ABSTRACT
A rapid competitive PCR method was de-
veloped to quantify DNA on the LightCy-
cler. It rests on the quantitative information
contained in the melting curves obtained af-
ter amplification in the presence of SYBR
Green I. Specific hybridization probes are
not required. Heterologous internal stan-
dards sharing the same primer binding sites
and having different melting temperatures to
the natural PCR products were used as com-
petitors. After a co-amplification of known
amounts of the competitor with a DNA-con-
taining sample, the target DNA can be quan-
tified from the ratio of the melting peak areas
of competitor and target products. The
method was developed using 16S rDNA frag-
ments from Streptococcus mutans and E. coli
and tested against existing PCR-based DNA
quantification procedures.
While kinetic analysis of real-time PCR is
well established for the quantification of
pure nucleic acids, competitive PCR on the
LightCycler based on an internal standard-
ization was found to represent a rapid and
sensitive alternative DNA quantification
method for analysis of complex biological
samples that may contain PCR inhibitors.
INTRODUCTION
Real-time PCR instruments allow a
detection of the produced dsDNA by
measuring the fluorescence signal pro-
duced when intercalating dyes such as
SYBR
Green I (Molecular Probes,
Eugene, OR, USA) or sequence-specif-
ic labeled probes (17). For a quantifica-
tion of initial template DNA, an exter-
nally standardized kinetic analysis
based on the fluorescence threshold cy-
cle number is established (8). However,
as any other PCR method using exter-
nal calibration, it rests essentially on
the assumption of equal amplification
efficiencies of sample and standards.
Hence, its accuracy and even applica-
bility depends critically on the purity of
the template because the presence of
PCR inhibitors that occurs frequently
in biological samples may compromise
results or even lead to wrong negatives.
This problem is solved by internal stan-
dardization using housekeeping genes
(6) or competitive PCR techniques that
are characterized by a co-amplification
of the target DNA and homologous
(1–4) or heterologous internal stan-
dards (13,15) using only one pair of
primers (9,16). A kinetic analysis of
competitive real-time PCR would re-
quire the design and optimization of
two different pairs of hybridization
probes, one pair specific for the wild-
type product and the other for the com-
petitor, which are labeled by different
fluorescent dyes, allowing a separate
analysis of one reaction at two different
wavelengths. The parallel application
of more than one pair of hybridization
probes requires a time-consuming opti-
mization of PCR conditions, and sever-
al probes often have to be tested before
reliable results are obtained. Such
methods have not yet been published.
To take advantage of the rapid am-
plification process together with the
fast and contamination-reducing, post-
PCR analysis on the LightCycler
(Roche Molecular Biochemicals,
Mannheim, Germany), in comparison
to analysis on agarose (2), temperature
gradient electrophoresis gels (5), or
HPLC (3), we developed a competitive
PCR method that uses the information
contained in the melting curves ob-
tained routinely after amplification by
following the fluorescence while slow-
ly heating up to 95°C. Although the ba-
sic concept of this approach has been
mentioned briefly already by Ririe et
al. (10), it has not yet been proved ex-
perimentally. In this paper, we show the
applicability and limitations of a quan-
tification of DNA with the LightCycler
using competitive PCR.
MATERIALS AND METHODS
Preparation of Competitors for
LightCycler PCR
A 224-bp DNA fragment of human
muscle fructose-1,6-bisphosphatase
cDNA (14) cloned in pGEM-T
(Pro-
mega, Madison, WI, USA)was used as
starting material for the heterologous
LightCycler competitors. For Strepto-
coccus mutans, the binding sites of the
forward primer Strmufo (5′-GGT-
Molecular Diagnostic Techniques
1382
BioTechniquesVol. 31, No. 6 (2001)
Research Report
Rapid Competitive PCR Using Melting Curve
Analysis for DNA Quantification
BioTechniques 31:1382-1388 (December 2001)
