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Monochrome Multiplexing in PCR by Photobleaching of Fluorogenic Hydrolysis Probes Friedrich Schuler, Martin Trotter, Roland Zengerle, and Felix von Stetten Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02960 • Publication Date (Web): 03 Feb 2016 Downloaded from http://pubs.acs.org on February 14, 2016

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Analytical Chemistry

Monochrome Multiplexing in PCR by Photobleaching of Fluorogenic Hydrolysis Probes Friedrich Schulera,b,*, Martin Trottera, Roland Zengerlea,b,c, Felix von Stettena,b a

b

Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany

Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany

c

BIOSS- Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

Keywords: PCR, multiplexing, photobleaching, fluorogenic probe, digital PCR, dPCR, Abstract Multiplexing in Polymerase Chain Reaction (PCR) is a technique widely used to save cost, sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels several methods for monochrome multiplexing have been suggested. However, they either pose restrictions to the amplifiable target length, the sequence or the melting temperature. To circumvent these limitations we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their

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different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as FAM) and Atto467N (emitting at the same wavelength as Cy5) were found to bleach significantly less than FAM and Cy5 i.e. the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100,000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover a 2-plex digital PCR reaction in 1 color channel was shown. In future the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as FRET probes and immunoassays.

Introduction Multiplex PCR was first developed in 1988 by Chamberlain et al. 1. The concept to distinguish different amplification products by fluorophores of different wavelength was developed in 19932, and has since been used in a variety of applications to save cost 3–6 and precious sample material. Much effort has been invested to further optimize multiplex PCR

7,8

. Multiplex PCR has been

used to detect viruses, 9,10 bacteria, 11 as well as antibiotic resistances of bacteria 12. The positive impact of multiplex PCR on clinical practice has been shown

13

. Moreover, it has been used to

monitor food quality 14, especially to detect genetic modifications 15,16. When one multiplex reaction mixture is used to detect more than one potential target, different methods are used to detect which target molecule has been amplified. The most common is the use of different fluorescently labeled probes 2 (including hydrolysis probes 17, molecular beacons


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and mediator probes 19). The wavelength of emission is detected to correlate the signal to the

amplified PCR product. However different other approaches exist, such as melting curve analysis 20,21

which relies on the fact that the melting temperature of the probe-DNA dimer depends on

the target length and sequence. Methods combining the two above mentioned techniques exist as well

22,23

. The use of unspecific intercalating dyes offers the advantage of sequence independent

melting curve analysis at one excitation wavelength. Hydrolysis probes17, also called TaqMan probes, are common in PCR. A short DNA oligonucleotide is labeled with at least one fluorescent dye and at least one quenching compound (usually each at one end of the oligonucleotide). As long as the fluorescent dye and the quencher are close to each other, Förster resonance energy transfer (FRET)

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transfers the energy of the

fluorescent dye to the quencher and the fluorescence is low. However, when the distance between the two increases due to probe digestion by the nuclease activity of the polymerase during PCR, the fluorescence signal increases (the probe is “dequenched”). A technique used in digital PCR (dPCR) is multiplexing by dilution of probes

25–27

. Probes

with different sequences and the same fluorophore are added at different concentrations. If the probe is depleted during PCR, the absolute fluorescence after PCR can be used to deduce which probe was dequenched during amplification. Another approach to the problem was taken by using different probes for the same target labeled with up to four fluorophores, one per probe similar to a barcode being assigned 28,29. Probes labeled with different fluorophores but the same sequence, are added to the reaction at a unique ratio, so that the fluorescence intensity of all four color channels can be compared and the relative fluorescence intensities are matched to one of the used ratios of fluorophores. Another approach applicable to SNP genotyping uses a more complicated double stranded probe design

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. More recently it was shown that different lengths


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of amplified target can be used in dPCR to distinguish targets

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31,32

. The duplex is stained

unspecifically and the fluorescence intensity of the completed reaction depends on the length of the duplex strand. Again the absolute fluorescence is used to distinguish different targets. Furthermore, distinguishing different fluorochromes by their lifetime has been proposed33,34. However since the lifetime of a fluorophore is in the nanosecond range it is impossible to distinguish differences in these time regimes with current PCR machines that usually integrate over several microseconds. Although prior studies have shown that there is a variety of multiplexing options, there is need to add a versatile approach to the current portfolio that poses no restrictions to the target length, sequence or melting temperature of the PCR amplicon. Typical laboratory equipment can detect 2-5 different color channels

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. However increasing the number of color channels increases

complexity since it has to be ensured that there is no spectral overlap between the channels but suitable fluorescent dyes exist to be used in the according spectral region. Usually the cost associated with additional color channels is high, compared to the base price of the instrument. Even if cost is not an issue, it is not practical to increase the number of color channels to very high numbers since most of the spectrum is already covered by existing channels. Therefore, additional multiplexing options need to be developed which do not rely on differentiation by color. In our study we used standard hydrolysis probe based PCR, including probes labeled with readily available dyes. In contrast to another method 20 it requires no special consideration when designing the assay and uses standard laboratory devices for processing. Moreover, it does not depend on the absolute fluorescence intensity after PCR completion as other do. Systems relying on absolute fluorescence intensity differences

28,29,32

require very carefully designed assays to

ensure that no bias in reaction efficiency interferes with the absolute measurements. In addition


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to that, the price and complexity of assays increase dramatically if more than one labeled probe is needed per target. In order to further increase the multiplexing opportunities, to make them independent of the afore mentioned interdependencies and to increase the number of targets that can be detected per color, the idea of our study was to use the decay of fluorescent molecules upon irradiation (photobleaching) to distinguish the amplification products (see Figure 1). We investigated whether the differences in photobleaching behavior between dyes would be large enough to reliably distinguish amplification of two target DNA sequences on the same color channel. The novel method would be independent of target length, sequence specific and would not rely on absolute fluorescence intensity differences. Since hydrolysis probes are very common 36 and can be labeled with a large variety of fluorophores they were chosen as demonstrator in our study.


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Figure 1 Schematic of the investigated monochrome multiplexing concept. Two target sequences (A and B) are amplified by duplex PCR. Target specific hydrolysis probes labeled with fluorophores that are identical in their emission wavelength, but different in their photobleaching behavior, are dequenched during formation of amplification products. Both show a signal increase in the same fluorescence channel. The final fluorescence level can be the same or different (as depicted here). After PCR an external light source is switched on and the fluorescence intensity decreases due to photobleaching. For slowly bleaching dyes such as Atto647N and Atto488 the relative decrease is small (∆A), for fast bleaching dyes such as Cy5 and FAM the relative decrease is large (∆Β).


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Experimental Section PCR workflow The hydrolysis probe 4-plex PCR reaction mixture consisted of 2x QuantiFast Multiplex master mix (Qiagen, Stockach, Germany), primers and probes were adapted from37 and used at 500 nM (except mecA forward and reverse primer which was used at 700 nM concentration, which improved the limit of detection) and 200 nM concentration respectively except mecC probe, which was used at 20nM concentration and DNA/RNAse free water (Invitrogen, Carlsbad, USA). Final volume was 10 µl per reaction. For sequences please refer to the Table S1. DNA templates used were synthetic sequences of the relevant regions, namely parts of mecA resistance gene, mecC resistance gene, nuc region and Panton-Valentine leucocidin (PVL) region. The PCR was performed in 0.1 ml tubes using a Rotor-Gene Q (Qiagen, Stockach, Germany). The cycling protocol consisted of 5 min at 95 °C followed by 40 cycles of 15 sec 94 °C and 40 sec 58 °C. The green and red channels were used for readout of FAM/Atto488 and Cy5/Atto647N respectively. For the subsequent fluorescence measurements during the bleaching procedure the same machine was used. For this, the cycling time was set to 1 sec at 28 °C, 15 “cycles” were programmed to allow for stabilization of temperature in the tubes. The machine was then paused, the sample removed from the holding ring. The sample was irradiated with a white LED (20 W 230 V LED (TechBox)) for 1 min. Subsequently the sample was transferred back to the machine for subsequent measurement. The process was repeated 6 times. dPCR workflow The TaqMan PCR reaction mixture consisted of Bio-Rad 2x ddPCR supermix for probes (BioRad, Munich, Germany), primers and probes (200 nm and 300 nm respectively) and DNA/RNAse free water. For sequences please refer to the Table S1. DNA templates used were


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B. subtilis DNA (extracted from B. subtilis culture) and E. coli DNA (Invitrogen, Carlsbad, Germany). The droplets were generated and treated according to manufacturer’s protocol using a Bio-Rad QX100 system. The cycling protocol consisted of 95 °C for 10 min and 40 cycles of 30 s at 95 °C and 60 s at 60 °C followed by 98 °C for 10 min and was performed in a Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad, Munich, Germany). Afterwards the droplets were transferred to a custom made read-out chamber on a microscope slide format polymer chip manufactured by Lab-on-a-Chip Design & Foundry Service of Hahn-Schickard (see Figure S2 for details). The fluorescence readout of the chip was performed in a LaVision Bioanalyzer (4F/4S, LaVision BioTec GmbH , Bielefeld, Germany) with an integration time of 150 ms. A mercury-vapor lamp filtered by 482 nm excitation filter and 536 nm emission filter was used as light source. Between the measurements the sample was irradiated with the scanner’s excitation light for 1 min. The sample was measured each minute to generate a decay curve. Bleaching for 10 minutes without measurements in between did not show any differences. Image analysis of the droplets was performed with ImageJ 38(NIH). For a verification experiment 4 different concentrations of E. coli DNA (~220, 440, 880, 1760 copies µl-1) were mixed with an unchanged concentration of background B. subtilis DNA (~200 copies µl-1). The template DNA was mixed with mastermix containing primers and probes for both targets and subjected to droplet digital PCR (ddPCR) reaction. For each concentration the fluorescence intensity of 150 droplets was measured before and after bleaching. Fluorescence measurements were performed each minute during a total bleaching period of 10 minutes. The intensity values at 0 min were used to distinguish positive droplets from negative droplets by a threshold. Afterwards the intensity values of each droplet were normalized with the value at 0 min set to 1. Subsequently, the normalized fluorescence at 10 min was used to distinguish


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droplets in which the Atto488 probe was dequenched (>75% of initial fluorescence intensity) and droplets in which the FAM probe was dequenched (99.9%. The data points include interand intra-experimental variability. 3 experiments with 4 replicates each were performed for each data point. Table 1 Final fluorescence of PCRs with fluorogenic probes bearing different fluorophores being activated. The fluorescence after bleaching for 6 minute in white light is compared to the initial fluorescence before bleaching. Both dyes were present in the concentration of 200 nM except Cy5 labeled probe which was used in 20 nM concentration. The DNA concentration was 105 cp. µl-1 in all cases. Dye

Final fluorescence

Atto488

88%

Atto488&FAM

40%

FAM

25%

Atto647N

95%


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Atto647N&Cy5

53%

Cy5

21%

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To account for samples that might have different concentrations of target DNA an experiment was designed for both color channels containing zero, low, medium and high concentrations of each target and every combination. It can be clearly seen, that the level of confidence for the nuc/PVL system is not as good as for the mecA/mecC system. This is most likely due to the relatively poor performance of the assay, as can be seen from real-time PCR curves (see SI). Moreover, it seems that the amplification of nuc target DNA fails if an excess of PVL target DNA is in the PCR mix and vice versa (compare Table S2). The dyes used for nuc and PVL, namely Atto488 and FAM, respectively, perform well when used with another assay in ddPCR (compare Figure 4). Table 2 Level of confidence with which the given combination can be distinguished from the corresponding experiment in which only one target was amplified. In all experiments the same 4plex assay was used. For each value 4 replicates were performed. Data points for 100 copies were not included as the amplification is unreliable at these concentrations (compare Table S2). For amplification plots see SI.

Monochrome multiplexing in digital PCR Since the basic concept was demonstrated in the experiments mentioned above the concept was also investigated as an additional multiplexing approach for dPCR. Since also in droplet


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digital PCRs one droplet might contain more than one target molecule three different populations of droplets were observed (compare Figure 4): those containing only dequenched Atto488, those containing only dequenched FAM and those containing both unquenched dyes. Using the decay rate of the fluorescence signals of individual droplets it was possible to deduct which probe was dequenched during ddPCR and which target (E. coli or B. subtilis DNA) was present in the droplet.


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Figure 3: Quantification of target DNA using monochrome multiplexing in droplet digital PCR (ddPCR). A) 4 samples with varying amounts of E. coli DNA (220, 440, 880, 1760 cp. µl-1) and constant amounts of B. subtilis DNA (200 cp. µl-1) were co-amplified. Amplification of E. coli DNA resulted in dequenching of a slow bleaching Atto488 labeled probe, while amplification of the B. subtilis DNA resulted in dequenching of a fast bleaching FAM labeled probe. Both, FAM and Atto488, emit light at comparable wavelengths and were discriminated by monochrome multiplexing method according to (A). The abscissa shows input copy numbers of E. coli DNA, the ordinate shows the E. coli DNA concentrations that were measured in 4 digital PCR experiments after the bleaching step. B) The same procedure was applied to 3 different concentrations of B. subtilis DNA (200, 632, 2000 cp. µl-1) and a constant background of E. coli DNA (600 cp. µl-1). 150 droplets were evaluated per experiment.


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Figure 4 Fluorescence decay in droplet digital PCR (ddPCR). Normalized decay of fluorescence intensity of >200 positive droplets from 3 different experiments (for each dye except “Atto488 and FAM”: ~50 droplets from 3 experiments) containing both FAM and Atto488 labeled probes, the named one being dequenched. The light source of the scanner was used for bleaching, which explains deviations in the half-life of the FAM signal.

The decay of fluorescence intensity of the dye is slower than in the previous tube experiments. This is most likely due to the different light source, since this time the excitation light of the fluorescence scanner was used instead of a separate white light LED. In order to verify that the number of target molecules is correctly quantified by this method four samples of a 2-fold dilution series of E. coli DNA were spiked with a comparable, constant amount of B. subtilis DNA and then evaluated in ddPCR (compare Figure 3A)). The measured concentration in the samples correlates well with the expected concentration. A comparable result is achieved when varying B. subtilis concentration with E. coli concentration kept constant as can be seen from Figure 3 B). This shows that droplets containing B. subtilis DNA that dequenched the FAM


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labeled probes during amplification were not wrongly interpreted as E. coli containing droplets and vice versa. Fluorescent images of the droplets containing FAM dequenched hydrolysis probes and Atto488 dequenched hydrolysis probes are depicted in Fig. 5.

Figure 5: Photobleaching in droplet digital PCR. A) Micrographs taken before and after 10 minutes of irradiation with white LED light. Bright droplets in the “before bleaching” images contain FAM dequenched dye, some are marked with red arrows for visualization. All bright droplets in the “after bleaching” images contain Atto488 dequenched hydrolysis probes since FAM fluorescence intensity is reduced to background level after 10 minutes of bleaching, as can be seen from the droplets marked with the arrows. The yellow numbers refer to the expected number of E. coli targets per µl. Each image shows the droplets after digital droplet PCR in the readout chip (compare Fig. S2). All of the droplets contain the same PCR mastermix, the target DNA is distributed statistically. B) Fluorescence intensity decay of a droplet with dequenched FAM probe over the course of 10 minutes (white numbers).


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Conclusion Multiplexing by photobleaching of fluorogenic probes has successfully been demonstrated in standard PCR and digital PCR reactions. A 4-plex PCR using only the green and red (FAM and Cy5) color channels has been shown. To demonstrate that the concept can be extended to other color channels a proof-of-principle was shown with fluorescent dyes from the yellow color channels (see Figure S1). In future, the principle of multiplexing by photobleaching may be extended to further fluorophores with the potential to double or even triple the degree of multiplexing in multiple color reactions. In order to find fluorophores with similar emission wavelengths and different bleaching behaviors a larger number of fluorophores should be evaluated and tested. In contrast to other multiplexing concepts, photobleaching poses no restrictions to the target length, sequence or melting temperature. It uses standard hydrolysis probes, standard cycling protocols and is compatible with other multiplexing strategies. Targets can be distinguished at the same color with a confidence > 99.9%, if it is known before the experiment, that only one of the possible targets is present per experiment and the targets are present in high concentrations of 100,000 cp. µl-1. If the possibility exists, that both targets are present in the same experiment or lower concentrations of targets are present, the level of confidence is reduced to >68%. The reduced confidence level is most likely due to the relatively poor performance of the chosen assay. Thus assay performance needs to be carefully studied when designing a 4-plex assay. In our experiments, two additional steps needed to be performed, the bleaching and the second measurement of the sample. However, integration of a light source for bleaching into the PCR or dPCR-devices could eliminate additional handling steps.


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Multiplexing by photobleaching might be especially interesting to isothermal amplification reactions, where cheap heating elements are used that offer no precise control over temperature and therefor make melting curve analysis impossible. Quantum dots are very resistant against photo bleaching and have a very narrow emission spectrum45 and could be used once they are available in standard PCR hydrolysis probes. The novel approach may also be expanded to other probe formats than hydrolysis probes, such as molecular beacons, QG probes44, or FRET probes, and to other assays than PCR, such as homogeneous immunoassays or cell based assays.

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Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Fax: +49 761 20373299 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources We gratefully acknowledge financial support from EU Framework 7 project “ANGELab“ Grant agreement number: 317635. ACKNOWLEDGMENT


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We want to thank Simon Wadle and Michael Lehnert for helpful discussions, Xuanye Wu for help in the lab and Marie Follo from the University Hospital Freiburg, Germany for her help with producing droplets, the Lab-on-a-Chip Design & Foundry Service of Hahn-Schickard for manufacturing of readout chips and Gerhard Birkle for help with the production of equipment. ABBREVIATIONS PCR, polymerase chain reaction; dPCR, digital PCR; ddPCR, droplet dPCR; DNA, deoxyribonucleic acid; LED, light emitting diode; FAM, 6-carboxyfluorescein; Cy5, cyanine 5; FRET, Förster resonance energy transfer

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FOR TOC ONLY:


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Monochrome Multiplexing in Polymerase Chain Reaction by

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