Detection chemistries used in realtime PCR

There are three basic methodologies commonly used in the detection of RNA or DNA targets by real-time PCR and all of them utilize fluorescent dyes. In each case, a low initial fluorescent signal is increased proportionally during each succeeding PCR cycle in tandem with the exponential increase in the DNA product(s) formed.

The simplest assay system involves the incorporation of a free dye into the newly formed double-stranded DNA product. The most used dye for this purpose in real-time PCR is SYBR® Green I (Molecular Probes/Invitrogen). The background fluorescence from SYBR® Green I when in solution as a free dye and stimulated by light of the appropriate wavelength is very low. The same is true for single-stranded nucleic acids at the concentrations used for real-time PCR. In contrast, as the double-stranded DNA product is formed, SYBR® Green I binds to the minor groove of the double-stranded DNA. The DNA-dye complex results in a dramatic increase in fluorescence output, when properly illuminated, of roughly 2,000 times the initial, unbound,

Cepheid

Corbett

Eppendorf

Roche

Roche

Stratagene

Techne

SmartCycler®

Rotor-Gene™

Mastercycler®

LightCycler® 2

LightCycler®

Mx®3005p

Quantica™

realplex4

480

4 LEDs

4 LEDs

96 LEDs

Blue LED

Xenon lamp

Tungsten-

Tungsten-

halogen

halogen

4 Silicon

4 PMTs

2 channel

Photodiodes

CCD camera

PMT

4 PMTs

photo-

PMTs

detectors

All wells

Through tube

96 fiber

Through tube

Plate

Whole plate

optic cables

scanning

450-650 nm

470-625 nm

470 nm

480 nm

450-615 nm

350-635 nm

460-650 nm

500-650 nm

510-665 nm

520-605 nm

530-710 nm

500-670 nm

440-610 nm

500-710 nm

4

4

4

6

6

5

4

16 indepen-

36/72-well

96-well plate

32 Capillaries

96/384-well

96-well plate

96-well plate

dent wells

rotors

plates

25 and

100-200 pl

10-50 pl

10-20 Ml/small

20-100 pl/96

25 pl

15-50 pl

100 pl

100 Ml/large

5-20 pl/384

Solid state

Forced air

Peltier

Heater & fan

Peltier

Modified

8-Peltier

heating/

heating/

Thermocycler

for cooling

Thermocycler

Peltier

forced air

cooling

thermocycler

cooling

Not stated

12-logs

9-logs

9-logs

8-logs

10-logs

9-logs

fluorescent signal (Figure 1.3). This assay system has a very good signal to noise ratio. The popularity of SYBR® Green I assays with real-time PCR users is due to three factors: 1) low cost for the dye; 2) ease of assay development, only a pair of primers is required; and 3) the same detection mechanism can be used for every assay. The down side is that every double-stranded molecule made in the reaction will generate a signal, such as primer dimers or inappropriate PCR products. This fact puts a high premium on good primer design and careful quality control during assay development. It also means that a hot-start DNA polymerase is a must to lessen or eliminate extra-assay signals. A new dye, EvaGreen™ (Biotium, Hayward, CA) has been presented for use in real-time PCR. EvaGreen™ costs more than SYBR® Green I but it is reported to show little PCR inhibition which can be a problem with high concentrations of SYBR® Green I, substantially higher fluorescence over SYBR® Green I, and greater stability at high temperatures. Whether these characteristics will be enough to entice users away from SYBR® Green I remains to be seen. A third new free dye, BOXTO (TATAA Biocenter, Gothenburg, Sweden), has come onto the market. Unlike SYBR®

Denature

Anneal

Extend

Figure 1.3

Figure 1.3

Graphical representation of the incorporation of SYBR® Green I dye resulting in an increase in fluorescent signal during the PCR. Free dye has very low fluorescence and will not bind to single stranded or denatured DNA. During primer annealing, a double-stranded structure is formed and SYBR® Green I dye is bound resulting in a dramatic increase in fluorescent signal. During primer extension by Taq DNA polymerase, the fluorescent signal increases proportionally to the number of SYBR® Green I dye molecules bound per double-stranded molecule. The process is repeated in each cycle with increasing total fluorescence.

Green I or EvaGreen™, BOXTO is optimally excited and emits light at higher wavelengths than SYBR® Green I or EvaGreen™. In principle then, it could be used in the same reaction with a FAM conjugated probe for the detection of primer-dimers or to perform a melt-curve on PCR products, neither of which is possible with a probe-based assay. The most recent additions to the DNA binding dye choices are LCGreen™ I and LCGreen™ PLUS (Idaho Technology, Salt Lake City, UT) and will be discussed in

Chapter 9. More details of the use of SYBR® Green I in real-time PCR can be found in Chapter 8.

There are many quite different dye-primer based signaling systems available for real-time PCR. They range from the very simple LUX™ (light upon extension, Invitrogen) primers to more complex externally and internally quenched primers to the very complex structure of scorpion primers. It is beyond the scope of this chapter to describe each of these primer-based systems (Bustin, 2004). The template specificity for the dye-primer-based assays is the same as for SYBR® Green I. The exception to that rule is the scorpion primer, where the signal generated by the primer is dependent on a complementary match with sequence located within the PCR amplicon. Dye-primer-based assays do allow for multiplexing which is not possible with SYBR® Green I, EvaGreen™ or BOXTO and they do not require the design of another, intervening, fluores-cently labeled probe (see probe-based assays below).

LUX™ primers contain a 4-6 base extension on the 5' end of the primer that is complementary to an internal sequence of the primer near the 3' end. The extension overlaps the position of a fluorescein dye (FAM or JOE) attached to a base near the 3' end of the primer. The design of LUX™ primers initially was a somewhat hit or miss proposition. However, new algorithms used by the program at the Invitrogen web site now make their successful design more assured for the investigator. The reporter dye signal is quenched when in close proximity to the double stranded stem formed within the primer while in solution. Quenching of the fluorescein signal is particularly efficient when in close proximity to guanidine residues. During the first PCR cycle, the LUX™ primer is incorporated into a new strand. At this time, the stem can still be in place. However, when a new complementary second strand is made, the stem and loop are made linear. This structural change results in an increase in fluorescent signal of as much as 10-fold over background (Figure 1.4).

Extended primer (dsDNA)

Hairpin primer Figure 1.4

Single-stranded primer

Hairpin primer Figure 1.4

Structure and mode of action of a LUX™ (light upon extension) fluorescently labeled primer. LUX™ primers are inherently quenched due to a designed, short self-complementary sequence added to the 5' end of the primer. The fluorescent moiety on the 3' end of the primer is quenched in this confirmation. Following the melting and subsequent annealing of the primer to the template, the primer is made linear and is incorporated into the new DNA strand leading to a significant, as much as ten-fold, increase in fluorescence.

Figure 1.5

Structures of the natural guanidine and cytosine bases showing hydrogen bonding compared with the hydrogen bonded iso-base structures. The iso-bases will only base pair with themselves and not the natural dG and dC residues nor dA, dT or dU. They are recognized as dNTPs and incorporated by DNA polymerase along with natural DNA bases into newly synthesized DNA.

A new primer-based assay system that is just coming onto the market is the Plexor system from Promega. The basis for this assay is the use of two isomers of the bases cytodine and guanine, iso-C and iso-G (Figure 1.5) (Sherrill et al., 2004). The iso-bases will only base pair with the complementary iso-base and DNA polymerases will only add an iso-base when the cognate complementary iso-base is present in the existing sequence. One primer is synthesized with a fluorescently labeled iso-C on the 5' end. The PCR master mix contains free iso-dGTP coupled to a dark quencher dye (DABSYL). As each amplification cycle progresses, the fluorescent signal from the free fluorescently tagged iso-C primers is progressively quenched as the labeled primers are incorporated into the growing amplified PCR products (Figure 1.6). The quenching is accomplished following the synthesis of the complementary strand bearing the iso-G-dark dye. Thus, unlike all of the preceding assays where the fluorescent signal goes up, in this assay the reporter signal goes down with increasing PCR cycles. For this reason, special software has been written to facilitate data analysis using the exported raw data file formats from several real-time instruments. One advantage of this system is the ability to multiplex a large number of assays with only primers using a universal quencher (iso-dGTP-DABSYL). Like all primer-based assay systems, the quality of the assay is completely dependent on the specificity of the primers. Therefore, good assay (primer) design will be critical for assay specificity. On the other hand, it may be easier to multiplex five assays in a reaction without the complexity of five probe sequences along with ten primer pairs. This new assay system will have to be tried and put to the test by the real-time community to see if it will become as ubiquitous as the SYBR® Green I and TaqMan® assays are now.

The third category of signaling systems for real-time PCR are those involving a third, fluorescently labeled oligonucleotide located between the primers called a probe. Unlike the two assay systems above that consist

Reporter isoC

isoG + Quencher

Reporter isoG + Quencher

Reporter

isoG + w Quencher

Figure 1.6

Graphic illustrating the operating principle underlying the Plexor® real-time PCR assay. One primer is labeled at the 5' end with an iso-dC and a fluorescent reporter dye. The primer binds to the complementary template and is extended. In the next cycle, a complementary strand is made during the PCR, using a second, unlabeled primer. An iso-dG-DABCYL (4-(dimethylamino)azobenzene-4'-carboxylic acid) base, present in the PCR master mix along with the four natural dNTPs, is incorporated as a complement to the iso-dC in the new strand, The DABCYL dark dye, now physically very close to the reporter, quenches the signal from the reporter dye.

solely of a pair of primers, probe-based assays have the added specificity of an intervening third (or fourth, see below) oligo called the probe. This is because the probe is the fluorescently labeled component of these assays. A further advantage of a probe-based assay is extraneous signals from primer dimers that will be detected by free dye or dye-primer-based assays are not detected by probe-based assays. Extra-assay DNA products larger than primer dimers will also not be detected. The only PCR amplicon that can be

Anneal lTaq TTTTTTi Extend

Figure 1.7

Graphical illustration showing fluorescent signal generation from a TaqMan® probe. When free in solution, the reporter fluorescent dye is quenched by a second molecule using FRET. As the reaction cools toward the annealing temperature, the probe binds to a complementary template sequence at a higher temperature than the primer on the same strand. During primer extension by Taq DNA polymerase, the probe is displaced and degraded by a 5' nuclease present in Taq DNA polymerase. Release of the reporter dye from the probe molecule and its proximity to the quencher dye allows the full signal of the reporter dye to be realized.

detected by a probe-based assay are those to which the primers and the probe are both able to bind simultaneously. There are five distinct probe-based assay systems in use today, TaqMan® probes, Molecular Beacons®, minor groove binding (MGB) probes, Locked nucleic acid (LNA) probes and hybridization probes. In each case, multiple reporter dyes can be used with a variety of quenchers (Table 1.3) to form efficient FRET pairs. Thus, there are many opportunities for multiplexing with probe-based assays.

TaqMan® or hydrolysis probes are linear oligonucleotides that have historically been labeled with a reporter on the 5' end and a quencher on the 3' end, although the opposite orientation can and has been used. When the reporter and quencher dyes are in close proximity in solution, the reporter signal is quenched. The efficiency of the quench is determined by the probe sequence which in turn determines how efficiently the ends of the probe associate in solution. When the probe anneals to its complementary target sequence, the two dyes are maximally separated and the reporter signal detected by the instrument. There are no good predictors in any software program for how well a probe will be quenched in practice. However, there is a direct and inverse correlation between probe length and quenching efficiency. For this reason, TaqMan® probes are kept to less than 30 bases in length.

It is essential that the probe anneal to the target strand sequence prior to the primers to ensure that signal can potentially be generated from every newly synthesized amplicon. Taq DNA polymerase binds and begins to extend the new strand very rapidly following primer binding. For this reason, hydrolysis probes are designed to have a Tm 9-10°C higher than their matched primers. Hydrolysis probes depend on the 5' nuclease activity of Taq DNA polymerase for their cleavage during new strand synthesis to generate a fluorescent signal from the newly freed reporter dye (Figure 1.7). The TaqMan® name comes from this hydrolysis step in an analogy to the action of the old computer game character, Pacman. New thermal stable DNA polymerases have been introduced from other genera of bacteria that do not contain 5' nucleotidase activity. They work well for PCR but cannot cleave hydrolysis probes.

Molecular beacons are similar to TaqMan® probes in that they are labeled on each end with reporter and quencher moieties and, like TaqMan® probes, their Tm is higher than the primers used for the assay. Unlike hydrolysis probes, molecular beacons do not depend upon probe cleavage for signal generation. Molecular beacons have a 4-6 base self-complementary sequence extension on each end. Thus, in solution, the ends of molecular beacons fold into a perfect stem structure bringing the reporter and quencher dyes close together forming a close FRET association. The close proximity of the two dyes leads to very efficient quenching of the reporter signal. During the annealing step, the probe becomes unfolded, the two dyes are separated and the reporter signal detected by the real-time instrument (Figure 1.8).

A minor groove-binding (MGB) protein bearing probes are hydrolysis probes with shorter lengths due to the MGB moiety. As the name implies, an MGB molecule is added to one end of the nucleic acid sequence, which increases the affinity and lowers the Tm requirements of the nucleic acid portion of the probe. There are two companies that sell MGB probes. Applied Biosystems sells a probe with the MGB component and quencher on the 3' end and the reporter on the 5' end of the nucleic acid sequence, similar to TaqMan® probes. Like TaqMan® probes, their MGB probes can be hydrolyzed by Taq DNA polymerase. The second company making MGB probes is Nanogen. They put the quencher and MGB component on the 5' end and the reporter on the 3' end of the probe. The MGB moiety effectively blocks hydrolysis of these probes. Like molecular beacons, signal is generated during the annealing step in the thermocycle. MGB probes from either company are used primarily for SNP (single nucleotide polymorphism) and allelic discrimination assays where high sensitivity to a single base mismatch is required. They can also be used for transcript analysis,

Figure 1.8

Illustration showing the mechanism of signal generation for a molecular beacon probe. Designed complementary sequences 4-6 bases in length at the 5' and 3' ends bring the terminally placed reporter and quencher dyes in very close proximity in solution leading to a much reduced reporter signal via FRET. Following the melting of the template and probe structures, probe molecules anneal to complementary sequences within the template as the temperature is lowered to the annealing set point, At this time, the molecular beacon is completely linear on the template, fully separating the reporter and quencher dyes and resulting in full reporter signal due to the loss of FRET. Unlike TaqMan® probes, molecular beacons do not require hydrolysis for signal generation.

particularly when the G/C content of the template is very low resulting in TaqMan® probes longer than 30 bases.

LNA probes are hydrolysis probes that are much shorter due to the higher binding affinity of locked nucleic acid bases to standard DNA (Goldenberg et al, 2005). LNAs are bicyclic DNA (or RNA) analogues (Figure 1.9). For this reason, LNA-based probes need to be only 8-9 bases long to achieve a Tm of 68-69°C under standard assay conditions. The most extensive use of LNA probes currently is by Exiqon. It has been able to take advantage of the redundancy of nanomeric sequences in the genome to design a relatively small number of LNA probes that can be used by the investigator to design

Base

Base

Locked nucleic acid

Locked nucleic acid

Deoxynucleic acid

Figure 1.9

The deoxyribose ring structures of locked nucleic acids (LNAs) versus the natural bases. The Tm values for oligonucleotides with LNA bases are much higher versus those with natural bases alone.

a real-time PCR assay for every gene in many different genomes, using their web-based software. For the human genome, a set of 90 LNA probes is sufficient for complete coverage of the known transcriptome. LNA probes, due to their short length, can be used effectively in SNP and allelic discrimination assays as well.

Unlike the other probe-based assays described above, hybridization probes consist of two oligonucleotides that anneal between the primers, each with a single fluorescent dye. This probe system uses FRET to generate, rather than to quench, the fluorescent signal. The more 5' probe oligo, closer to the forward primer sequence, has a donor dye on the 3' end and the second probe molecule has an acceptor dye on the 5' end. When the probes anneal, the two dyes are brought into close proximity to one another, only 1-3 bases apart. The detector dye (a fluorescein dye) absorbs energy from the light source and the resulting emitted energy is transferred to the second dye (a rhodamine dye) by FRET. It is the emitted signal from the acceptor dye that is detected by the real-time instrument and recorded (Figure 1.10). Hybridization probes were designed for and are used primarily in the first Roche LightCycler® although they are not limited to those instruments. This assay system works well but has the drawback of requiring an extra probe molecule, which adds to the cost and complicates assay design.

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