Practical and theoretical principles underlying realtime PCR

RNA quantification begins with the making of cDNA (complementary DNA) by reverse transcriptase. There are two kinds of RT enzymes readily available on the market, AMV (Peters et al., 2004) and MMLV (Gerard et al., 1997). AMV is a dimeric protein from the avian myeloblastosis virus. MMLV is derived from the Moloney murine leukemia virus and is a monomeric protein. Both enzymes have RNase H activity, which is the ability to degrade RNA in an RNA-DNA hybrid. However, AMVs have higher RNase H activity compared to MMLV enzymes. In both enzymes, the RNase H activity can be separated from the RNA-dependent DNA polymerase activity by mutagenesis. More importantly, AMVs are more processive than MMLV enzymes. That is, they can incorporate more nucleotides per unit enzyme per template molecule. The temperature optimum for the native AMV enzyme is higher than that for MMLVs; 42°C versus 37°C. Cloned variants of both enzymes have pushed the temperature limits much higher: 58°C for AMVs and 55°C for MMLV modified enzymes. From this description, you most likely would deduce that engineered AMV enzymes would be best suited for making cDNAs from RNA samples prior to real-time PCR. However, in practice it is the engineered MMLVs that work best for this purpose. It is not clear why this is true but it has been proposed that although the high temperature of the PCR kills the polymerase activity of both reverse transcriptases, the DNA binding capability remains and may present a physical barrier to Taq polymerase during the PCR. The higher temperature stability and dimeric nature of AMV may be the reason it is less suited for use in real-time PCR. A comparison of the most commonly used reverse transcriptase enzymes is given in Table 1.1 kindly prepared by Dr. Duncan Clark, GeneSys Ltd.

Both of these enzymes have been shown to be inhibitory to PCR in a concentration dependent manner. For this reason it is important to keep the amount of reverse transcriptase as low as possible while still achieving optimal cDNA synthesis. In practice, less reverse transcriptase is required for assay-specific primers than for assays primed by either oligo-dT or random primers because the total amount and complexity of the cDNA made is much reduced. It should be pointed out that cDNA will still be made even if no primers are added to the reverse transcriptase reaction. This is caused by self-priming due to secondary structure within the RNA template.

There are three ways to prime a reverse transcriptase reaction: oligo-dT, random primers or assay-specific primers (Winer et al., 1999; Calogero et al., 2000). There are pros and cons for each type and in the end, the best choice turns out to be tied to the format of the subsequent PCR. Oligo-dT has been used extensively because it will prime primarily mRNAs. However, not all messenger RNAs have poly-A tails and there are poly-A tracts within some RNA sequences. The largest problem with oligo-dT priming is that it limits the selection of the following PCR assay to a region near the poly-A tail of the mRNA. For transcripts that are very long, some are over 15 Kb in length, the assay has to be placed within the 3' UTR (3' untranslated region) of the transcript. Although this will result in high transcript fidelity for the target of interest, it means that the assay most likely will not cross an exon/exon junction. For some transcripts, the 3' UTR sequence can be very A/T rich and thus not very conducive for the design of a PCR assay. The use of random primers removes the 3' UTR bias as cDNA is made throughout the transcript sequence. However, random primers will also make cDNA from ribosomal and transfer RNAs, which makes a much more abundant and complex cDNA population thus increasing the risk of false priming during the PCR. Further, it is possible that priming will be initiated within the PCR amplicon (the sequence amplified during PCR) by one of the random primers, eliminating that molecule from possible detection. A third alternative is the use of assay-specific primers. In this case, the reverse primer for the following PCR is used to prime cDNA synthesis in the reverse transcrip-tase reaction. This ensures, in theory, that every potential transcript

Table 1.1 Commonly used reverse transcriptases

Organism

Molecular weight

Number of amino acids

Single chain or su bun its

Extension rate RNaseH activity

Half life at 37°C (minus template primer)

Half life at 50°C (minus template primer)

Half life at 50°C (plus template primer)

MMLV

Moloney Murine

Leukemia virus 75 kDa 671

Single

MMLV RNaseH-(equivalent to Superscript I)

Moloney Murine

Leukemia virus 56 kDa 503

Single

Yes No (C-terminal deletion of full length enzyme)

435 mins

3 mins

3 mins

MMLV RNaseH-(equivalent to Superscript II)

Moloney Murine

Leukemia virus 75 kDa 671

Single

MMLV RNaseH-(equivalent to Superscript III)

Moloney Murine

Leukemia virus 75 kDa 671

Single

No (single No (single point mutation) point mutation)

390 mins

6 mins

10 mins

>435 mins

220 mins

Avian myeloblastosis virus

2 kb-4 kb/min Yes, strong

110 mins

2 mins

15 mins

Thermus thermophilus

94 kDa

Two Single

63 kDa alpha chain and 95 kDa beta chain -heterodimer

targeted by the PCR assay will have a corresponding cDNA synthesized. Further, the length of each cDNA has only to be the length of the amplicon. For any real-time PCR assay, the maximal length of the amplicon should be, at most 250 bases long.

From the previous discussion, the use of assay-specific primers would appear to be the best choice, and it is for many applications. However, the use of assay-specific primers has two main drawbacks. First it uses more sample RNA as only one assay can be run from each cDNA reaction and second, it cannot be used if a multiplex (more than one real-time assay run in the same PCR) reaction is contemplated. Therefore, the appropriate priming method must be determined by the investigator based on the amount of template available and the kind of real-time assay that is planned.

The business end of real-time PCR detection is, of course, the polymerase chain reaction. The heart of the modern PCR is the addition of a thermostable DNA polymerase. The most commonly used enzyme is from Thermus aquaticus or Taq. Wild type Taq is a 5'-3' synthetic DNA-dependent DNA polymerase with 3'-5' proof reading activity. It also has 5'-nuclease activity. Although wild-type Taq can be purchased commercially, the enzyme most commonly used for real-time PCR is a cloned version of the enzyme that has been mutated to remove the 3'-5' proof reading activity. Taq is a DNA-dependent DNA polymerase and is a very processive enzyme compared to other commercially available thermostable DNA-dependent DNA polymerases, which accounts for its popularity for use in real-time PCR. Table 1.2, kindly supplied by Dr Duncan Clark, GeneSys Ltd., lists comparative information for the most commonly used thermostable DNA polymerases for real-time qPCR (quantitative PCR) as well as Pfu from Pyrococcus furiousus used for standard PCR applications as a reference. The list is not intended to be totally inclusive of all available thermostable DNA polymerases.

There are two versions of Taq commercially available, the engineered polymerase and a hot-start enzyme. Hot-start enzymes may have added components that keep the polymerase from working until they are inactivated at a high temperature over a period of 1-15 minutes. Common additives are either one or more antibodies directed against the active site of the polymerase or one or more heat labile small molecules that block polymerase activity. Also, there are enzymes with an engineered mutation(s) that requires heating for enzymatic activation. Hot-start enzyme activation is accomplished by the high temperature (94°-95°C) of the first denaturing step in the PCR cycle. The difference among them is in how long the enzyme must stay at the high temperature for significant enzymatic activation to occur.

A hot-start enzyme is important for some PCRs as most, if not all, the false priming occurs within the first cycle while the components are being heated past the annealing temperature for the first time. Until the reaction mixture reaches the lowest temperature of the thermocycling program, usually 55° to 60°C, primers can bind and prime new strand synthesis more easily from the incorrect sequence causing false priming. Once a primer has initiated false priming, the sequence of the primer is incorporated into the rogue sequence. Should the other primer also manage to false prime from

Table 1.2 Commonly used DNA polymerases for PCR

Taq DNA polymerase

Tth DNA polymerase

Stoffel fragment

KlenTaq fragment

Pfu DNA polymerase

Organism

Thermus

Thermus

Thermus

Thermus

Pyrococcus

aquaticus YT1

thermophilus HB8

aquaticus YT1

aquaticus YT1

furiosus

Molecular weight

94 kDa

94 kDa

61 kDa

63 kDa

90 kDa

Number of amino acids

832

832

544

555

775

Single chain or subunits

Single

Single

Single

Single

Single

Extension rate

2 kb-4 kb/min

2 kb-4 kb/min

2 kb-4 kb/min

2 kb-4 kb/min

1 kb-2 kb/min

Reverse transcriptase

Minimal/low

Yes, Mn2+ dependent

Minimal/low

Minimal/low

No

activity

Half life @ 95°C

40 mins

20 mins

80 mins

80 mins

>4 hrs

Processivity

50-60 bases

30-40 bases

5-10 bases

5-10 bases

15-25 bases

5'-3' exonuclease

Yes

Yes

No

No

No

activity

3'-5' exonuclease

No

No

No

No

Yes

activity

Incorporates dUTP

Yes

Yes

Yes

Yes

No

Extra A addition

Yes

Yes

Yes

Yes

No

Real versus ideal amplification

20 Cycle no.

Figure 1.1

Graphical representation of the fluorescent signals from an ideal verses an actual reaction over 40 cycles of real-time PCR. In an ideal PCR, there are two phases: a baseline where the signal is below the level of instrument detection followed by a persistent geometric increase in fluorescence that continues over the remaining cycles of the experiment. However in an actual PCR, there are four phases. As in the ideal reaction, there is a baseline followed by a geometric phase. However, the amplification becomes less than ideal leading to a linear phase and finally a plateau where no further increase in signal occurs over the remaining signals. ♦ - Ideal PCR, • - Simulated real amplification curve.

the rogue sequence, an unintended PCR product will result as both primer sequences have been incorporated into the PCR product. However, if the DNA polymerase is not active when the reaction is first heating, there will be no opportunity for false priming to occur at that time. This does not mean that there will never be false priming with hot-start enzymes but it will greatly reduce the number of falsely amplified products. Hot-start enzymes are important when the complexity of the DNA or cDNA population being amplified is very high. Examples are genomic DNA or cDNA made from oligo-dT or random primers. Members of our laboratory have empirically shown that cDNA made from assay-specific primers does not require a hot-start Taq.

Initially, the running of an RT-PCR or PCR experiment was akin to a black box. Reagents were put into a tube with the template, run using the appropriate program on a thermocycler and, if everything went well, a band would appear at the appropriate place on a gel following electrophoresis, at the size of the expected PCR product. With the advent of real-time PCR, it has become possible to look into the tube during the process and 'see' what is happening to the template as it is amplified through multiple PCR cycles. As a result, we have learned a lot about how the reactants, templates and primers affect the final outcome.

Cycle

Figure 1.2

Cycle

Figure 1.2

Sample dilution series of an oligonucleotide standard over a 7-log range, from a real-time PCR experiment. Each amplification curve illustrates the four phases of a polymerase chain reaction experiment: baseline, signal being made but not detectable by the instrument; geometric, detectable signal with maximal PCR efficiency; linear, post-geometric with slowly declining PCR efficiency; and plateau, no or very little new product made.

Prior to embarking on a real-time experiment, it is useful to acquire an understanding of the basic principles underlying the polymerase chain reaction. The amplification of any template is defined by four phases: 1 -baseline; 2 - exponential; 3 - linear and 4 - plateau. The baseline phase contains all the amplification that is below the level of detection of the realtime instrument. Although there is no detectible signal, exponential amplification of the template is occurring during these cycles. The exponential phase is comprised of the earliest detectible signal from a polymerase chain reaction where the amplification is proceeding at its maximal exponential rate. The length of this phase depends on the template concentration and the quality of the real-time assay. In an ideal reaction there are 2 complete molecules synthesized from every one template available in the exponential phase. This is the definition of an assay that is 100% efficient. In the linear phase, the amplification efficiency begins to taper off. Figure 1.1 shows an ideal amplification curve that remains at 100% efficiency versus a more realistic one showing a linear and plateau phase. As can be seen, the linear phase is also a straight line but amplification is no longer 2 products from every 1 template molecule in each cycle but rather degrades to 1.95 from 1 and gradually declines in template replication efficiency further until the fourth phase, the plateau, is reached where amplification rapidly ceases for the remaining cycles of the experiment. Figure 1.2 shows the amplification curves for a real-time PCR assay over a 7-log dilution series of synthetic oligonucleotide DNA template. Note that all the curves are parallel to one another showing that the amplification for each template dilution has the same kinetics. The four phases of amplification can clearly be seen within each individual curve.

It is not clear why the PCR goes through the linear and plateau phases. There are more than adequate reactants to sustain the PCR at an ideal rate for many more cycles than are typically seen in a 40 cycle experiment. There has been speculation in that there are 'toxic' products built up after so many cycles and these lead to a decline and cessation of product amplification. It is more likely that the highly amplified product concentration in the reaction favors association over dissociation and the denaturation of the DNA polymerase over a large number of cycles both lead to a slow down and eventual cessation of amplification. An alternative to exponential amplification, LATE PCR (linear after the exponential PCR) (Sanchez et al., 2004; Pierce et al., 2005), adds one primer at a reduced concentration, but comparable Tm, compared to the other primer so that the PCR proceeds at an exponential rate until it reaches a detectable level. The reaction then runs out of one primer and proceeds by copying one strand only each cycle using the remaining primer. This is called linear amplification. By slowing the reaction to a linear amplification, the occurrence of the plateau phase is greatly delayed to non-existent.

It is worth taking a moment to discuss what happens during the first two cycles of any PCR. If the template is cDNA or a single stranded DNA oligo, the first cycle of the PCR will make every molecule at least partially double stranded, depending on the length of the template and the length of time in the cycle for DNA synthesis. The main point is there is no amplification occurring during the first PCR cycle in this case. On the other hand, if the template is double stranded, like genomic DNA or a double-stranded DNA viral genome, amplification will commence during the first cycle, as both primers will have a template for synthesis. This is particularly important to keep in mind if one is using a double-stranded standard to quantify a single-stranded cDNA template.

All the real-time instruments on the market today are based on the detection of a fluorescent signal. The increase in fluorescence is directly proportional to the increase in the amplified product during the PCR. Fluorescent molecules absorb light as photons within a narrow wavelength range of light. The wavelength at which the dye absorbs light maximally is called the excitation wavelength for that molecule. Following excitation, the molecule is pushed to a higher energy state. This higher energy state is transient and short lived. The excited molecule rapidly decays, falling back to the ground energy state. When this occurs a photon of light is emitted at a longer wavelength. The light that is released is at the emission wavelength. This shift between the excitation and emission wavelengths is called a Stoke's shift (Lakowicz, 1983). For every fluorescent dye, there is an optimal excitation and emission wavelength. A fluorescent molecule can be excited or detected in a narrow range of wavelengths around these optima. Fluorescent molecules with the greatest Stoke's shift are the most desirable as they allow the cleanest separation of the excitation from the emitted wavelengths of light.

A major requirement for any fluorescent assay is that the initial and final signal intensities have as large a difference as possible. This is called the assay delta. All fluorescent assays used for real-time PCR achieve this delta by utilizing FRET (fluorescence resonance energy transfer) (Selvin, 1995). FRET requires two molecules that can interact with one another, at least one of which must be capable of fluorescence. The fluorescent component is called the donor and the second molecule is called the acceptor. During FRET, the donor fluorescent dye is excited by an external light source at or near its optimal excitation wavelength and then emits light at a shifted, longer wavelength (Stoke's shift), as described above. Instead of being detected by the instrument, the emitted light is used to excite an acceptor molecule, which is in close physical proximity. The acceptor molecule absorbs the emitted light energy from the donor dye, effectively quenching the donor signal. The wavelength emitted by the donor molecule must be near the absorbance maximum of the acceptor molecule. The acceptor molecule may or may not emit light. If light is emitted by the acceptor, it will be further shifted and a longer wavelength from that emitted by the donor. The acceptor signal will be detected by the real-time instrument, but it will not be recorded as a reporter signal by the software. FRET depends on the donor and acceptor molecules being in close proximity (10-100 A) and falls off with the sixth power base 10 of the distance between the two molecules. The other major requirement is that the excitation wavelength of the acceptor be close to the emitted wavelength of the acceptor dye (Didenko, 2001). Some of the most common donor and acceptor (reporter and quencher) dyes currently used in real-time PCR are listed in Table 1.3.

There are three classes of fluorescent molecules used in real-time PCR. The three types are defined by their function within an assay. The first is the donor dye and is usually called the reporter. The fluorescent signal from the reporter is the one that is monitored during the course of the experiment. Second, is the acceptor or quencher molecule and is responsible for the initial quenching of the reporter signal. Last is the reference dye. The reference dye is common to all reactions, does not interact with the assay components and is used to normalize the signal from well to well in software.

In theory, any fluorescent dye can be a reporter in an assay. The one used most commonly is 6-FAM (6-carboxy fluorescein). This dye is efficiently excited at 488 nM, the wavelength produced by the argon-ion lasers of the original ABI real-time instrument, the now retired 7700, and the current ABI 7900. However, 6-FAM has remained the first choice for other instruments as well because it can be easily conjugated to oligonucleotides and gives a strong signal. Another reporter in wide use today is SYBR® Green I. Unlike 6-FAM, SYBR® Green I is a free dye in the PCR and works by providing a dramatic increase in fluorescence when bound to double stranded DNA. Examples of some of the many assay chemistries available for realtime PCR will be discussed in Section 1.6.

Quencher molecules can be fluorescent dyes or any molecule that can absorb light energy within the appropriate wavelength range. The original quencher dye used with the 6-FAM reporter was TAMRA (6-carboxy-tetra-methylrhodamine). When coupled to the ends of an oligonucleotide, the FAM signal is effectively quenched by the close proximity of TAMRA, due to oligo folding, while in solution. There are also dark dye quenchers that, as the name implies, quench the reporter signal but emit no light. The first of these was DABSYL (4-(dimethylamino)azobenzene-4'-sulfonyl chloride). More recently, other dark quenchers have come onto the market. Biosearch Technologies markets a series of black hole quenchers (BHQs); Integrated DNA Technologies has two forms of Iowa Black, and Nanogen (formerly Epoch Biosciences) uses its Eclipse dark quencher. It has long been recognized that fluorescein dyes are quenched by guanidine residues. This is the working principle behind the LUX system from Invitrogen (see below) and why probes with fluorescein reporters should never begin with a G residue.

The purpose of a reference dye is to monitor the fluorescence signal from each well and correct for any well-to-well differences in detection efficiency within the instrument. This was particularly important for the ABI 7700, which has a unique fiber optic cable delivering and receiving the excitation and emitted light for each of the 96 wells. The mechanism of sending and receiving light from the plate is quite different in the host of real-time instruments available today. Many still use a reference dye to ensure that the signal from each well of the plate is balanced during data analysis. Depending on the light path used by the instrument and the quality of the thermocycler used, there can be plate 'edge effects' which will require well-to-well normalization. However, if the 'edge effects' become too pronounced, differences between outer and internal wells cannot be normalized by a reference dye. The most common reference dye is ROX (6-carboxy-X-rhodamine). ROX can be found as a short 6-FAM/ROX FRET-based oligo (ABI), a free dye conjugated with glycine (Invitrogen) or as a polyethylene glycol conjugate sold as Super-ROX (Biosearch Technologies). ROX was initially used when 6-FAM was the sole reporter dye. With multiplexing becoming more common, it should be pointed out that ROX does not have to be the dye used for well-to-well normalization as ROX emits light in the middle of the valuable red spectrum. In theory, any dye with a unique spectral signature compared to the ones in the assay could be used. However, it would have to be available in a format that kept it in solution. The Bio-Rad instruments normalize the signal using a second plate with a fluorescein dye prior to the real-time run instead of an internal reference dye in the master mix on the experimental plate.

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