Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
Citation: Kang M-J, Yu H, Kim S-K, Park S-R, Yang I (
Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
Min-Jung Kang 0
Hannah Yu 0
Sook-Kyung Kim 0
Sang-Ryoul Park 0
Inchul Yang 0
M. Thomas P. Gilbert, Natural History Museum of Denmark, Denmark
0 1 Center for Bio-Analysis, Korea Research Institute of Standards and Science , Daejon , Republic of Korea, 2 Department of Bio-Analytical Science, University of Science and Technology , Daejon , Republic of Korea
Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, 22.1%, and 213.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.
Funding: This work was supported by the Basic Research Projects of Korea Research Institute of Standards and Science, Development of Protein Measurement
Standards. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Quantification of trace amounts of DNA is of special
importance in certain analytical applications where the
concentration of a target DNA is very low or only limited amounts of
samples are available for analysis. Forensic DNA analysis,
detection and quantification of pathogenic agents, and
quantification of residual DNA impurity in foods and biopharmaceutical
products are typical examples . Due to technical difficulties
concerning quantification of trace-level DNA, special guidelines
are often suggested to minimize analytical uncertainties and
achieve a standard of best practice for the quantification of
tracelevel DNA . Certain regulatory guidelines also describe
acceptable quantities and technical requirements for analysis of
contaminating DNA in foods and drugs. The Food and Drug
Administration (FDA) guidelines suggest that the acceptable
residual amount of host cell DNA in biopharmaceutical drugs
should be below 100 pg/dose, while the acceptable limit of host
cell DNA allowed by the European Union (EU) is up to 10 ng/
Many different methods for quantification of DNA have been
developed and applied for specific uses. UV spectrophotometry
reading absorbance at 260 nm is the most common laboratory
approach for quantification of DNA. The ordinary UV
spectrophotometry is considered effective for 550 mg/mL DNA while a
significantly improved sensitivity of 1 pg/mL DNA by use of a
microliter-sample measuring device was reported . However, it
is hard to achieve such a high sensitivity in quantification of
practical samples containing only trace amounts of DNA since
they would be generally not very concentrated to the level of 1 pg/
mL. In addition, contamination of nucleotides, RNA, and proteins
will significantly interfere with the UV absorbance-based
quantification of DNA [8,9]. Fluorescence-based techniques are also
widely used for quantification of DNA. These methods show much
higher sensitivity and accuracy compared with UV
spectrophotometry for the quantification of DNA . However, the
fluorescence-based method was also subject to interferences by
contaminants, and was reported to be not effective for
quantification of DNA samples lower than 50 pg/mL [9,11].
Several other methods were developed for a specific purpose
regarding the quantification of an extremely low level DNA,
especially for quantification of residual host cell DNA in
biopharmaceuticals [3,12]. The hybridization method relies on
radio isotopic or chemiluminescent detection of DNA hybridized
to random and sequence-specific probes [13,14]. Another method
known as the threshold method utilizes antibody-mediated
detection and quantification of DNA captured by single-strand
binding protein (SSB) . Both the hybridization method and the
threshold method are capable of quantifying picogram levels of
DNA. These methods are advantageous in that they can quantify
DNA in a sequence-independent manner and are applicable to
universal DNA species. However, they also have disadvantages
involving a relatively long analysis time, labor-intensiveness, and
complicated procedures .
Another common platform for analysis of a trace amount of
DNA is PCR [17,18]. Due to the extreme sensitivity and simplicity
of experimentation, PCR technology has become the first
laboratorial choice both for qualitative and quantitative analysis
of DNA. Although sequence-specificity is an incomparable merit
of PCR technology, it also involves several important limitations
with regard to quantitative analysis of DNA. PCR will amplify and
quantify only a specific target DNA, and not the whole DNA
content. The quantity of the whole DNA content therefore cannot
be measured directly by PCR, but could only be estimated
indirectly from the quantity of a specific target DNA. The
sequence-specificity of PCR also limits the applicability of the
method only to DNA samples containing more than one
genomeequivalent amounts. The quantification limit by ordinary PCR will
then be 3 pg or above for human genomic DNA, although
quantification of femtogram amounts of DNA from viruses and
bacteria could be relatively easily achieved . New approaches
of amplifying multi-copy genes such as rDNA genes and Alu
repeats have been applied to overcome the limited sensitivity of
ordinary PCR [20,21]. Substantially improved quantification
sensitivities of 1 picogram human DNA and 300 femtogram
CHO cell DNA have been reported respectively by PCRs of Alu
repeats and rDNA genes [20,22]. However, those multi-copy PCR
methods are not universally applicable to all DNA species due to
species-specificities of the target genes. In this regard, we aimed to
develop a sensitive and universal method for quantification of
trace amounts of DNA which could enable accurate quantification
of femtogram levels of DNA independently of their species.
Results and Discussion
Optimization of real-time DOP-PCR conditions
A real-time degenerate oligonucleotide primed PCR
(DOPPCR) strategy was designed to achieve a quantitative estimation of
trace-level DNA samples. The DOP-PCR strategy enables the
whole genome amplification of a DNA sample regardless of its
origin and sequence . The DOP-PCR strategy has two
potential advantages compared with ordinary PCR methods: its
sequence independence, which enables universal applicability of
the method for amplification of any arbitrary DNA species, and a
potential sensitivity that is not limited by the requirement for one
genome-equivalent amount of DNA as a template. Then in theory,
the DOP-PCR method could successfully produce amplicons from
a sub-genomic amount of DNA even if the sequences and origins
of the target DNA are not known. Therefore, we postulated that
DOP-PCR combined with the real-time PCR format could enable
universal quantification of sub-genomic amounts of target DNA of
DOP-PCR primers were first optimized specifically for our
realtime PCR strategy. Typical DOP-PCR primers are composed of
three distinct sequence elements: an anchoring sequence at the 39
end, a random sequence in the middle, and a tag sequence at the 59
end. Among the three sequence elements, the anchoring sequence
and the random sequence were thought to directly affect the
amplification efficiency of DOP-PCR. Therefore, those regions were
the main targets for optimization of primers for the quantitative
DOP-PCR method. The most important criterion in evaluation of
quantification performances from different real-time PCR
conditions was the linearity of a standard curve which would be reflected
as a regular spacing of amplification profiles of serially diluted
standard DNA samples. Another important consideration was the
limit of quantification that could be successfully amplified and
distinguished from the no template control sample. During
optimization of primers, quantification performances of real-time
DOP-PCR experiments using different primers were evaluated by
two major criteria, linearity of the standard curve generated from
serially diluted standard DNA samples and the limit of quantification
Amplification profiles obtained from usages of various different
degenerate primers are shown in Fig. 1. Amplification profiles
from three distinct primers with different GC contents of
anchoring sequences were compared (50%, 100%, and 33% GC
contents in Fig. 1A, 1B, and 1C, respectively). The primer with a
50% GC content exhibited the best amplification profiles
compared to the others. Amplification profiles of even intervals
from 8 ng80 fg DNA standards were obtained using the 50%
GC-content primer (Fig. 1A), while poor spacing between
amplification profiles (Fig. 1B) or insufficient sensitivity (Fig. 1C)
were resulted by using the other primers. The 39 anchoring
sequence determines the frequency, sequence preference, and
strength of base pairing in priming of the primers to template
DNA. Then, the low GC-content primers will easily dissociate
from templates at the elongation temperature of PCR due to weak
base pairing. The easy dissociation of primers from templates
could result in the decreased initiation and maintenance rate of
polymerization, and subsequently lowered amplification efficiency
in PCR. Uneven intervals between amplification profiles of serially
diluted standard samples obtained from the high GC-content
primer seem to be caused by excessive primer dimerization and
subsequent nonspecific amplification, which resulted in
indiscriminating amplification profiles. Another 50% GC-content primer
with a different anchoring sequence of TGTTGC showed similar
amplification patterns to those shown in Fig. 1A (data not shown).
Therefore, it is apparent that an anchoring sequence with a 50%
GC content is the best choice to achieve consistent and sensitive
amplification profiles in the real-time DOP-PCR.
The length of the random sequence in the middle of the DOP
primer is also an important determinant of DOP-PCR efficiency
because it affects the frequency and strength of priming. It was
expected that a shorter random sequence in the primer will result
in more frequent but lesser strong priming of primers to templates
during PCR. Completely opposite results of lesser frequent but
stronger priming of primers were expected by using a longer
random sequence in the primer. Therefore, the length of the
random sequence should also be optimized. Real-time
amplification profiles using primers of different random sequences are
presented in Fig. 1 (4, 6, and 8 bases in Fig. 1D, 1A, and 1E,
respectively). Random sequence of 6 bases (N6) exhibited the best
performance showing even intervals and high sensitivity (Fig. 1A),
while uneven spacing of amplification profiles (Fig. 1D) and
insufficient sensitivity (Fig. 1E) were resulted from the use of 4
bases (N4) and 8 bases (N8) of random sequences, respectively.
Based on these results, we concluded that a primer with a 50% GC
content in the anchoring sequence and 6 bases of a random
sequence in the middle would be the best choice for performing
real-time quantitative DOP-PCR. The concentration of the
primer in DOP-PCR was also optimized. Use of a lower
concentration of the DOP primer resulted in decreased sensitivity
while a higher concentration exhibited uneven spacing of
amplification profiles (data not shown). It seems that the decreased
sensitivity by use of a low-concentrated primer had resulted from
the decreased frequency of priming due to insufficiency of primers
while disproportional amplification profiles by use of a
highconcentrated primer were caused by increased dimer formation
and subsequent nonspecific amplification during the DOP-PCR.
It should be noted that the 80-ng sample produced an
apparently different amplification profile that did not accord with
Figure 1. Optimization of primers for real-time DOP-PCR. Amplification profiles of real-time DOP-PCR were obtained by using various
degenerate primers. Serially diluted human placental DNA samples ranging from 80 fg to 80 ng and a no-template control (NTC) were amplified. The
primers used were Tag-N6-ATGTGG (A), Tag-N6-CCGCCC (B), Tag-N6-ATTTCG (C), Tag-N4-ATGTGG (D), Tag-N8-ATGTGG (E), and a combination of
TagN6-ATGTGG and Tag-N6-TGTTGC (F).
those of the other standard samples even under the optimized
DOP-PCR condition (Fig. 1A). The apparently discordant
amplification profile indicated that DNA was amplified under an
apparently different amplification kinetics in the 80-ng DNA
sample, so that the quantification strategy employed in the current
real-time DOP-PCR could not be extended to that level of DNA.
Non-negligible levels of fluorescence signals were persistently
observed in the no template control (NTC) samples. Those signals
might have resulted from an increased rate of primer dimerization
owing to random sequences in the primer and subsequent
increased non-specific amplification. It could have also resulted
from amplification of tiny amounts of contaminating DNA in the
PCR reagents, especially in the Taq polymerase. In any case, the
limit of the quantification by the optimized real-time DOP-PCR
was not further extended below 80 fg, since amplification profiles
from 80 fg or lower samples were not distinguishable from that of
NTC. It is also noteworthy that a combination of the two best
primers (50% GC contents and 6 random sequences) did not
produce distinguishably better amplification profiles than those by
single best primers (Fig. 1F). Therefore, we used only one primer
seen in Fig. 1A for the remaining real-time quantitative
Application of DOP-PCR to different species of DNA
To assure the general applicability of the method to diverse
DNA samples, DNA samples of different origins and different
complexities were tested. Amplification profiles and their relevant
calibration curves of serially diluted standard DNA samples from
human, calf, E. coli, and lambda phage are presented in Fig. 2. The
figures represent typical examples of amplification profiles and
their relevant standard curves from six independent experiments.
All four DNA species resulted in the similar patterns and exhibited
even intervals between amplification curves of serially diluted
DNA standards. All showed successful amplifications of 80-fg
templates, which were clearly distinguishable from those of the
notemplate controls (NTC). However, all amplification profiles of the
80-ng DNA samples regardless of their origins exhibited certain
distinctive patterns which did not accord with those of other
template amounts. This observation implied that the phenomenon
of disproportional amplification of the 80-ng DNA in DOP-PCR
was not caused by a sequence-dependent mechanism, but simply
by a quantity-dependent mechanism. It is supposed that too high
rates of self-annealing between denatured templates and
subsequent interferences with normal primer annealing for PCR have
led to the discordant amplification patterns in the 80-ng template
The theoretical basis for quantification of DNA by real-time
PCR resides in the assumption that amounts of amplified DNA are
proportional to the amounts of template DNA in pre-saturation
stages of amplification. Such a proportionality and repeatability of
real-time PCR would be represented by a calibration curve
calculated from a set of serially-diluted standard DNA samples.
Therefore, the validity and accuracy in quantification of DNA by
the current real-time DOP-PCR were evaluated by the calibration
curves themselves. All standard curves exhibited a good linearity
(R2 values from 0.995 to 0.999) for diverse DNA species ranging
from 80 fg to 8 ng. Data for 80 ng were omitted from the plotting
of standard curves since inclusion of those data severely impaired
the linearity of standard curves. The good linearity of standard
curves strongly supports the conceptual validity concerning
quantification of DNA by DOP-PCR for 80-fg to 8-ng DNA
samples. Furthermore, the low variability of data not only among
triplicate reactions, but also among six independent experiments,
indicated the high consistence and reproducibility of the
quantitative real-time DOP-PCR. For example, in the analysis
of HPD, the standard deviations of threshold cycle (Ct) values from
six independent experiments were 0.30, 0.10, and 0.44 for the
8ng, 80-pg, and 80-fg DNA samples, respectively (data not shown).
Those variabilities in Ct values respectively correspond to 17.9%
((100.30/4.1821)6100%, where 4.18 is the average intervals of Ct
values between 10-fold diluted DNA samples), 5.7%, and 27.4% of
variabilities in assigning DNA quantities based on the standard
curve. Then, the measurement uncertainties in quantification of
DNA by real-time DOP-PCR are expected to be of those levels.
However, the accuracy, the analytical precision from multiplicated
reactions in a single experiment, and the measurement uncertainty
from multiple independent experiments will also be influenced by
various other experimental parameters such as quality of the
template DNA, fidelity of PCR instruments, and proficiency of
experimenters. Then the practical accuracy and the practical
measurement uncertainty could be more or little variable
depending on those experimental parameters in quantification of
real samples in the fields.
The slope of a standard curve is mathematically correlated to
PCR efficiency according to the equation E = 1021/slope21, where
E is the PCR efficiency . A 100% efficiency corresponds to a
slope value of 23.32. Slopes of the real-time DOP-PCR
experiments ranged from 23.9 to 24.1 depending on the species
of template DNA. These slope values correspond to amplification
efficiencies of 70 to 80% in DOP-PCR which are lower than those
in ordinary real-time PCR. This means that about 20 to 30% of
template DNA molecules failed to serve as successful templates to
produce new DNA molecules in each PCR cycle. This might have
resulted from decreased priming efficiency of perfect match
primers to templates by competition with partially complementary
primers. Since random sequences are included in the DOP
primer, there will exist excessive amounts of partially
complementary primers, which could compete and interfere with the perfect
match primers for priming. Another possibility is self-annealing of
template DNA molecules. In contrast to ordinary PCR, all
amplicons have the same primer sequences at the both ends except
for random sequences. Therefore, the possibility of self-annealing
among denatured template molecules would be greatly elevated in
DOP-PCR, which could lead to decreased amplification efficiency.
However, in spite of the lower amplification efficiencies of
DOPPCR, standard curves of all DNA species exhibited
interexperimental variations less than 0.5 as Ct values that correspond
to variabilities about 32% in the calculated amounts of DNA from
six independent experiments (data not shown). This suggested that
the real-time DOP-PCR is mechanistically stable and consistent so
that an accurate and consistent quantification of DNA in a
sequence-independent manner could be achieved.
Determination of absolute DNA quantity
To investigate the accuracy and the measurement uncertainty of
the real-time DOP-PCR method for quantification of DNA, three
laboratory-prepared HPD samples were blind-tested. Several
other real-time PCR approaches widely used in biomedical
researches and forensic works were also performed in parallel
for comparison. They included real-time PCR approaches for the
specific single-copy target (TH01) and the primate-specific
multicopy gene, Alu (Yd6 and Yb8). Amplification profiles of three test
samples (solid lines) and 80 fg8 ng standard samples of HPD
(dotted lines) are shown in Fig. 3. Real-time PCRs for Yd6 (Fig. 3C)
and TH01 (Fig. 3D) exhibited an apparent lack of sensitivity by
failing to amplify 800-fg or lower standard DNA samples, while
the DOP-PCR (Fig. 3A) and the PCR for Yb8 (Fig. 3B) showed
successful amplification of all standard samples. These results were
Figure 2. Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from
human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng
and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of
one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.
consistent with the previously reported sensitivities of real-time
PCR analyses targeting TH01 and Yd6 [22,25]. It is interesting to
note that the sensitivity obtained by real-time PCR of Yb8 was
improved to 80 fg in our experiments, while the previous study
had reported a sensitivity of 1 pg . The conclusion of Walker et
al. was based on the observation that although amplification of
100-fg DNA was successful, the amplification profile was not
clearly distinguishable from that of the no-template control.
Therefore, it is noteworthy that a careful optimization of
previously reported PCR conditions could result in a further
improved sensitivity. The same sensitivities were observed in
amplifications of the test samples. Clear and consistent
amplification profiles of all three test samples were obtained by the
DOPPCR and the Yb8-PCR, while indiscrete or no amplification
profiles for the test sample of 400 fg (U3) were obtained by the
TH01- and Yd6-PCR approaches, respectively.
Estimated quantities of test samples by the real-time PCR
approaches were compared with gravimetric reference values
(Table 1). As expected from the amplification profiles, all four
PCR approaches resulted in satisfactory quantification
performances for the test sample of 400 pg (U1). Differences between the
measured values and the gravimetric reference values were 7.4%
(DOP-PCR), 20.2% (Yb8), 22.2% (Yd6), and 15.6% (TH01). The
analytical precisions represented by coefficients of variation (CV)
from three independent measurements were 12.6% (DOP-PCR),
1.8% (Yb8), 16.2% (Yd6) and 12.2% (TH01). These results
Figure 3. Quantification of laboratory-prepared HPD samples by different real-time PCR approaches. Three laboratory-prepared test
samples of HPD were quantified by different real-time PCR approaches: real-time DOP-PCR (A), real-time PCR for the multi-copy Alu, Yb8 (B), the
multi-copy Alu, Yd6 (C), and the single-copy TH01 (D). Test samples (solid lines) were amplified in parallel with six standard HPD samples ranging from
80 fg to 8 ng (dotted lines). Gravimetric reference values of the test samples were 403 pg (U1), 4.10 pg (U2) and 418 fg (U3).
*Values obtained from gravimetric dilutions of HPD. SD: standard deviation. ND: not determined.
indicated that all four real-time PCR approaches would be
similarly adequate for quantification of 400-pg levels of DNA.
However, completely different results were obtained for the test
samples U2 and U3. Real-time PCR of TH01 and Yd6 resulted in
inaccurate estimates or no estimates at all, while the DOP-PCR
and the Yb8-PCR exhibited stable and accurate quantification
capabilities. Specifically for example of the DOP-PCR, differences
between measured values and reference values were 22.1%, and
213.9% with analytical precisions of 15.5%, and 11.3% for the
4pg and the 400-fg samples, respectively. These values of accuracies
and precisions from the DOP-PCR-based measurements were
correlated well with the measurement uncertainties predicted by
the mathematical analyses of standard curves themselves (5.7
27.4%). Therefore, based on the results, it could be concluded that
the real-time DOP-PCR is a stable and accurate method for
quantification of DNA ranging from 80 fg to 8 ng. Besides, the
real-time PCR for Yb8 also produced very good estimations of the
4-pg and 400-fg DNA samples. Accuracies of 212.9% and 23.1%
and precisions of 6.7% and 7.6% were obtained by the Yb8-PCR
respectively for the 4-pg and 400-fg DNA samples. They were
comparably sensitive and accurate results with those by the
DOPPCR. However, it should be reminded that although the
performance of Yb8-PCR was similar with that of the current
DOP-PCR method for quantification of sub-picogram amounts of
DNA, the Yb8-PCR is of limited applicability only to human and
The overall results presented in this paper confirm that the
realtime whole genome amplification method adopting the DOP-PCR
strategy is highly stable and accurate for quantification of a wide
range of DNA samples from 80 fg to 8 ng. This effective range
covers one genome-equivalent amounts of DNA of most
mammalians. Therefore, this method would be particularly
effective for quantification of a sub-genomic amount of DNA to
which ordinary PCR approaches were not generally applicable.
Furthermore, the method is universally applicable to a variety of
DNA species, regardless of their origins and availability of the
sequence information. However, in spite of the universal
applicability of the method to various sources of DNA samples,
it should be noted that the same species of standard DNA with the
target DNA should be used to achieve a highly accurate
quantification. Uses of different species of standard DNA could
result in significantly lowered quantification accuracy. For
example, a 4-pg HPD sample had been quantified as 2.7 pg,
2.5 pg and 1.3 pg by uses of CTD, E. coli DNA and lambda DNA
as standards, respectively while an accurate estimate of 3.8 pg was
obtained by use of the same species of HPD standard(data not
Based on these results, we suggest the real-time DOP-PCR as a
universal method for sensitive and accurate quantification of
subpicogram amounts of DNA. We also believe the method have a
potential to be robustly applied in analyses of forensic DNA
samples, residual DNA impurities in foods and
biopharmaceuticals, and in molecular diagnostics.
Materials and Methods
Genomic DNAs from four different species were used as
templates for PCR analysis: human placental DNA (HPD, Sigma),
calf thymus DNA (CTD, Invitrogen), E. coli DNA (extracted from
BL21 strain), and lambda phage DNA (NEB). HPD was
fragmented by sonication to an average size of 3,000 bp and
absolutely quantified by ICP-OES . Calibration standards for
real-time PCR were prepared by gravimetric ten-fold serial
dilutions of HPD in TE buffer. Test samples for in-house blind
tests were prepared also by gravimetric dilutions of HPD.
Concentrations of CTD, E. coli DNA, and lambda phage DNA
were determined by measuring UV absorbance at 260 nm.
Absorbance of HPD was measured in parallel to obtain a
fit-forpurpose UV extinction coefficient for calculation of concentrations
of other DNA species from their measured absorbances. The final
values of DNA concentrations were then calculated by applying
the obtained fit-for-purpose UV extinction coefficient to their
measured absorbances instead of applying the simple equation of
50 mg/mL for an absorbance of 1.
All real-time PCR reactions were performed in triplicate runs.
Each reaction mixture was prepared as 50 mL and aliquotted into
three 15 mL run-replicates for PCR. Reproducibility of real-time
PCR was confirmed by three to six independent experiments
Sequence (59 to 39)
prepared and performed on different days. Real-time DOP-PCR
was performed in a 15 mL reaction volume containing 80 ng80 fg
of template DNA, 12 mM of primers ( 2 mM for single degenerate
primer PCR and 1 mM each for double degenerate primer PCR),
and the 2X SYBRH premix EX Taq mixture (Takara) using a
StepOneTM real-time PCR instrument (Applied Biosystems). Data
were collected and analyzed using the built-in StepOneTM
Software V2.1. Primers used are described in Table 2. Degenerate
oligonucleotide primed PCR (DOP-PCR) was performed
following the previously described procedures . In brief, after initial
denaturation for 10 minutes at 95uC, five low stringency cycles of
94uC for 60 seconds, 32uC for 90 seconds, ramping to 72uC over
a 3-minute period, and 72uC for 180 seconds were performed.
Next, forty high stringency cycles of 94uC for 60 seconds, 62uC for
60 seconds and 72uC for 120 seconds were performed. Reactions
were completed by final extensions for 7 minutes at 72uC
Fluorescence was read only during the high stringency cycles.
Primers for DOP-PCR were boiled for 5 minutes at 95uC right
before use to eliminate pre-formed primer dimers. Cycling
conditions for the TH01 gene or Alu element were as follows:
an initial denaturation for 3 minutes at 95uC and 40 cycles of
94uC for 30 seconds, 55uC for 30 seconds, and 72uC for
30 seconds. Primer concentrations for real-time PCR of the
TH01 and the Alu element were 0.67 mM.
Conceived and designed the experiments: IY M-JK. Performed the
experiments: M-JK HY. Analyzed the data: M-JK IY S-KK S-RP.
Contributed reagents/materials/analysis tools: M-JK HY. Wrote the
paper: M-JK IY S-KK S-RP.
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