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Quantitative PCR and Digital PCR Detection Methods

Introduction

Fluorescent dyes or probes are included in PCR mixes to monitor the change in DNA amplicon concentration as the reaction proceeds. Several popular detection methods, that are well established for the indirect measurement of template in qPCR, are discussed in this chapter. In addition, Dual-labeled Probes and some DNA-binding dyes have been shown to work well in digital PCR (dPCR).

dsDNA-Binding Dyes

Double-stranded DNA (dsDNA) binding dyes function as intercalating and/or minor groove binding agents and emit detectable fluorescence when bound to dsDNA but have a very low background when free in solution. Therefore, fluorescent signal intensity increases proportionately to the quantity of amplicon present. Double-stranded DNA-binding dyes are popular because they are a low cost detection option and do not require additional design considerations.

SYBR® Green I Dye

This dye is the most popular dsDNA-binding dye and has a long history of use in molecular biology. When free in solution, with only single-stranded DNA (ssDNA) present, SYBR Green I dye emits a signal of low intensity (Figure 5.1, Table 5.1). As the PCR progresses and the quantity of dsDNA increases, more dye binds to the amplicons and hence, signal intensity increases (see the animation on the following web page sigma.com/sybr-animation). However, since the dye binds to all amplified products indiscriminately, artefacts such as those resulting from primer dimers or nonspecific binding of the primers also contribute to the overall fluorescence. This can make it difficult to obtain accurate quantification, especially at low template concentrations. However, a post-PCR melt curve analysis can help determine reaction specificity1 (Figure 5.2).

 

Action of SYBR Green I Dye

Figure 5.1. SYBR Green I dye cycles between an unbound (denatured) and a bound (annealing through extension) state as the reaction progresses and signal intensity increases as the quantity of amplicons increase.

 

Table 5.1. Spectral Characteristics of SYBR Green I Dye.

Name Excitation Maximum (nM)a Emission Maximum (nM)a
Reporter Dye
SYBR Green 1 497 520

aWhen complexed to dsDNA

 

Example Melt Curve Analysis

Figure 5.2. Example Melt Curve Analysis. dsDNA-binding dyes bind reversibly, so fluorescence intensity decreases as the reaction temperature increases above the melting temperature (Tm). In conjunction with controls, this type of analysis enables the detection of nonspecific products which melt at different temperatures than the specific product.

 

Probes

In all applications of qPCR, the amplification reaction is driven by specific forward and reverse primers. However, unlike assays relying on detection using dsDNA-binding dyes, probe detection systems do not include a free dye but rather a third oligonucleotide (and sometimes a fourth) conjugated to a reporter dye and/or a quencher moiety.

Dual-Labeled Probes

Dual-Labeled Probes (also known as hydrolysis or alternatively TaqMan® probes) are used in the 5’ nuclease assay2,3, which is the most popular probe detection chemistry (Figure 5.3; also see the animation of the following web page: sigma.com/probe-animation). A Dual-Labeled Probe is a single-stranded oligonucleotide that is labeled with a reporter dye and a quencher moiety. The reporter is located at the 5’ end and the quencher at the 3’ end. The quencher absorbs the natural fluorescence emission of the reporter, usually by Forster-type energy transfer, more commonly referred to as fluorescence resonance energy transfer (FRET). After amplification from the forward primer, the Taq DNA polymerase encounters the probe. The 5’ exonuclease activity inherent in the Taq DNA polymerase then separates the 5’ reporter from the 3’ quencher (Table 5.2, Figure 5.3), which provides a fluorescent signal that is proportional to the amplicon yield.

The hydrolysis probe assay is specific and accurate for quantification of low copy number targets. Specificity can be improved even further by the inclusion of modified nucleotides such as LNA® in the probe (as described below). LNA modified probes are particularly useful for differentiating between single nucleotide polymorphisms (SNPs) or other similar sequences. Dual-Labeled Probes require careful design (see PCR/qPCR/dPCR Assay Design) and are generally more expensive than dsDNA-binding dyes. Furthermore, while amplification of nonspecific products may remain undetected, side reactions may cause the overall reaction to be less efficient and so assays containing probes may still benefit from optimization (see Sample Purification and Quality Assessment).

 

Mechanism of Dual-Labeled Probes

Figure 5.3. Mechanism of Dual-Labeled Probes. Taq DNA polymerase extends the primer situated on the same strand as the probe until it reaches the probe position. The inherent exonuclease activity hydrolyzes the probe from 5’ to 3’, which releases the reporter dye into solution and thereby causes an increase in fluorescence. The measured fluorescence signal is directly proportional to the amount of target DNA.


Table 5.2. Spectral Characteristics of Common Dual-Labeled Probe Reporter Dyes.

Name Excitation
Maximum (nm)
Emission
Maximum (nm)
Compatible
Quencher
Reporter Dye
6-FAM™ 494 515 BHQ®-1, TAMRA
JOE™ 520 548 BHQ-1, TAMRA
TET™ 521 536 BHQ-1, TAMRA
Cal Fluor® Gold 540a 522 541 BHQ-1
HEX™b 535 555 BHQ-1, TAMRA
Cal Fluor Orange 560b 540 561 BHQ-1
TAMRA™ 555 576 BHQ-2
Cyanine 3 550 570 BHQ-2
Quasar® 570c 548 566 BHQ-2
Cal Fluor Red 590d 565 588 BHQ-2
ROX™ 573 602 BHQ-2
Texas Red® 583 603 BHQ-2
Cyanine 5 651 674 BHQ-3
Quasar 670e 647 667 BHQ-3
Cyanine 5.5 675 694 BHQ-3
a JOE/TET alternative
b VIC® alternative
c Cyanine 3 alternative
d TAMRA alternative

e Cyanine 5 alternative

Most real-time PCR thermal cyclers have multiple detection channels enabling flexibility in the choice of probe labels. It is critical to select the reporter dyes that are compatible with the detection channels for the instrument and ensure that the correct filters and calibration are in place. When multiplexing, reporter combinations are required that are as distinct as possible from each other to minimize optical cross talk. Typical reporters include: FAM, HEX, Texas Red and Cyanine 5. Under identical conditions, it is usual to observe differences in emission intensity from different reporters. For this reason, it is advisable to analyze the data from each reporter combination independently (using different threshold settings as appropriate for the probe emission).

Molecular Beacons

Molecular Beacons (also known as hybridization probes) are single-stranded probes that are held in a hairpin-loop conformation (20–25 nucleotides) by complementary stem sequences (4–6 nucleotides) at each terminus4 (Figure 5.4). The hairpin-loop is complementary to the template and the hydrogen-bonded stem sequences allow the 3’ quencher to suppress the fluorescence of the 5’ reporter (Table 5.3) when the probe is free in solution.

 

Mechanism of Molecular Beacons

Figure 5.4. Mechanism of Molecular Beacons. Molecular Beacons hybridize to their specific target sequence causing the hairpin-loop structure to open, separating the 5’ reporter from the 3’ quencher. As the quencher is no longer in proximity to the reporter, fluorescence emission takes place. Unlike Dual-Labeled Probes, the mechanism of detection of Molecular Beacons does not rely on degradation during the reaction. The measured fluorescence signal is directly proportional to the amount of target DNA.


Table 5.3. Spectral Characteristics of Common Molecular Beacons Reporter Dyes.

Name Excitation
Maximum (nm)
Emission
Maximum (nm)
Compatible
Quencher
Reporter Dye
6-FAM™ 494 515 BHQ®-1, DABYCL
Fluorescein 495 520 BHQ-1, DABYCL
JOE™ 520 548 BHQ-1, DABYCL
TET™ 521 536 BHQ-1, DABYCL
HEX™ 535 555 BHQ-1, DABYCL
Cyanine 3 550 570 BHQ-2, DABYCL
ROX™ 573 602 BHQ-2, DABYCL
Texas Red® 583 603 BHQ-2, DABYCL
Cyanine 5 651 674 BHQ-3, DABYCL
Cyanine 5.5 675 694 BHQ-3, DABYCL


LightCyler® Probes

A LightCycler Probe or FRET system (also known as dualhybridization probes) consists of a pair of single-stranded fluorescent-labeled oligonucleotides5,6 (Figure 5.5). Oligo Probe 1 is labeled at the 3’ end with a donor fluorophore dye and Oligo Probe 2 is labeled at the 5’ end with one of a few available acceptor fluorophore dyes (Table 5.4). The free 3’ hydroxyl group of Oligo Probe 2 must be blocked with a phosphate group to prevent DNA polymerase extension.

 

Mechanism of LightCycler FRET Probes

Figure 5.5. Mechanism of LightCycler FRET Probes. During the annealing step, the primers and both of the probes hybridize to their specific target regions, bringing the donor dye into close proximity to the acceptor dye (the probes are usually spaced 1 to 5 nucleotides apart). When the donor is excited by light from the realtime PCR instrument, energy is transferred by FRET from the donor to the acceptor. The emission wavelength for the dye on the acceptor probe is detected. The increase in fluorescence signal is directly proportional to the amount of target DNA.

 

Table 5.4. Spectral Characteristics of LightCycler Acceptor and Donor Fluorophore Dyes.

Name Excitation
Maximum (nm)

Emission
Maximum (nm)

Oligo Probe 1: Donor Fluorophore
3’ Donor Fluorophore (Fluorescein) 495 520
Oligo Probe 2: Acceptor Fluorophore/Reporter Dye
5’ Acceptor Fluorophore
(LC Red 610, 640, 670, and 705)
N/A 610, 640, 670,
and 705


Scorpions® Probes

Scorpions® probes are available in two forms; uni-probe and bi-probe. The uni-probe consists of a stem-loop structure, similar to a Molecular Beacon but this is attached to the forward primer with a PCR blocker located between the two oligo sections7 (the blocker prevents Taq DNA Polymerase from extending the primer (Figure 5.6A, Table 5.5).

The bi-probe structure is a duplex with the 5’ reporter, probe sequence, PCR blocker and forward primer on one strand and the 3’ quencher on the other strand; the quencher strand is duplexed with the reporter strand (Figure 5.6B).

 

Mechanism of Scorpions® Probes

Figure 5.6. Mechanism of Scorpions® Probes. The uni-probe A) has all probe components on one strand, whereas the bi-probe B) has the probe components on two strands. Both formats of Scorpions® Probes include the forward PCR primer. This forward primer is extended to become part of the newly formed amplicon. During annealing/extension, the probe sequence in the Scorpions® hybridizes to the template, which separates the reporter from the quencher and thereby results in a fluorescent signal. As the tail of the Scorpions® and the amplicon are part of the same strand, the detection interaction is intramolecular and therefore more rapid than other probe detection systems. The template is typically chosen to be between 5 and 50 bases from the 3’ end of the Scorpions® primer. Both the uni-probe and bi-probe Scorpions® require a separate reverse primer.

 

Table 5.5. Spectral Characteristics of Common Scorpions® Reporter Dyes.

Name Excitation
Maximum (nm)
Emission
Maximum (nm)
Compatible
Quencher
Uni-Probe and Bi-Probe: Reporter Dye
6-FAM™ 494 515 BHQ®-1, DABYCL dT
JOE™ 520 548 BHQ-1, DABYCL dT
TET™ 521 536 BHQ-1, DABYCL dT
HEX™ 535 555 BHQ-1, DABYCL dT
Rhodamine Green™ 504 532 BHQ-1, DABYCL dT
Oregon Green® 488 490 514 BHQ-1, DABYCL dT
Oregon Green 514 489 526 BHQ-1, DABYCL dT
TAMRA™ 555 576 BHQ-2, DABYCL dT
Cyanine 3 550 570 BHQ-2, DABYCL dT
ROX™ 573 602 BHQ-2, DABYCL dT
Texas Red® 583 603 BHQ-2, DABYCL dT
Cyanine 5 651 674 BHQ-3, DABYCL dT
Cyanine 5.5 675 694 BHQ-3, DABYCL dT

 

Quenchers

Most probe detection systems require a quencher moiety. Some of those used in the original probes structures were acceptor fluorescent dyes, e.g., TAMRA, which work well with FAM but are not suitable for other dyes. As a reporter dye itself, TAMRA produces fluorescence and therefore can result in a poor signal-to-noise ratio. For this reason, dark quenchers which emit heat instead of light, such as Black Hole Quencher® (BHQ®) were developed by Biosearch Technologies. The choice of dark quenchers as popular alternatives to dye molecules provides quenching over a wide range of wavelengths and opens the possibility of multiplex reactions containing a larger number of target/probe combinations.

Onyx Quencher™ (OQ™) is a proprietary dark quencher from Sigma-Aldrich. Four derivative versions (OQA, OQB, OQC and OQD) are available. As shown in Table 5.6, the four Onyx Quenchers are compatible with a variety of popular reporter dyes.

The data presented in Figure 5.7 show amplification of an artificial template that was derived from a synthetic oligo for the optimization of a Schistosoma mansoni target assay8. Detection was using a FAM-labeled probe with a comparable dark quencher, CDQ (A) or OQA (B). From these data, it is evident that both the CDQ and the OQA have equivalent performance, with similar background fluorescence and similar Cq values for analyzed data from template of the same concentration.

In summary, OQ is equivalent in performance to CDQ and importantly, is available license, restriction and royalty free for any application. This makes the Onyx Quencher an excellent and cost effective choice for the development of commercial kits and reagents for molecular diagnostics that contain qPCR probes.

Table 5.6. Onyx Quencher or OQ is Compatible with a Variety of Common Reporter Dyes.

Name Compatible Quencher
Reporter Dye
FAM, HEX, TET, JOE, Rhodamine 6G OQA
TAMRA, Cyanine 3 OQB
ROX, Texas Red OQC
Cyanine 5, Cyanine 5.5 OQD

 

Dilutions of an artificial oligo were amplified using 250 nM primers

Figure 5.7. Dilutions of an artificial oligo were amplified using 250 nM primers and the amplicon detected with a Dual-Labeled Probe at 200 nM. A) The probe was labeled with FAM and quenched with CDQ or OQA (as indicated) and the raw fluorescence data are shown. B) The probe was labeled with FAM and quenched with CDQ or OQA and the baseline corrected data are shown. There are no significant differences between the performances of either probe.

Nucleic Acid Analogs

A number of modifications can be used to produce oligonucleotides with altered biochemical properties. These modifications generally confer a higher Tm which can be manipulated to provide improved specificity. This allows for assay design in regions of challenging sequence or when a single oligonucleotide is required to detect several sequences, e.g., all serotypes of a virus.

Locked Nucleic Acid™ (LNA®)

LNA® is an RNA analog base (Figure 5.8) that yields enhanced sensitivity and specificity when incorporated into probes9. Probes with LNA bases have greater thermal stability and therefore hybridize more strongly to the template. Each LNA base may increase the Tm of the probe by up to 8 °C10, which makes LNA a powerful tool in SNP discrimination assays11 (a single base mismatch has a greater destabilizing effect on duplex formation than without LNA), multiplexing (allows for simpler Tm optimization) and problematic target sequences (LNA probes can be shorter, which allows them to be designed around problems such as AT- or GC-rich regions, repetitive sequences, or sequences with significant secondary structure).

LNA® also provides protection against nuclease digestion making it suitable for in vivo use12.

 

Comparison of LNA and DNA nucleotide structures

Figure 5.8. Comparison of LNA and DNA nucleotide structures. LNA differs from DNA in that LNA contains ribose with a methylene bridge between the 2’ oxygen and 4’ carbon, which ‘locks’ the ribose in the 3’ endo conformation, whereas DNA contains 2’-deoxyribose with no methylene bridge.

 

Zip Nucleic Acid (ZNA®)

ZNA-modified oligos have been demonstrated to increase hybridization affinity for their targets by decreasing the electrostatic repulsions resulting from the polyanionic nature of nucleic acids13. This is achieved by conjugating spermine derivatives as cationic moieties (Z units) to an oligonucleotide (Figure 5.9). ZNA molecules are versatile as the number of Z units is variable and can be placed throughout the oligonucleotide: 5’, 3’ or internal. By selecting the number of cationic units, the global charge of the ZNA molecule can be modulated.

 

ZNA structure and mechanism

Figure 5.9. ZNA structure and mechanism. A) Structure of oligonucleotide with Z unit at the 5’ end. The number and placement of the units is variable. B) ZNA functions by reducing the charge repulsion between the oligonucleotide and target and hence, zips up the molecules.

 

ZNA oligos are reported to be easy to design and specific, offer improved performance at high annealing temperatures, effective at very low concentrations, provide robust amplification in AT-rich regions and efficient under short cycling conditions14.

Summary

There are many considerations when selecting a detection method for a particular application. While SYBR Green I dyes and Dual-Labeled Probe assays are popular and generally work well, there are situations in which the other detection systems might be preferable. Table 5.7 can be used as guidance during early assay development.

 

Table 5.7. Relative Effectiveness of Detection Methods for Common Applications.

Application SYBR
Green I
Dual-
Labeled
Probes
Molecular
Beacons
LightCycler
Probes
Scorpions®
Probes
Mass Screening XX        
Microarray Validation XX X      
Multiple Target Genes/
Few Samples
X X      
SNP Detection     X X XX
Allelic Discrimination     X X XX
Pathogen Detection X X X X XX
Multiplexing   XX XX X XX
Viral Load
Quantification
  X X X XX
Gene Expression
Analysis
X XX XX XX XX
Gene Copy
Determination
  X X X XX
End-point
Genotyping
    XX   XX
In vitro Quantification     XX    

Blank spaces indicate that the detection method is not recommended; X = good performance; and XX = better performance.

References

  1. Ririe, K.M., Rasmussen, R.P., Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154-160
  2. Holland, P.M., Abramson, R.D., Watson, R., et al. Detection of specific polymerase chain reaction product by utilizing the 5’----3’ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 1991; 88: 7276-7280
  3. Heid, C.A., Stevens, J., Livak, K.J., et al. Real time quantitative PCR. Genome Res 1996; 6: 986-994
  4. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 1996; 14: 303-308
  5. Wittwer, C.T., Herrmann, M.G., Moss, A.A., et al. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 1997; 22:130-138
  6. Bernard, P.S., Ajioka, R.S., Kushner, J.P., et al. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am J Pathol 1998; 153: 1055-1061
  7. Whitcombe, D., Theaker, J., Guy, S.P., et al. Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 1999; 17:804-807
  8. PCR Technologies: Current Innovations. 3 ed. CRC Press; 2013
  9. Costa, J.M., Ernault, P., Olivi, M., et al. Chimeric LNA/DNA probes as a detection system for real-time PCR. Clin Biochem 2004; 37: 930-932
  10. Petersen, M., Wengel, J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 2003; 21: 74-81
  11. Johnson, M.P., Haupt, L.M., Griffiths, LR. Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. Nucleic Acids Res 2004; 32: e55
  12. Wahlestedt, C., Salmi, P., Good, L., et al. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci U S A 2000; 97: 5633-5638
  13. Noir, R., Kotera, M., Pons, B., et al. Oligonucleotide-oligospermine conjugates (zip nucleic acids): a convenient means of finely tuning hybridization temperatures. J Am Chem Soc 2008; 130: 13500-13505
  14. Moreau, V., Voirin, E., Paris, C., et al. Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription. Nucleic Acids Res 2009; 37: e130.