
parts of a gene or different genes are
amplified in separate PCRs. Since the
downstream primer of the first
template and the upstream primer of
the second template are designed to
contain com- plementary sequences,
the two parts of the gene or the
different genes can be linked together
by subsequently ampli- fying the two
different PCR products in one reaction
tube. In similar approach- es, internal
standards can also be con- structed by
PCR amplification of a mixture of
religated PCR products (1,37). Finally,
if the sequence of the specific template
contains one or two internal restriction
sites, these sites can be used to obtain
a construct with an in- sertion or a
deletion (3,11,70,72,104).
Differences between wild type and
standard can also be obtained by
incor- poration of restriction sites
(2,6,35,38, 59,68,88). Following PCR,
the stan- dards containing such
restriction sites are digested with the
respective restric- tion enzyme. Since
the digested prod- ucts are smaller in
size, they can easily be distinguished
from the wild-type template and can
be analyzed separ- ately.
Exchange of nucleotides in specific
templates is another way to construct
competitive internal standards (54,75,
78,106). After competitive PCR, the
wild-type fragment and competitive
in- ternal standards containing
nucleotide exchanges can be identified
following differential hybridization
with probes specific for wild-type
sequences. itive internal standards is to use a
A nonspecific spacer gene or spacer
DNA. These competi- tors have
b nucleotide sequences totally different
a from the wild type, except for wild-
s type primer sequences attached to their
i 5 ends. Such approaches have been
c used for quantitation of cyto- chrome
a P450 by using the glutathione
l transferase  gene as the spacer se-
l quence for an internal standard (96) or
y for quantitation of glycerinaldehyde 3-
phosphate dehydrogenase with an in-
d ternal standard based on the v-erb B
i oncogene (85). Nonspecific spacer se-
f quences are also frequently used to
f construct multicompetitor standards
e containing priming sites for various
r genes of interest. Such
e multicompetitor standards can be
n constructed by direct- ly linking
t together several primer sites and then
attaching them to a spacer se- quence
a (4,7,52,86,99).
p Finally, generation of competitive
p standards by amplification of multiple
r DNA fragments from cellular material
o in a PCR with low-stringency anneal-
a ing conditions has been described
c (34,94). This procedure results in a va-
h riety of different-sized PCR products,
the most suitable of which is purified
t and used as internal standard in a
o quan- titative competitive PCR assay.
The competitive templates con-
t structed in one of the ways described
h above are usually loaded on a gel,
e sepa- rated, excised and extracted.
They can then be used directly in a
c competitive PCR assay (14,48,85), but
o more often, they are cloned into
n plasmids, prefer- ably by the help of
s additionally intro- duced restriction
t sites (e.g., References 9, 61, 72, 104
r and 110).
u Cloning of competitive standards
c into plasmids usually involves enzy-
t matic reactions such as digestion with
i specific restriction enzymes,
o generation of blunt ends and ligation
n (e.g., Refer- ences 61, 70, 88 and 92).
In addition to these conventional
o strategies, other simpler procedures
f have been devel- oped. Should a
plasmid containing the wild-type
c sequence be available, this plasmid
o can be used directly as an in- ternal
m standard after introducing a dele- tion
p into the wild-type sequence as
e described by Zarlenga et al. (108).
t Fur-

Vol. 21, No. 2 (1996) BioTechniques 269

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