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Articles |
1
Institut für Biochemie, FB 08, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
a Author for correspondence. Fax 49-641-9935409; e-mail Meinhard.U.Hahn{at}chemie.bio.uni-giessen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used the LightCyclerTM real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection.
Results: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior).
Conclusions: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon’s genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The LightCyclerTM, one of the commercially available real-time PCR systems, is a rapid thermocycler with online fluorescence detection, in which the PCR is carried out in glass capillaries. Heating and cooling are by temperature-controlled airflow (8)(12). The capillaries are placed in a rotation-symmetric chamber to ensure homogeneous temperature distribution. For quantification, unknown samples are amplified in parallel with external calibrators. For all reactions, the threshold cycle (CT) values are determined according to the algorithm described by Higuchi et al. (1). The CT is the fractional cycle number at which the fluorescence signal reaches an arbitrary but defined threshold value within the early exponential phase of the reaction. CT values are proportional to the logarithm of the initial copy numbers of the target, which are used to determine the initial copy numbers of unknown samples. The accuracy is critically dependent on the amplification efficiency, which must be the same for the DNA calibrator and the unknown sample DNA.
We performed LightCycler PCR studies on human tumor DNA samples for the precise quantification of allelic imbalances of the human tumor suppressor genes p53 and p16, as well as the oncogene ErbB-2. We found that the p16 and ErbB-2 target sequences were amplified with unexpected variations. Although the standard deviations for fivefold measurements per sample were very small (<10%), the mean values of some samples were obviously inaccurate by a factor of 2 or 3, apparently depending on the position of the sample-containing capillaries within the sample carousel. Here we describe our systematic analysis of this phenomenon. Our results suggest that the denaturation of the sample DNA during the early phase of PCR is critical for some targets and, therefore, likely to be influenced by minute temperature differences in the thermostated reaction chamber.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For restriction, 1 µg of genomic DNA was incubated for 3 h at 37 °C with 5 U of HaeIII (USB) in a total volume of 50 µL in the buffer recommended by the supplier (3.3 mmol/L Tris-acetate, pH 7.9, 1 mmol/L magnesium acetate, 6.6 mmol/L potassium acetate, 0.01 g/L bovine serum albumin).
pcr
Primers were obtained from MWG, and hybridization probes
(12) were from TIB MOLBIOL. The lengths of the amplified
sequences were 125 bp (p53 and p16) and 117 bp
(ErbB-2), respectively. All oligonucleotide sequences (Table 1
) were checked with the program Oligo 5.0 (National Biosciences)
for absence of false priming sites, formation of primer dimers,
primer/probe hybrids, and secondary structures.
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The LightCycler system from Roche Diagnostics GmbH was used for
amplification and data collection. The sample carousel of the
instrument has a capacity of 32 capillaries. All reactions were carried
out in a total volume of 10 µL per capillary. Each reaction mixture
contained 0.5 g/L bovine serum albumin; 6 mmol/L
Mg2+; 0.5 µmol/L each primer; either 0.2
µmol/L each probe or a 1:30 000 dilution of SYBR Green; 0.2 mmol/L
dATP, dCTP, dGTP, and dTTP; 0.5 U of Taq DNA polymerase; and
2000 copies of the DNA target in 1x Taq PCR buffer (10
mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.3 at 20 °C). The reaction
composition was optimized for a high signal-to-noise ratio. All
biochemicals were obtained from Roche Diagnostics. A total reaction
mixture for 32 reactions was made at 4 °C and distributed into the
precooled capillaries.
The standard amplification protocol consists of an initial denaturation step at 95 °C for 30 s, followed by 45 amplification cycles at 95 °C for 0 s, 55 °C for 5 s, and 72 °C for 10 s (temperature ramp was constant at 20 °C/s). For some experiments, genomic DNA was incubated in a boiling water bath for 10 min before the PCR reaction mixture was prepared. For hybridization probes, measurements were taken at the end of the annealing phase at 55 °C; for SYBR Green, measurements were taken at the end of the extension phase at 72 °C. After PCR, each amplification reaction was checked for the presence of nonspecific products by native polyacrylamide gel electrophoresis.
data evaluation
The raw data were evaluated with the LightCycler software, Ver. 3.
For experiments with hybridization probes, the CT
values were calculated from the fluorescence signal ratio of the
acceptor and donor fluorophores (channel 2/channel 1), using the
threshold method (8). For SYBR Green,
CT values were calculated using the data of
channel 1.
The melting behavior of the genomic template was calculated for DNA
segments of 1 kb (± 500 bp around the amplicons), using the
computer program Melt94 (13).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). With some targets, however, a broad range of scattering of
the CT values was observed (Fig. 1B
). In our
studies, we found two such susceptible targets (p16 and
ErbB-2), whereas other targets tested (p53,
HBB, IGF-1, GAPDH, and
PBGD) did not show this effect. The CT
values depended on the position of the capillary in the carousel and
showed a sinusoidal distribution when plotted vs the capillary position
(Fig. 1
, C and D). This phenomenon was observed on three of seven
LightCycler instruments tested (all of them checked to be within the
technical specifications) and was independent of the source of the
template DNA (isolated from different individuals), the isolation
protocol, and the amount of target DNA (within
103–104 haploid genome
equivalents) used (data not shown).
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Signal generation with SYBR Green yielded a sinusoidal scattering pattern of CT values that was the same as that obtained with hybridization probes (data not shown), demonstrating that problems encountered with susceptible targets are independent of the detection format, including the temperature segment for fluorescence detection (at 55 °C after the annealing phase and at 72 °C after the extension phase). The sinusoidal scattering of the CT values was also observed for different primer pairs, all hybridizing in the vicinity of the target sequence ErbB-2, indicating that the effect was not attributable to special properties of the primers (data not shown).
Because the sequential measurement of 32 samples takes 6 additional
seconds, it could be that the systematic distribution of
CT values is a function of the time interval
spent at the measuring segment. The same distribution of
CT values was obtained for different annealing
times (5, 10, and 20 s at 55 °C) and annealing temperatures
(50, 55, and 60 °C for 5 s) for hybridization probes and
different elongation times (10 and 20 s) for SYBR Green (data not
shown). In addition, variation of the denaturation time (0, 5, and
10 s) had no influence on the results (data not shown), indicating
that the reaction mixture is heated to the specified temperature even
without holding it.
In contrast, with increasing denaturation temperature the amplitude of
the scattering decreases, but the temperature cannot be chosen
sufficiently high to suppress the scattering completely because
temperatures >96 °C produce noisy amplification curves with a
shorter exponential phase, which are not suitable for accurate
quantitative evaluation (data not shown). This temperature dependency
indicates that the denaturation step is a possible source of the
scattering of the CT values. By melting curve
analysis with the LightCycler we could show that the p16-,
ErbB-2-, and p53-specific PCR products melt at
86 °C, far below the applied denaturation temperature. Therefore,
we checked whether the melting behavior of the genomic DNA template
caused these effects. We used purified p16-specific PCR
product as well as HaeIII-digested genomic DNA as template.
The restriction enzyme HaeIII has recognition sites near the
target sequence, producing a 298-bp restriction fragment containing the
p16 amplicon. With both restricted templates, the
CT values showed no scattering (Fig. 1D
). This
was also demonstrated for ErbB-2 (data not shown).
Incomplete denaturation of the genomic DNA template in the first few
cycles, in which the amplification of the genomic target contributes
considerably to the total amplification, can decrease the amplification
efficiency in the first cycles and eventually shift the
CT values toward higher cycle numbers. Therefore,
enhancement of the denaturation temperature in the first few cycles
should shift the CT values of susceptible, but
not those of nonsusceptible, targets toward lower cycle numbers. Using
a temperature profile with various denaturation temperatures in the
first five PCR cycles only and a constant denaturation temperature of
95 °C in all consecutive cycles, we could show this effect for the
susceptible target p16 but not for the nonsusceptible target
p53 (Fig. 2
), demonstrating that melting of the genomic template at the
beginning of the PCR is critical for accurate quantification. We have
not systematically explored how many cycles at increased temperature
are required to get an optimum result; this should be done when working
out a protocol for a given template/primer system.
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The position-dependent scattering of CT values for the susceptible target p16 was also completely eliminated when the DNA sample was boiled for 10 min before the PCR reagents were mixed and the target was amplified. When a denaturation temperature of 95 °C was used during all PCR cycles, the samples without the preboiling step showed a sinusoidal scattering of the results, leading to a variation of the CT between 23.0 and 24.5 cycles (SD = 0.5 cycles), whereas the CT values of the preboiled samples were independent of the positions (20.5 ± 0.1 cycles). When a denaturation temperature of 98 °C was used for the first five cycles (all following cycles, 95 °C), the samples that had not been boiled showed the same results as the boiled ones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); in
contrast, p53 and the reference genes tested
(IGF-1, HBB, PBGD, and
GAPDH) did not show this scattering. This effect was
observed on three of seven instruments tested. On those LightCyclers
showing position-dependent scattering of the CT
values, the effect was highly reproducible and was independent of
variations in annealing times and temperatures as well as denaturation
times, whereas enhancement of the denaturation temperature decreased
the scattering of the CT values (Fig. 1C
). The
observed sinusoidal CT pattern and the influence
of the denaturation temperature on the CT
scattering of identical samples led us to the conclusion that
presumably very minor temperature differences exist within the rotation
symmetric thermal chamber of the instrument.
We hypothesized that genomic regions comprising the target sequences of
the susceptible genes do not melt completely at 95 °C and,
therefore, are amplified with a lower efficiency in the first PCR
cycles. In that case, very small temperature differences within the
reaction chamber can lead to the observed scattering of
CT values. This hypothesis is supported by the
results of experiments using restricted genomic DNA as well as PCR
product as template, which in contrast to the untreated genomic DNA
gave accurate results without any systematic scatter. In addition,
inspection of the susceptible target gene sequences (p16 and
ErbB-2) with the software program MELT94 (13)
showed a region 100 bp away from the amplicon with a calculated
melting temperature of 10 to 15 °C higher than that of
the surrounding sequence. The sequences surrounding the other amplicons
tested, 500 bp upstream as well as downstream, do not have such regions
with higher melting temperatures. Presumably, these regions with
increased melting temperatures, located in the vicinity of the
p16 and Erb-B2 amplicons, are likely to make
these targets susceptible to inaccurate quantification. If this is the
case, then alteration of the reaction conditions that lower the melting
temperature of the DNA during PCR should ameliorate the results, in
particular the initial denaturation temperature (e.g., initial boiling
of template DNA for complete melting) as well as reductions in the
ionic strength (no KCl instead of 50 mmol/L KCl) and
Mg2+ concentrations (2–4 mmol/L
MgCl2 compared with the 6 mmol/L
MgCl2 used here)
(7)(19)(20), or addition of organic
solvents (e.g., dimethyl sulfoxide, formamide, glycerol, and betaine).
Increasing the initial denaturation temperature, of course, is the
reaction condition most easily to change.
In one series of experiments, we tested the classical way to achieve complete denaturation of the genomic template DNA (19)(20): boiling of the genomic DNA samples before PCR. This is effective because after melting of complex (e.g., human) genomic DNA, the complementary strands will not rehybridize except at highly repetitive sequences, and even there incorrectly. Indeed, when we used this boiled template DNA, the systematic scattering of CT values was completely eliminated. It is an advantage of this procedure that the polymerase and other reagents do not suffer high thermal stress, but a disadvantage that rare clinical samples, often available only in small amounts, have to be handled in an additional manual step, which has the risk of contamination of the samples by amplicons.
In an additional series of experiments with DNA that had not been boiled, the denaturation temperature was increased (>96 °C) during all PCR cycles. The amplitude of the scattering decreased, but the resulting amplification curves were noisy and not suitable for accurate quantitative evaluation. This is probably attributable to the thermal stress and denaturing of the DNA polymerase and other reaction components (e.g., dNTPs) or damage to the fluorophores.
To avoid both the additional boiling step and excessive thermal stress
during PCR, we used native DNA samples and limited the
increased denaturation temperatures to the first five cycles of PCR.
This should be sufficient to improve the melting of the genomic target
in those early cycles, where the genomic DNA contributes significantly
to the total amount of amplifiable DNA. Indeed, this modified
temperature profile yielded good signal profiles and a decrease of the
CT values for both susceptible targets,
p16 (Fig. 2
) and ErbB-2 (data not shown), the
same effect that was observed when boiled template DNA was used. To
exclude systematic effects of temperature differences, we used
different LightCycler instruments that gave a homogeneous,
non-sinusoidal CT pattern. The experiment in Fig. 2
shows that minor temperature effects can be resolved sensitively with
such LightCycler instruments. On the basis of the amplitude of the
sinusoidal pattern (1.5 cycles at a denaturing temperature of
95 °C; see Fig. 1C
) and the slope of the plot
CT vs denaturing temperature
(Tden; 0.5 cycles/ °C; see Fig. 2
) we estimated the range of temperature differences in the sample
carousel during denaturation to be approximately ± 1.5 °C,
which is in reasonable agreement with the technical specification of
the LightCycler system (± 1.0 °C capillary temperature precision
over 30 s and over all capillary positions at 95 °C) given in
the LightCycler Operator’s Manual, Ver. 1.2.
Therefore, even with PCR instruments working within their technical
specifications, quantification of some targets can yield less precise
results when the conditions are not optimized to achieve complete
melting of the target DNA. The reliability of the results cannot be
derived by the error or correlation coefficient of the calibration
curve: if, in our case, the capillaries containing the calibrators are
placed at the first positions of the sample carousel, the resulting
calibration curves with up to 10 values will have small errors, e.g.,
with mean CVs <10% (Fig. 1
, C and D), whereas the
CT values of subsequently placed samples will be
less precise because of the position-dependent effect. We suppose that
this phenomenon is general and likely to be observed with other
real-time PCR cyclers, especially for parallel sample processing that
depends on an absolutely homogeneous temperature distribution over all
sample positions. Indeed, there is circumstantial evidence from the
literature that similar problems had been encountered before, although
not explicitly discussed. In the report by Raggi et al.
(18), the quantification of MYCN using TaqMan
probes on an ABI Prism 7700 machine was compared with a conventional
competitive quantitative PCR that is known to be a very accurate
technique. In Fig. 3 of that report (18), two relatively
distinct groups of samples with relative MYCN frequencies of
1 and 4, quantified by competitive PCR, are shown. The same samples,
measured with the ABI Prism 7700, vary within a factor of 4 (from
0.5 to 2 and from 2 to 8, respectively), although the calibration
curves have coefficients of correlation >0.997.
The difficulties described above may also occur if a thermocycler with position-dependent temperature differences is used for quantitative competitive PCR of a susceptible genomic target in competition with an internal standard lacking the genomic flanking sequences of the amplicon containing higher melting domains. Therefore, we conclude that the source of the inaccuracy described here has to be considered in general for accurate quantification of copy numbers when quantitative PCR techniques are used.
Whereas a constant denaturation temperature during all cycles usually is chosen (14)(15)(16)(17)(18), we recommend boiling the template DNA before PCR as described in the literature (19)(20) or use of a high denaturation temperature (>97 °C) during the first few cycles to avoid imprecise quantification as a result of the effects described here. But even with such a modified PCR temperature profile, we recommend testing the system for position-dependent effects before quantitative measurements. Furthermore, it should be possible to eliminate these minor temperature anomalies in the LightCycler with a permanently rotating sample carousel (in addition to rotating the air with a fan) because within a symmetrically formed chamber the temperature need not be the same at every position in the chamber, for example, because of differences in the heat capacities of the walls.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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