In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements

J. Biochem. Biophys. Methods 65 (2005) 97–105

www.elsevier.com/locate/jbbm

In situ monitoring of polymerase extension rate and

adaptive feedback control of PCR by using

fluorescence measurements

Sudip Mondal*, V. Venkataraman

Department of Physics, Indian Institute of Science, Bangalore 560012, India

Received 5 August 2005; received in revised form 29 September 2005; accepted 12 October 2005

Abstract

Real time PCR detection systems based on fluorescence detection from intercalating dyes (such as

SYBRR Green I) typically take only single point measurements during every cycle to quantify the

amplification. In this process key information about enzymatic kinetics is lost. In this work we measure

SYBR Green I fluorescence intensity every 0.5 s within a cycle during PCR in polypropylene tubes.

We observe that the intensity during the extension cycle increases while the template is being extended.

Results obtained for different lengths are used to estimate an in vitro polymerase activity rate of

Thermus aquaticus and Thermus brockianus. An important practical consequence of this result is that

the extension time of each PCR cycle can be individually optimized while the reaction is in progress.

We demonstrate this idea of adaptive feedback control and show that the total number of cycles and

total time required to reach maximum fluorescence is reduced as compared to conventional PCR.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Real-time PCR; SYBR Green I; Intra-cycle fluorescence; In situ extension rate; Adaptive feedback control

1. Introduction

Real time polymerase chain reaction (PCR) has undergone an immense development

through the last decade after it was first suggested and demonstrated by Higuchi et al. using

ethidium bromide [1,2]. Conventional PCR followed by gel electrophoresis is replaced

by real time PCR mainly because it is: (i) easy to automate and incorporate in a device,

0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jbbm.2005.10.002

* Corresponding author. Tel.: +91 80 2293 2316; fax: +91 80 2360 2602.

E-mail address: [email protected] (S. Mondal).

S. Mondal, V. Venkataraman / J. Biochem. Biophys. Methods 65 (2005) 97–10598

(ii) makes the whole process faster, and (iii) reduces the number of steps of handling and

hence minimizes chances of contamination. Fluorescent probes used to monitor in vitro

DNA amplification are (i) double stranded-DNA (ds-DNA) specific dyes and (ii) DNA

sequence specific oligonucleotide probes. The most commonly used ds-DNA intercalating

dye is SYBRR Green I (SG) in both research and commercial RT-PCR protocols. This is

because it is relatively inexpensive and selective to ds-DNA compared to single stranded

DNA (ss-DNA) by ~11 fold [3]. SG fluorescence sensitivity is limited by binding to

non-specific products, which can be overcome by melting curve analysis. For example in

the case of amplifying multiple DNA fragments in multiplex PCR [4,5], the final

products can be distinguished by their characteristic melting temperatures. Similarly

complex DNA melting profiles with A/T- and G/C-rich clusters give rise to multiple

transition peaks [6,7]. SG fluorescence has been used to monitor DNA amplification

during PCR in plastic reaction tubes [8,9], glass capillary tubes [10–15], miniaturized

devices [16,17] and hand held diagnostic devices [18].

Since DNA polymerase plays an important role in PCR, attempts have been made to

understand every reaction mechanism such as polymerase binding, effect of buffer

components, extension along the template and release of polymerase from the fully extended

template. DNA polymerization catalyzed by Klenow Fragment of DNA polymerase I (KF)

and Thermus aquaticus (Taq) DNA polymerase has been examined by gel electrophoresis of

rapidly quenched products [19–22], quartz microbalance [23,24] and time resolved

fluorescence spectroscopy [25–27]. These techniques have some drawbacks: for example,

rapid quench technique requires isotope labeling, quartz microbalance requires immobiliza-

tion of the template and fluorescence technique requires fluorophore labeling. Hence they

have not been used to monitor polymerase activity during PCR. In this paper we show that

real time continuous fluorescence data obtained during the extension phase within a PCR

cycle can be used to directly infer polymerase extension rates. Though attempts have been

made previously to collect complete fluorescence spectra in real time [1,10,15,28,29], very

little was concluded from those measurements. In present work we demonstrate how the

intra-cycle fluorescence can be used to adapt the cycling parameters to optimize extension

stay time. This can reduce the number of cycles required to reach the maximum fluorescence

by considerable amount.

2. Materials and methods

2.1. DNA samples and primers

DNA amplification was performed using DyNAmok SYBRR Green qPCR kit (Finzymes, Espoo,

Finland) containing modified Thermus brockianus (Tbr) DNA polymerase, SYBR Green I fluorescent dye,

optimized PCR buffer, 5 mM MgCl2, dNTP mix including dUTP. A 20-AL amplification mixture was

prepared using 10 AL of 2�qPCR kit, 1 AL of template solution, 0.2 AM of both upstream and downstream

primers. In order to test the effect of another polymerase without isolating the Tbr from the mix, 2 U of Taq

polymerase, supplied in 20 mM Tris–HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5%

TweenR-20, 0.5% Igepal and 50% Glycerol (Bangalore Genei, Bangalore, India) was added wherever

mentioned. We used Prostatin C1 (PC1, 346 bp) and TGFh1 (999 bp) cDNAs cloned in the plasmid

BlueScript (Stratagene, USA) as template at concentration of 1 ng/AL for PCR. The above templates were

selectively amplified using the same primer set T3 (5V-ATTAACCCTCACTAAAGGGA-3V) and T7 (5V-TAATACGACTCACTATAGGG-3V) to produce 502 bp and 1155 bp size, respectively. Genomic DNA

purified from S. marcescens (Bangalore Genei) at concentration 80 ng/AL, amplified using forward (5V-

S. Mondal, V. Venkataraman / J. Biochem. Biophys. Methods 65 (2005) 97–105 99

AGCAGGGATGACCAACTC-3V) and reverse (5V-GCGGATCCTCTTGCCAAAGAGAGAAT-3V) primers

produced 800 bp products.

2.2. Amplification in commercial thermocycler

Amplification was carried out for volumes ranging from 1 to 25 AL of PCR mixture in low profile tube

using PTC-150 MiniCycler (MJ Research, Waltham, Massachusetts, USA). Apart from a change in the

fluorescence intensity, the results obtained were independent of the reaction volume. Here we report the

results only for 3 AL. The tubes were used without caps and the hot bonnet to collect maximum

fluorescence from the top. The mixture was overlaid using 6 AL of mineral oil (M-5904, Sigma, St. Louis,

MO, USA) to avoid evaporation at high temperatures. The sample was initially incubated at 95 8C for 1 min

followed by 35 cycles of thermal cycling (denatured at 95 8C for 15 s, annealed at 50 8C for 15 s, extended

at 72 8C for 1 min) and a final extension step at 72 8C for 5 min. During the optimization experiment of

extension time, the stay time was fixed at 40 s, 1, 2, or 4 min, or varied manually according to the

fluorescence as mentioned in the text. In order to record the thermal profile a 2�2.3 mm 100 V Platinum

resistor was used as a temperature sensor. The sensor was dipped inside an adjacent tube containing 10 ALof mineral oil.

2.3. Fluorescence detection system

An optical detection system for fluorescence measurement was made for SG dye. SG emits fluorescence

at 520 nm when excited at 470 nm. The fluorescence setup was mounted on a stage with vertical movement

to focus on the sample. It consists of a high power blue light emitting diode (LED) (Roithner Lasertechnik,

Vienna, Austria) mounted on a heat sink attached to a cooling fan. The LED was driven by a LED current

driver (Roithner Lasertechnik). The light was focused using an aspheric glass lens (Roithner Lasertechnik)

and filtered by excitation band-pass filter of 450–490 nm (Carl Zeiss, Germany). The filtered blue light was

reflected and guided perpendicularly to the reaction chamber by a dichroic beam-splitter (Carl Zeiss) and

focused using a 10� objective (Carl Zeiss). Emitted fluorescence was collected by the same objective,

guided vertically through the beam-splitter (Carl Zeiss), filtered using green emission filter (Carl Zeiss) and

focused on to a silicon photo detector by a lens. The photo current was recorded using a SR830 Lock-In

Amplifier (Stanford Research Systems, Sunnyvale, CA, USA). Lock-In detection technique was used since

it is very sensitive and removes background noise. The amplifier was locked on to an internal frequency of

190 Hz that was used to modulate the LED driver. The resistance of the platinum temperature sensor inside

the dummy tube with mineral oil was recorded using Keithley 196 system DMM, and converted to

temperature later. All data was recorded and stored in a PC for further processing.

3. Results

3.1. Continuous intra-cycle fluorescence monitoring

We start with amplification of PC1 DNA in polypropylene tubes using a commercial

thermocycler. In Fig. 1a we show a typical curve of detector current, which is proportional to

fluorescence intensity, as a function of cycle number for three samples (one positive and two

negative controls). The data is recorded after the extension step in every cycle. All three samples

were loaded in 1% agarose gel. The results agree with the fluorescence measurements, i.e., only

the lane corresponding to the positive control gives a bright band in the gel.

Instead of single point fluorescence in every cycle the detector signal is recorded

continuously with a delay of 0.5 s. Time evolution of fluorescence for PC1 DNA for 35 cycles

is shown in Fig. 1b. The fluorescence trace as a function of time follows the temperature profile,

as observed by earlier workers [1,10]. The amplification can be clearly observed as an increase

Fig. 1. Continuous intra-cycle fluorescence monitoring. (a) Single point fluorescence data of DNA amplification carried

out in polypropylene tubes without template (square), with non-specific template (circle) and a positive sample (triangle).

(b) Continuous fluorescence of SYBR Green I monitored every 0.5 s during PC1 amplification.


in the overall fluorescence envelope after the 25th cycle. In negative control (without template or

with non-specific DNA) fluorescence follows the thermal profile but the overall envelope does

not change significantly, indicating that there is almost no formation of ds-DNA over PCR

cycles. The fluorescence at the denaturation temperature for all the cycles remains almost

constant since no ds-DNA is present for the SG dye to bind. This fluorescence data is used to

monitor the background drift. Upon excessive cycling of PCR the fluorescence envelope starts

decreasing with every cycle corresponding to smear developed in the gel photograph [30].

Single point fluorescence data can be obtained from a continuous data by picking up the

fluorescence value in every cycle at an appropriate temperature, which will generate a plot

similar to Fig. 1a.

3.2. In situ monitoring of polymerase activity

We now examine the data of Fig. 1b more closely. Fig. 2 compares the extension steps of few

cycles in the beginning of the reaction with that in the exponential phase, when the fluorescence

envelope starts rising. During the extension steps of the early ground phase, where no

amplification happens, fluorescence remains constant with time as long as the temperature is

held constant (Fig. 2b). At the beginning of the exponential phase, the fluorescence at the

extension step shows an increase for few seconds and saturates until the temperature is changed

to the next step (Fig. 2d). It should be noted that in both phases, the temperature profile remains

the same (Fig. 2a and c). The fluorescence increase in the extension step is observed to be a

maximum in the exponential phase and the trend agrees well with the overall envelope. In the

negative control no such feature is observed in the extension step throughout the cycling. Since

the increase in fluorescence is observed during the extension step, it can depend on kind of

polymerase, dNTPs concentration, molar ratio of polymerase to primed site, the length to be

extended, PCR buffer etc. Since the concentration of the reaction components change with every

Fig. 2. Comparison of fluorescence data for initial and intermediate cycles. The fluorescence for 16th, 17th and 18th

cycles during the ground phase (b) and 25th, 26th and 27th during exponential phase (d) are shown as a function of time

with corresponding thermal profiles (a and c).


cycle during amplification, they are difficult to control. So we have chosen to study the

dependence of this feature on the nature of polymerase and the length of the template.

Fig. 3 shows increasing fluorescence features in extension step during three typical PCR

cycles with two different template sizes, PC1 (Fig. 3a) and TGFh1 (Fig. 3b). Extension

temperature is maintained at 72 8C, optimum for the polymerase activity as specified by the

supplier, for both of them. The horizontal axes are offset to start both figures from zero at an

arbitrary point in a cycle. The polymerase used for both of these is modified Tbr DNA

polymerase. Fig. 3c and d compares the same two templates using a different kind of

Fig. 3. Comparison of extension steps for two different template lengths PC1 (a) and TGFh1 (b). The reactions were

carried out using Thermus brockianus polymerase. Comparison of extension steps for same two template lengths PC1 (c

and TGFh1 (d) carried out using extra Thermus aquaticus polymerase added to Thermus brockianus master mix.

)

Table 1

Polymerase extension rate using SYBR Green I fluorescence

Polymerase DNA Length (bp) Average time (s) Rate (npsa)

Thermus brockianus PC1 502 13.1 36.8

TGFh1 1155 30.9 36.7

PC1 502 9.7 49.7

Thermus aquaticus S. Mar. 800 15.6 49.9

TGFh1 1155 19.1 59.4

a nps=nucleotide per second.


polymerase, Taq that has higher polymerase activity, added in excess to the mix containing Tbr.

Similar data was obtained from amplification of S. marcescens using PCR mix with extra Taq.

The ratio of the template size to the rise time is a measure of the polymerase activity rate. The

values are summarized in Table 1 for the two kinds of polymerase.

3.3. Adaptive control and optimization of extension time

By monitoring the fluorescence rise time, we observe that the total time required to extend

the template molecules is not the same for all PCR cycles. In order to optimize the reaction

time we manually terminate the extension step immediately after the fluorescence saturates.

We compare this technique of variable cycle time with the standard fixed time PCR of

TGFh1 using Tbr. In Fig. 4, normalized fluorescence is plotted against cycle number for the

variable time PCR experiment as well as standard PCR with 40 s, 1, 2 and 4 min fixed

extension times. The difference between the maximum and minimum fluorescence in a cycle

(at annealing and denaturation temperature, respectively) is defined as D. The minimum

value of D (DMIN) occurs during the starting cycle while the maximum (DMAX) is reached

when the fluorescence envelope saturates. For the standard fixed time PCR experiment, the

number of cycles to reach the maximum is less if the extension time is longer. However the

total reaction time also increases. In the case of variable time PCR both number of cycles

and total reaction time is minimized. All amplified products were loaded in 1% agarose gel

Fig. 4. Optimization of reaction time during PCR of TGFh1 cDNA. Amplification was carried out with extension times

of 40 s (square), 1 min (circle), 2 min (triangle), 4 min (inverted triangle) and variable time (diamond) for 50, 40, 31, 27

and 30 cycles, respectively. During variable time PCR the extension time was kept at least 40 s and forced to proceed to

next step when the fluorescence saturated.

Table 2

Optimization of extension time using intra-cycle fluorescence data

Extension time Total time (s) Total cycle

40 s 6640 50

1 min 6176 40

2 min 6747 31

4 min 9170 27

Variable 5670 30


to give same band intensity. Table 2 compares the relevant parameters for these two kinds of

experiments.

4. Discussion

Herein we demonstrate a fast real time fluorescence setup suitable for SG fluorescence. The

setup, unlike conventional PCR fluorescence machines, monitors fluorescence continuously

throughout the PCR cycles. Single point fluorescence eliminates the contribution from non-

specific products by taking data at a single high temperature and at the same time removes the

thermal effect of SG fluorescence. But in doing so it loses important and easily available

information regarding the DNA polymerase, namely the activity rate. During extension, the

primer annealed to the ss-DNA template is extended by the polymerase. The number of base

pairs increases with time and finally forms the ds-DNA product. We expect that the number of

SYBR Green I dye molecules intercalated between the base pairs also increase with time. This

should lead to an increasing fluorescence, which saturates after the product is formed. In an

earlier report [29], Ederhof et al. observed a similar fluorescence rise during extension phase and

attributed it to the same mechanism. However they did not see the saturation and therefore could

not conclude anything quantitative regarding the polymerase activity. This mechanism is

consistent with the fact that the time interval during which the rising fluorescence is observed

increases with template size and decreases with the polymerase activity. The fluorescence in

some of the cycles after the increase shows a dip that may be due to background drift. Assuming

the number of polymerase molecules to be higher compared to template number, which is true at

least at the onset of the exponential phase, the time required for the extension can be used to

extract polymerase activity rate. Here we have measured, for the first time, the rate of

polymerase activity using fluorescence rise in situ during PCR cycles that are comparable to

earlier results [20,21].

Although denaturation and annealing are supposed to happen almost immediately once the

appropriate temperatures have been reached, primer extension is not instantaneous. Extension

time is the rate-limiting step in faster protocols of DNA amplification. Elongation time can differ

depending on the template length, type of polymerase, dNTP concentration, salt concentration,

PCR buffer pH etc. Since these are dynamic numbers, their values change in every cycle and

hence the extension times are expected to change accordingly. Although previous workers have

hinted at the possibility of using continuous fluorescence to monitor and optimize reaction times

[10,11,31], our work shows how this can be implemented, at least for the extension step. The

amplification of TGFh1 cDNAwith different extension time demonstrates the dynamic nature of

the polymerase activity. In case of lesser stay time during extension the polymerase activity is

unfinished before it is changed to next step and hence requires more number of cycles to reach

the peak fluorescence. With higher hold time, the extension is finished sooner in the initial cycles


and the fluorescence remains unchanged for rest of the stay time. Overall fluorescence reaches

the peak value earlier but in the process total time taken is longer compared to the above case.

Hence in optimized condition, extension time is varied in every cycle for the polymerase to

finish extension of all the templates. The temperature is allowed to change to next step when the

fluorescence reaches a constant value. The total number of cycles and total reaction time, in this

reaction, is reduced by 40% and 14%, respectively when compared to conventional PCR with

40 s extension time. The improvement can be more substantial in case of longer templates.

Reducing the number of cycles minimizes degradation of polymerase activity since the total

exposure time to high temperature is decreased.

Many efforts have been expended to understand the detailed mechanisms of PCR in order to

increase the speed of the process and simultaneously miniaturize the reaction. One major

advantage in miniaturization is that the rate of heating and cooling can be very fast, lowering the

total time of analysis. In miniaturized devices where the surface to volume ratio is very high,

surface effects becomes important. The adverse interactions between the inner surface of the

microchip and components like template and polymerase are increased [32,33]. Surfaces with

different chemical behavior might lead to different degree of polymerase activity. Our work

suggests that intra-cycle fluorescence monitoring along with adaptive control can be used even

in microchips to track the polymerase activity and optimize cycle number.

5. Simplified description of the methods and its applications

Intra-cycle SYBR Green I fluorescence is recorded continuously during PCR using Lock-In

technique. Fluorescence was measured every 0.5 s during PCR using two different kinds of

polymerase and three different template sizes. The method can be used to track in situ

polymerase extension activity during PCR. Continuous data can be used for adaptive feedback

control of individual cycle parameters to optimize the reaction time.

Acknowledgements

We thank Dr. Debjani Pal, Ms. Saroj Rathore and Ms. E.K. Rosalind for electronics design.

We are grateful to Professor P. Kondaiah, Ms. Prathibha Ranganathan and Mr. Anil Mukund

Limaye for supplying the DNA samples PC1 and TGFh1 for the experiment. We thank

Professor V. Natarajan for his help in fluorescence setup. This work was supported by BRNS,

Department of Atomic Energy, and the Life Science Research Board (LSRB), Government of

India.

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In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements