Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2004
Electronic Supplementary Information
An Efficient Method to Perform Milliliter-Scale PCR Utilizing Highly
Controlled Microwave Thermocycling
Kristina Orrlinga, Peter Nilssona, Mats Gullberg*b, Mats Larhed*a
a
Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala
Biomedical Centre, Uppsala University, P. O. Box 574, SE-751 23 Uppsala, Sweden.
b
The Beijer Laboratory, Department of Genetics and Pathology, Rudbeck Laboratory, SE-751
85 Uppsala, Sweden.
Microwave-assisted PCR
General: All microwave-heated thermocyclings were performed in an Optimizer EXP
provided from Personal Chemistry. Microwave-transparent borosilicate glass vials (EmrysTM
process vials) and Teflon-coated magnetic stirring bars were used in all PCR amplifications.
The instrument software allowed individual programming of time, temperature, pressure, initial
power, maximum power and cooling. The power regulation could be adjusted according to the
microwave absorption level of the reaction mixture and was set to “very high” in all PCR
experiments. Cooling was performed with compressed air of room temperature. It should be
noted that the efficiency is somewhat dependent on the temperature of the air, and shorter or
longer cooling times can come in question.
All PCR reactions were conducted using 160 nM Frw (AGACCTTGGGATACTGCACGG)
and Rew (CCATTCCACAGCCTGGCACT) primers, 320 µM dNTP, 20 ng/µL BSA, Taq
polymerase, and human genomic DNA (Boehringer Mannheim, Germany) in a buffer of 2.9
mM MgCl2, 50 mM KCl and 10 mM Tris-HCl, pH 8.3. Real-time PCR quantification of the
amount of human genomic DNA added revealed that around 10 000 copies of the target
sequence were added per reaction. The amplified sequence is a 53 bp fragment from the human
chromosome 13.
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Supplementary material (ESI) for Chemical Communications
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2.5 mL Microwave-assisted PCR:
The 2.5 mL reaction mixture and a magnetic stirring bar were added to a 2.0-5.0 mL process
vial. The vial was sealed with a septum and positioned into the microwave cavity. The
microwave instrument was programmed to heat for 45 s at 90 ºC and 175 W initial power, cool
for 50 s and finally heat for 85 s at 60 ºC with 15 W maximum power during the very first
thermocycle. The 32 subsequent thermocycles were programmed to heat for 35 s at 88 ºC, cool
for 50 s and heat at 60 ºC for 85 s with 15 W as maximum power. The resulting temperature
profiles are depicted in Figure 1. The overall process time was 1 h and 34 min. Duplicates were
prepared, amplified and analyzed (see Figures 3 and 4). One negative control with buffer
without gDNA was treated with the same procedure.
Fluoroptical temperature readings:
The fluoroptical measurement instrument was a Nortech GC68A006 provided by Fibronic
inc. The instrument was calibrated at three points – boiling pure water (100 ºC), boiling dry
tetrahydrofuran (67 ºC) and a temperature cell with a crystallizing compound (26.65 ºC). The
instrument measurements had to be corrected with the equation Tactual = 0.9757  Treading +
0.8145.
The fluoroptical probe was inserted into the 2.0-5.0 mL process vial through a drilled hole in
the security lid and a punched hole in the vial septa. The probe was introduced after the heating
started, thus there is no readings from the fluoroptical probe during the first heating cycle in
Figure 1, showing the simultaneous readings from the fluoroptical probe and the infrared
pyrometer.
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Figure 1. 2.5 mL-Scale Temperature Profiles
Using both IR-Pyrometry and a Fluoroptic Probe
100
90
80
Temp (°C)
70
60
50
40
Readings: IR-sensor
Readings: FO-probe
30
20
10
0
0
1000
2000
3000
4000
5000
6000
Time (s)
15 mL Microwave-assisted PCR:
The 15 mL reaction mixture and a magnetic stirring bar were added to a 10-20 mL process
vial. The vial was sealed with a septum and positioned into the microwave cavity. The
microwave instrument was programmed to heat for 60 s at 90 ºC and 300 W initial power, cool
for 80 s and finally heat for 85 s at 60 ºC with 15 W maximum power during the first
thermocycle. The settings for the successive second cycle were 50 s of heating at 88 ºC, 80 s of
cooling and 85 s of heating at 60 ºC with 15 W maximum power. It was shown that the cooling
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time was not sufficiently long, thus the cooling step was prolonged to 90 s in the third cycle. To
ensure good cooling and a stable temperature at 60 ºC the cooling period was extended with 5
additional seconds in the 30 subsequent thermocycles. The sample was prepared, amplified and
analyzed (see Figures 3 and 4). The resulting IR-temperature curve is presented in Figure 2.
Figure 2. 15 mL-Scale Microwave-Assisted
Thermocycling Using IR-Pyrometry
100
90
80
Temp (°C)
70
60
50
40
30
20
10
0
0
2000
4000
6000
8000
Time (s)
The overall process time was 2 h and 7 min.
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Agarose gel-electrophoresis:
Samples were withdrawn from completed microwave-assisted PCR mixtures. Together with a
G-50 purified PCR product obtained using traditional heating the samples were applied to a 4%
agarose gel containing ethidiumbromide. For reference two ladders, 50 bp and 100 bp, were
used. After electrophoresis the gel was UV-illuminated and a photograph was taken, Figure 3,
(inversed colors). The samples were as follows; Lane 1: G-50 purified PCR product. Lane 2
and 4: PCR sample from two independent 2.5 mL microwave-assisted PCRs. Lane 3: PCR
sample from a negative control reaction using microwave-assisted PCR were no genomic DNA
had been added to the PCR mixture. Lane 5: A sample from the 15 mL microwave-assisted
PCR was loaded. The difference in intensity between the products loaded in Lane 2 and 4
corresponds to a difference in amplification efficiency of 96% and 92% respectively (Table 1).
Figure 3. Analysis of Microwave Heated PCR by Ethidiumbromide
Stained 4% Agarose Gel
Capillary electrophoresis:
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For enhanced size determination and to quantify the amount of obtained PCR product all
reactions were run on an Agilent BioAnalyzer 2100 using the DNA1000 chip-kit. In Table 1
the average results from two runs are presented for each sample. The corresponding
electropherograms can be seen in Figure 4A-D. The first and last peaks in each
electropherogram are internal markers used for calibration and normalization between samples.
Graph A is from 2.5 mL microwave-assisted PCR sample 1 (Figure 3, Lane 2), B from 2.5 mL
microwave-assisted PCR without gDNA added (no PCR product-peaks detected, Figure 3,
Lane 3), C from 15 ml microwave-assisted PCR (Figure 3, Lane 5) and D is a DNA ladder used
for size determination and quantification of the samples. Based on the capillary electrophoresis
results, an estimation of amplification efficiency was calculated for each reaction (Table 1).
Table 1
Sample
Lane Size (+/-5 bp)
Amount (ng/µl) Conc. (nM) Amplification(%)
2.5 mL Microwave-PCR
2
51
0.94
27
96
2.5 mL Microwave-PCR
4
51
0.31
9
92
15 mL Microwave-PCR
5
52
1.0*
29
approaches 100
53
0.175
5
NA
G-50 purified PCR product
* A smaller band, around 43 bp, were found to be present at 0.34 ng/µl. This is most likely a
primer-dimer product, total amount of product was thus 1.34 ng/µl.
Figure 4A-D. Normalized Electropherograms From Bioanalyzer DNA
Chip
A) 2.5 mL Microwave-PCR (Lane 2)
Prov 1
1
0,8
0,6
0,4
Prov 1
0,2
0
35
45
55
65
75
85
95
105
6
-0,2
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B) No Template 2.5 mL Microwave-PCR (Lane 3)
Prov 2
1
0,8
0,6
0,4
Prov 2
0,2
0
35
45
55
65
75
85
95
105
-0,2
C) 15 mL Microwave-PCR (Lane 5)
Prov 4
1
0,8
0,6
0,4
Prov 4
0,2
0
35
45
55
65
75
85
95
105
-0,2
D) Reference DNA Ladder for Quantification and Size-Determination
Ladder
1
0,8
0,6
0,4
Ladder
0,2
0
35
45
55
65
75
85
95
105
7
-0,2
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2004
Real-time PCR with traditional heating:
Samples were diluted a hundred thousand times in pure water and 2.5 µL of the diluted
sample was added to 22.5 µL PCR mixture containing the same concentration of reagents as in
the microwave experiment but with the addition of SYBR Green (Molecular Probes). The
thermocycling program used were 95 °C for 15 seconds followed by 60 °C for 60 seconds for
40 cycles followed by a melting curve. The latter to ensure that correct product had been
formed. A dilution series of purified PCR product was utilized to construct a standard curve
which was employed for quantification of product from the microwave-assisted PCR (Figure
5). Presented in Table 2 are the averages of three independent real-time PCR reactions for each
sample (ABI 7000). Using the formula obtained from the standard curve the amount of
molecules put into the reaction was calculated. Knowing that the initial amount of starting
material was 10000 copies, the amount of amplification after 33 cycles were also calculated.
Based on formula [1], the amplification efficiencies of the different reactions were calculated.
No account has been taken to the possibility that the reactions have reached plateau-phase
before 33 cycles. If so, the real amplification-efficiency during exponential-phase will be
higher than the number we report here. In order to determine this, however, analysis of amount
product after different number of microwave-assisted PCR cycles has to be performed. The
results obtained using real-time PCR analysis correlates well, albeit slightly lower yields are
detected than when using direct detection with capillary electrophoresis.
[1]
Yield = Starting amount  (1 + amplification efficiency)number of cycles
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Supplementary material (ESI) for Chemical Communications
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Figure 5. Real-Time PCR of a Dilution Series of Purified PCR Product
.
Table 2
Sample
Lane Ct
Molecules
Amplification
Efficiency
2.5 mL Microwave- PCR 2
19.65  0.46
220712
2.21109
92%
2.5 mL Microwave- PCR 4
20.96  0.34
84187
8.42108
86%
15 mL Microwave- PCR
18.35  0.31
5
570226
5.710
9
98%
9